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Sharma, Saurabh, Tejraj P. Kale, Lingaraj J. Balihallimath, and Abhishek Motimath. "Evaluating Effectiveness of Axiostat Hemostatic Material in achieving Hemostasis and Healing of Extraction Wounds in Patients on Oral Antiplatelet Drugs." Journal of Contemporary Dental Practice 18, no. 9 (2017): 802–6. http://dx.doi.org/10.5005/jp-journals-10024-2130.
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ABSTRACT Aim The aim of this study was to evaluate the efficacy of Axiostat Hemostatic Dental dressing in achieving hemostasis postextraction and determining its effect on pain and healing of the extraction wound, compared with control, i.e., conventional method of extraction in patients on oral antiplatelet therapy. Materials and methods Totally, 40 patients on oral antiplatelet drugs were included in the study and overall 80 extractions were done applying split mouth study design, without altering patient's drug regime. Extraction sites were divided into two groups: Group I received Axiostat Hemostatic Dental Dressing (study site), and group II received conventional method; pressure pack with sterile gauze under biting pressure followed by suturing if required (control site) was used to attain hemostasis. Results Extraction sites treated with Axiostat Hemostatic Dressing achieved hemostasis earlier (mean 1 minute 13 seconds) compared with control sites (mean = 14 minutes 1 second), which was also statistically significant (p < 0.001). Postoperative pain was considerably lower and significantly better healing was seen in the study group (p < 0.001) compared with the control. Conclusion Axiostat demonstrated to be an effective hemostatic agent that considerably lessens the bleeding time in patients on oral antiplatelet drugs postextraction. In addition, it even offered minimal postoperative pain and improved healing of the extraction wound. On comparing the results of this study with our study on HemCon Dental Dressing, Axiostat Dental Dressing (ADD) is found to be as effective and at par in achieving hemostasis in patients on oral antiplatelet therapy. Clinical significance The past few decades have seen an upsurge in use of low-dose aspirin either alone or in combination with other drugs. When these patients require dental/maxillofacial treatment, earlier concept of stopping these medications is associated with increased risk of thromboembolic event. The present study highlights an alternative approach using ADD which aids in quick hemostasis, accentuates healing, and reduce postoperative pain. How to cite this article Sharma S, Kale TP, Balihallimath LJ, Motimath A. Evaluating Effectiveness of Axiostat Hemostatic Material in achieving Hemostasis and Healing of Extraction Wounds in Patients on Oral Antiplatelet Drugs. J Contemp Dent Pract 2017;18(9):802-806.
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Chandra, Rakesh K., David B. Conley, and Robert C. Kern. "The Effect of FloSeal on Mucosal Healing after Endoscopic Sinus Surgery: A Comparison with Thrombin-Soaked Gelatin Foam." American Journal of Rhinology 17, no. 1 (January 2003): 51–55. http://dx.doi.org/10.1177/194589240301700109.
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Background The optimal form of nasal packing after endoscopic sinus surgery (ESS) still has not been established. Although wide variations exist among sinus surgeons, the goals are adequate hemostasis, rapid healing, and patient comfort. Preliminary studies indicated that FloSeal (FS), a novel absorbable hemostatic paste used as a nasal pack, was associated with minimal postoperative discomfort and effective hemostasis. This study was designed to evaluate the effects of this agent on mucosal healing in ESS. Methods Twenty consecutive patients underwent bilateral ESS. For each patient, one ethmoid cavity was randomized to receive FS and the other received thrombin-soaked gelatin foam. The extent of granulation tissue and adhesion formation was evaluated at 6–8 weeks after surgery. Results No significant differences were observed between the FS and the thrombin-soaked gelatin foam groups with respect to the preoperative Lund-Mackay score, extent of surgery performed, or need for additional nasal packing. However, the FS group showed clear trends toward increased granulation tissue (p = 0.007) and adhesion (p = 0.006) formation. Conclusion: Absorbable hemostatic agents are associated with a high degree of patient comfort and provide hemostasis comparable with traditional techniques. Different materials may induce differential patterns of mucosal healing, potentially affecting the ultimate result of ESS.
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Mattox, Shayna L., Kimberly J. Hammersmith, Jin Peng, Paul S. Casamassimo, and Janice A. Townsend. "Absorbable Hemostatic Pack Effect After Primary Incisor Extraction: A Pilot Study and Introduction of a Novel Scale to Assess Post-Operative Bleeding." Journal of Clinical Pediatric Dentistry 45, no. 2 (April 1, 2021): 67–73. http://dx.doi.org/10.17796/1053-4625-45.2.1.
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Objectives: This pilot study compared hemostatic pack (HP) application with no intervention following extraction of maxillary primary incisors in healthy children for effect on bleeding time and influence of patient or tooth variables utilizing a novel scale for assessment of bleeding following extraction. Study Design: A novel scale was created to assess bleeding after extraction. This scale was utilized in a randomized, split mouth study of healthy children ages 2–7 years old requiring extraction of at least 2 primary maxillary incisors under general anesthesia. One extraction site was randomly assigned to receive HP and the other had no hemostatic measures. Post-operative bleeding was rated at 2, 10, and 15 minutes post-extraction. Other variables recorded included age, sex, periapical radiolucency, presence of fistula, swelling, discoloration, intraoral stabilization device used, and vital signs at two time intervals. Pre-operative radiographs were reviewed for root resorption and periapical radiolucency. Results and Conclusions: Twenty-five patients provided 50 teeth. Hemostatic pack had a significant effect on reducing bleeding at each time point and that effect did not change over time. Age, sex, tooth pain, post-extraction heart rate, blood pressure, discoloration, amount of resorption, and presence of a periapical radiolucency had no significant effect on bleeding. The proposed bleeding scale had good intra-rater reliability and could be useful in future studies, once validated.
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Arakeri, Gururaj, and Peter A. Brennan. "Povidone-Iodine and Hydrogen Peroxide Mixture Soaked Gauze Pack: A Novel Hemostatic Technique." Journal of Oral and Maxillofacial Surgery 71, no. 11 (November 2013): 1833.e1–1833.e3. http://dx.doi.org/10.1016/j.joms.2013.07.025.
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Amer, Mohamed Zaghlool, Samah I. Mourad, Ahmed S. Salem, and Ehab Abdelfadil. "Correlation between International Normalized Ratio values and sufficiency of two different local hemostatic measures in anticoagulated patients." European Journal of Dentistry 08, no. 04 (October 2014): 475–80. http://dx.doi.org/10.4103/1305-7456.143628.
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ABSTRACT Objectives: The management of patients receiving oral anticoagulant therapy (OAT) undergoing minor oral surgeries is controversial. This study was designed to evaluate the correlation between International Normalized Ratio (INR) values and the sufficiency of two different local hemostatic measures in controlling postextraction bleeding in anticoagulated patients. Materials and Methods: One hundred and sixty patients receiving Warfarin OAT were included in this study. Patients were selected so that 80 patients have INR values of ≤2, whereas the remaining patients have the INR values ranging from 2 to 3. Forty patients were then randomly selected from each category to form two equal groups. Forty-five patients who had never been on OAT were selected as a negative control group (group 1). Failure to achieve hemostasis using a pressure pack was managed using either tranexamic acid (group 2) or Ankaferd Blood Stopper (ABS) (group 3). Results: The INR values of patients included in group 2 and 3 ranged from 1.5 to 3, with a mean of 2.2. No significant difference was recorded between the use of either tranexamic acid or ABS in achieving hemostasis in anticoagulated patients with INR values ranging between 2 and 3 (P = 0.93). Conclusion: Based on our findings, ABS is a hemostatic agent of good efficacy. The effect of ABS in controlling post-extraction bleeding in anticoagulated patients with INR values ≤3 is comparable to tranexamic acid with no evidence to support the superiority of tranexamic acid over ABS.
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Kameswaran, Mohan, S. Raghunandhan, and John K. Thomas. "A Prospective Double-Blinded Randomized Controlled Study Comparing the Efficacy of a Novel Biodegradable Synthetic Polyurethane Foam (Nasopore) vs Standard Polyvinyl Acetate Sponge (Merocel) as Packing Material after Functional Endoscopic Sinus Surgery: The First Indian Experience." An International Journal Clinical Rhinology 7, no. 3 (2014): 105–11. http://dx.doi.org/10.5005/jp-journals-10013-1208.
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ABSTRACT Background In Indian clinical practice, conventional nasal packing for hemostasis after routine rhinological surgery is usually performed with Vaseline (paraffin) gauze, and rarely with glove-finger packs or tamponade balloons. These materials are tedious to pack and cause discomfort to the patient on removal. Newer nasal packs which have recently emerged in the Indian scenario are found to be more user-friendly, equally effective for hemostasis and less traumatic to the operated nasal mucosa. Most rhinologists today, prefer to use polyvinyl acetate sponge packs (Merocel/Ivalon) for tamponade after nasal surgery. These packs are very effective but non-absorbable and need to be removed which does not augur well with many patients postoperatively. The recent entry of a biodegradable synthetic polyurethane foam (Nasopore) as an alternative nasal packing material, has evoked new interest, which initiated this study. Study method This prospective randomized double-blinded controlled study was aimed to compare the clinical efficacy and patient comfort level, while using Merocel and Nasopore as packing material after functional endoscopic sinus surgery (FESS). This study included thirty adults who were diagnosed with moderate to severe bilaterally comparable chronic rhinosinusitis, who underwent FESS under general anesthesia and received size-matched nasal packs randomly - Merocel on one side and Nasopore on the other. The assessment of clinical efficacy of both packs with regards to ease of packing, hemostasis, pressure effects, infections and adhesions was done with a Diagnostic Nasal Endoscopy at first postoperative day, first week and fourth week after surgery. All Merocel packs were removed on the first postoperative day. Patient comfort levels for both packs were recorded with a standard symptom questionnaire marked on a visual analogue scale of ten and the results were statistically compared between the two groups. Results Comparable outcomes were found while using Merocel or Nasopore with regards to ease of nasal packing and control of postoperative bleeding. There was a statistical difference in the hemostatic property between the two materials in the immediate postoperative period. Five out of 30 patients developed reactionary bleeds with Nasopore, which required repacking with same material within the first 24 hours, but no further bleeds were noted. Two out of these five patients on the first postoperative day had migration of Nasopore toward the choana and had to be repacked with additional Nasopore. Sequential postoperative nasal endoscopy revealed that Nasopore is more mucosal friendly with lesser incidence of adhesions, synechiae, infection and edema, with better biocompatibility and safety. The major success with Nasopore was found to be, the fact that no pack removal was necessary, which immensely improved patient satisfaction and willingness to use the material when compared to Merocel. This was proved by the patient's symptom questionnaire which showed significant benefits of Nasopore over Merocel with regards to compliance and comfort levels. Conclusion Nasopore is a novel biodegradable synthetic material which is clinically as efficacious and patient-friendly as Merocel and is suitable for postoperative nasal packing after functional endoscopic sinus surgery. The clinical benefits of Nasopore and its outcomes among patients as recorded in our study, stands proof to support Nasopore as a successful packing material in rhinological surgery. How to cite this article Raghunandhan S, Kameswaran M, Thomas JK. A Prospective Double-Blinded Randomized Controlled Study Comparing the Efficacy of a Novel Biodegradable Synthetic Polyurethane Foam (Nasopore) vs Standard Polyvinyl Acetate Sponge (Merocel) as Packing Material after Functional Endoscopic Sinus Surgery: The First Indian Experience. Clin Rhinol An Int J 2014;7(3):105-111.
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Hasan, Sajid, Mahmuda Akhter, Saeed Hossain Khan, Dilruba Sharmin, Md Manjurul Karim, and Rifat Rahman. "Evaluation of post-extraction bleeding in patients taking low dose aspirin." Update Dental College Journal 9, no. 1 (April 27, 2019): 32–36. http://dx.doi.org/10.3329/updcj.v9i1.41204.
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Background: Acetylsalicylic acid (ASA) generically known as Aspirin is an analgesic, antipyretic, anti-inflammatory and also an antiplatelet drug. In order to avoid excessive bleeding and to be on the safer side, dentists have traditionally advised their patients to stop taking aspirin before extraction of teeth although this surgical procedure can be done without cessation of aspirin intake.
Objective: The purpose of the study was to assess the necessity of interrupting aspirin therapy prior to dental extraction.
Materials and Methods: A cross sectional study was conducted in November 2015 at outpatient department of dentistry, BIRDEM Hospital, Dhaka. Sample of 50 patients who took low dose aspirin (75mg) once daily were purposely selected for this study. The blood pressure of all the subjects was recorded preoperatively. The extractions were done atraumatically under local anesthesia using 2% lidocaine with 1:100,000 epinephrine. A gelatin sponge piece was placed in socket and closed by atraumatic silk. The subjects were instructed to apply pressure pack with sterile gauze for 30 min. Evaluation was done in every 10 minutes for 30 minutes.
Results: Among 50 patients, 82.0% patients were suffering from IHD. Simple extraction was done in 92.0% of patients while the remaining extractions were done surgically. 68% was managed by pressure pack and gelatin sponge while 26.0% were managed by pressure pack only. According to Post-extraction bleeding, it was found that the bleeding time was 10 min in case of 94% patients while only 2% showed 30 minutes of bleeding time.
Conclusion: The study revealed that it is not necessary to alter or stop aspirin therapy and local hemostatic measures are sufficient to control bleeding. Therefore it can be assumed that extraction can be done without cessation of low dose aspirin and avoiding the life threatening issues.
Update Dent. Coll. j: 2019; 9 (1): 32-36
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Rajamani, Santhosh Kumar. "Effect of Topical Nasal Decongestants on Nasal Peak Flow Rates in Adults Suffering from Acute Sinusitis." Bengal Journal of Otolaryngology and Head Neck Surgery 26, no. 2 (August 29, 2018): 86–90. http://dx.doi.org/10.47210/bjohns.2018.v26i2.265.
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INTRODUCTION
In this research we study the effect of Nasal decongestant Xylometazoline 0.1% solution on Serial measurements of Nasal peak flow rates in a cohort of patients who were suffering from Acute Sinusitis.
CASE SERIES
A population of 90 patients were chosen from our regular Out-patient clinics who were suffering from Acute Sinusitis based on a Clinical diagnostic criterion. A baseline Nasal peak Flowmetry was done before and this was followed by common Decongestant Xylometazoline 0.1% solution spray application, followed by serial readings of Nasal peak Flowmetry done after 10 minutes, 25 minutes, 60 minutes, 120 minutes,240 minutes and 360 was taken then plotted and analysed.
DISCUSSION
From the A.U.C Curves it can be inferred that Maximum decongestant action of Xylometazoline 0.1% solution is seen 1 hour after application and the raise in decongestant reaches a plateau by 2 hours. Readings remain elevated from baseline well 6 hours post decongestion.
CONCLUSION
Patients who are prescribed Xylometazoline 0.1% solution are advised that maximum relief from congestion would be obtained around 1 to 2 hours after application and hence effect would decrease. Surgeons who use Xylometazoline 0.1% solution for nasal packing must proceed with the Surgery within 1 hour of application of the pack to obtain maximum hemostatic and decongestant benefit of this drug.
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Da Luz, Luis, Jeannie L. Callum, Andrew Beckett, Henry T. Peng, Paul Engels, Neil Parry, Homer Tien, Avery Nathens, Bruce A. Schwartz, and Keyvan Karkouti. "Fiirst-2: Prospective, Randomized Study Comparing Administration of Clotting Factor Concentrates with Standard Massive Hemorrhage Protocol in Severely Bleeding Trauma Patients." Blood 136, Supplement 1 (November 5, 2020): 6. http://dx.doi.org/10.1182/blood-2020-141329.
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Introduction: Acute trauma coagulopathy (ATC) is associated with severe hemorrhage. ATC etiology is often multifactorial, with acquired fibrinogen deficiency and consumption of clotting factors recognized as major factors. The FiiRST-2 study will determine the impact of early co-administration of fibrinogen concentrate (FC) and prothrombin complex concentrate (PCC) on the total number of allogeneic blood products (ABPs) transfused compared to the current standard of care (ratio-based plasma resuscitation). Methods: FiiRST-2 is a multicenter (8 Canadian centers), pragmatic, randomized, parallel-control, superiority trial with a two-armed, two-stage design with an adaptive interim analysis. Research ethics board approval has been obtained and the study complies with the Declaration of Helsinki. Trauma patients >16 years old at risk of massive hemorrhage will be randomized to receive FC + PCC or standard of care (ratio-based plasma resuscitation + FC in response to low fibrinogen levels) (Table 1) until all units in the second massive hemorrhage protocol (MHP) pack have been administered, or earlier if the MHP is terminated. The primary endpoint is to demonstrate superiority with respect to the number of ABP units (RBCs [red blood cells] + plasma + platelets) transfused within 24 hours of arrival at the trauma bay/emergency department. Secondary endpoints include RBC units transfused within 24 hours, incidence of thromboembolic events, and ventilator-free days up to Day 28. Safety endpoints include all adverse events (AEs) and serious adverse events (SAEs) up to Day 28. It is projected to enroll 360 patients depending on the results of the Interim analysis. Results: An interim analysis will be performed after 120 patients have completed the study. The study is expected to start late 2020 and enrollment is expected to require about 2 years. Conclusions: FiiRST-2 will determine if early hemostatic therapy with FC + PCC is superior to standard MHP packs in bleeding trauma patients. Results could have a major impact on clinical practice and improve management and outcomes in this high-risk group of patients. Disclosures Da Luz: Octapharma Canada: Research Funding. Callum:Octapharma: Research Funding; Canadian Blood Services: Research Funding. Schwartz:Octapharma: Current Employment. Karkouti:Canadian blood services: Research Funding; Octapharma: Research Funding. OffLabel Disclosure: PCC and fibrinogen concentrate for acquired coagulopathic bleeding (off-label in the USA)
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Maharjan, Shova, AK Jha, RR Joshi, AS Rijal, KK Shrestha, and A. Dhungana. "Comparison of Polyvinyl Acetate Sponge and Medicated Ribbon Gauge Nasal Pack following Nasal Surgery." Nepal Medical College Journal 21, no. 1 (March 31, 2019): 31–34. http://dx.doi.org/10.3126/nmcj.v21i1.24847.
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Nasal occlusive dressings are routine after nasal surgeries to arrest hemorrhage, to prevent septal hematoma, and to prevent postoperative adhesions. However, patients describe nasal packing and its removal as their worst experience. Various types of nasal packs are available. Medicated ribbon gauge is the traditional form of nasal pack which consists of an open-mesh cotton as a carrier whereas “Polyvinyl Acetate’ sponge is a compressed dehydrated material, an improvised one which increases in size and compresses blood vessels when rehydrated with normal saline. As Polyvinyl acetate sponge is smooth and spongy, it causes less pain and abrasion while in-situ and removal. This was a prospective comparative study done in tertiary hospital of Nepal. Patients were subjected to either polyvinyl acetate sponge or ribbon gauge nasal pack following nasal surgery. Comparisons were made in terms of pain score, maintenance of hemostasis and wound healing. There were 154 patients in the study with 104 males and 50 females. The pain score when nasal pack was in-situ was similar in both groups whereas it was lesser in the polyvinyl acetate group on its removal. However, bleeding and adhesion were found to be similar. Crust formation was less in polyvinyl acetate group. Six synaechia were noted in ribbon gauge group only. Pain was significantly less during removal of polyvinyl acetate pack.
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Shikani, Alain H. "Use of Antibiotics for Expansion of the Merocel® Packing following Endoscopic Sinus Surgery." Ear, Nose & Throat Journal 75, no. 8 (August 1996): 524–28. http://dx.doi.org/10.1177/014556139607500811.
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Sinus packs that are used for hemostasis and the prevention of synechiae following endoscopic sinus surgery frequently get colonized with bacteria from the infected sinus, which increases the risk for infection. This study was conducted in an attempt to characterize the bacterial flora of the chronically infected sinus, and to evaluate whether direct installation of antibiotics into the sponge inhibits bacterial growth. Bacteria were cultured from 89% of the sinuses at surgery, and in 67% of the cases, the same bacteria were recovered from the sinus dressings one week later. When the Merocel® sinus dressing was expanded with a combination of polymyxin, neomycin and hydrocortisone at the time of surgery, a 36% decrease in bacterial growth was observed, along with a decrease in the severity of pain associated with removal of the pack. This supports the concept of using an antibiotic for the expansion of the sponge following endoscopic sinus surgery.
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McIntosh, David, Allison Cowin, Damian Adams, and Peter-John Wormald. "The Effect of an Expandable Polyvinyl Acetate (Merocel) Pack on the Healing of the Nasal Mucosa of Sheep." American Journal of Rhinology 19, no. 6 (November 2005): 577–81. http://dx.doi.org/10.1177/194589240501900608.
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Background There is a lack of knowledge about the healing of the nasal respiratory mucosa after endoscopic sinus surgery (ESS). Nasal packs often are placed after ESS in an attempt to improve hemostasis and reduce adhesion formation. Most nasal packs need to be removed in the postoperative period. This is uncomfortable for the patient and the affect of these packs on the healing process is unknown. Methods We have standardized the sheep as a suitable animal model to examine the healing of the nasal epithelium after ESS. The nasal mucosa of sheep was wounded under endoscopic guidance and either packed with expandable polyvinyl acetate–based pack (Merocel), which was removed at the 5th postoperative day, or left unpacked to serve as control. Serial biopsies of the wounded area were taken every 28 days, up to 112 days postwounding, for examination using light and scanning electron microscopy. Results There was no significant difference in the rate of reepithelialization between the packed and control sides of the sheep (p > 0.05). There was no significant difference in the total amount of surface cilia coverage between the packed and control sides at any time points (p > 0.05). There was no significant difference in the maturity of the cilia between the packed and control sides at any time points (p > 0.05). Conclusion The use of Merocel packing postoperatively neither impairs nor promotes wound healing in the postoperative period.
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HAYASHI, Matsuno, Emi INAGAKI, Sachiko MORITA, Makiko MANO, and Atsuko AMANO. "Effects of Cold Pack on the Uterine Contraction and Hemostasis Just after Labor." Journal of Japan Academy of Midwifery 9, no. 1 (1995): 38–42. http://dx.doi.org/10.3418/jjam.9.38.
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Davis, Jessica P. E., and Stephen H. Caldwell. "Healing gone wrong: convergence of hemostatic pathways and liver fibrosis?" Clinical Science 134, no. 16 (August 2020): 2189–201. http://dx.doi.org/10.1042/cs20191102.
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Abstract Fibrosis results from a disordered wound healing response within the liver with activated hepatic stellate cells laying down dense, collagen-rich extracellular matrix that eventually restricts liver hepatic synthetic function and causes increased sinusoidal resistance. The end result of progressive fibrosis, cirrhosis, is associated with significant morbidity and mortality as well as tremendous economic burden. Fibrosis can be conceptualized as an aberrant wound healing response analogous to a chronic ankle sprain that is driven by chronic liver injury commonly over decades. Two unique aspects of hepatic fibrosis – the chronic nature of insult required and the liver’s unique ability to regenerate – give an opportunity for pharmacologic intervention to stop or slow the pace of fibrosis in patients early in the course of their liver disease. Two potential biologic mechanisms link together hemostasis and fibrosis: focal parenchymal extinction and direct stellate cell activation by thrombin and Factor Xa. Available translational research further supports the role of thrombosis in fibrosis. In this review, we will summarize what is known about the convergence of hemostatic changes and hepatic fibrosis in chronic liver disease and present current preclinical and clinical data exploring the relationship between the two. We will also present clinical trial data that underscores the potential use of anticoagulant therapy as an antifibrotic factor in liver disease.
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Bhosale, Archana, Kavya H. S., Yogeshwar Sadashiv Nandanwar, and Ayesha Ansari. "Pelvic pressure packing for intractable obstetric and gynaecological hemorrhage in a tertiary care hospital." International Journal of Reproduction, Contraception, Obstetrics and Gynecology 7, no. 12 (November 26, 2018): 4956. http://dx.doi.org/10.18203/2320-1770.ijrcog20184947.
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Background: Haemorrhage is one of the most common complication of any surgery. Haemorrhage can be arterial, venous or capillary ooze. Massive haemorrhage if not timely managed may lead to fatal consequences. There are various medical and surgical methods to control haemorrhage. This study aims to achieve hemostasis with the help of pelvic pressure pack in Obstetric and Gynaecologic surgeries when standard methods are failed and to evaluate efficacy of simple and modified technique of pack preparation.Methods: This is an observational study of 11 cases conducted over a period of 4yrs. This study reports modification of standard packing techniques which overcomes some of its limitations. Here the pack was used in different gynaecologic and Obstetric cases, where intractable haemorrhage was the major problem and standard methods to control haemorrhage had failed. Here a simple foley’s catheter rolled with condom and filled with normal saline was used to prepare a pack and kept over the bleeding surface. This specific pack will adopt the shape of the body cavity it is inserted into, thereby causing pressure tamponade against bleeding surfaces. Pack was removed after 48-72 hours of insertion. Postoperative control of bleeding, patient stability and morbidity were studied.Results: The pelvic pressure pack successfully controlled bleeding in 100% of cases without any morbidity and mortality.Conclusions: In the contemporary management of post-hysterectomy or adhesiolysis induced uncontrolled pelvic bleeding and venous oozes, the pelvic pressure pack appears to be valuable and effective option, affording correction of coagulopathy and further stabilization. We believe all Obstetricians and Gynaecologists should be familiar with this simple safe and cheap potentially lifesaving technique.
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Mokal, Nitin, and Navadeep Chavan. "Modified safe technique for circumcision." Indian Journal of Plastic Surgery 41, no. 01 (January 2008): 47–50. http://dx.doi.org/10.1055/s-0039-1699226.
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ABSTRACTWe have used surgical gauze under the prepuceal skin as a pack in 20 cases prior to marking incision for circumcision. The prepuceal adhesions were first dissected and seperated. The method allows a stable, well-supported prepuceal surface for marking incisions and avoids injuries to the glans. Because the prepuceal surface is taut and stable, hemostasis is easier and quicker and the operating time is reduced.
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Ereth, Mark H., Gregory A. Nuttall, Paula J. Santrach, Jacinta T. Klindworth, William C. Oliver, and Hartzell V. Schaff. "The Relation between the Platelet-activated Clotting Test (HemoSTATUS) and Blood Loss after Cardiopulmonary Bypass." Anesthesiology 88, no. 4 (April 1, 1998): 962–69. http://dx.doi.org/10.1097/00000542-199804000-00016.
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Background Platelet dysfunction is one of several major causes of bleeding after cardiopulmonary bypass. A timely, simple, point-of-care determinant of platelet function recently became available for clinical use. Adding platelet-activating factor to conventional activated clotting time methods (platelet-activated clotting test [PACT]) produces rapid results (<15 min) and may yield a measure of platelet responsiveness and whole-blood procoagulant activity. Methods Blood samples were drawn from 100 patients after cardiac surgery on their arrival in the intensive care unit for PACT, platelet count, prothrombin time (PT), and activated partial thromboplastin time (aPTT). Cumulative blood loss at 4, 8, and 12 h after arrival in the intensive care unit and perioperative transfusion requirements were quantitated. Coagulation tests and mediastinal blood loss were compared using the Spearman rank test and Pearson correlation. The sensitivity and specificity of the laboratory tests for predicting blood loss were analyzed using the receiver operating characteristic method. Results The PT was the only test that correlated with blood loss at 4, 8, and 12 h. The PACT did not correlate with blood loss at 4, 8, or 12 h, nor did the PACT correlate with the PT or the aPTT. The sensitivity and specificity of the PACT were less than those of the PT in predicting blood loss. Only the PT correlated with platelet and fresh frozen plasma transfusion. Conclusions The PT correlated with blood loss and transfusion requirements and was superior to PACT, aPTT, and platelet count for predicting excessive blood loss after cardiopulmonary bypass.
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Payne, Joshua, Trent Bickel, and Sandeep Gautam. "Figure-of-eight sutures for hemostasis result in shorter lab recovery time after ablation for atrial fibrillation." Pacing and Clinical Electrophysiology 41, no. 8 (June 27, 2018): 1017–21. http://dx.doi.org/10.1111/pace.13405.
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Thomas, Ike, Jathin Sam Thekkethil, Ramesh Chandra Kapoor, Tina Thomas, and Pramod Thomas. "A Novel Technique of Using Sponge as Post-Operative Nasal Packing." Bengal Journal of Otolaryngology and Head Neck Surgery 26, no. 1 (April 28, 2018): 23–28. http://dx.doi.org/10.47210/bjohns.2018.v26i1.152.
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Introduction
In an effort to find a comparable but less expensive nasal pack with the qualities of MerocelR,this study was aimed at comparing the clinical efficacy and patient comfort level, while simultaneously using MerocelR and commercially available sponge as packing material in the same patient.
Materials and Methods
This study included those patients who underwent septoplasty, turbinoplasty or FESS and nasal packing was done randomly with MerocelR and commercially available sponge (polyurethane foam) on the same patient. Patients shared their experience on the symptom questionnaire on the first post-operative day and they underwent sequential diagnostic nasal endoscopy to assess the endoscopic status of the nasal cavity, which were documented meticulously.
Result
The post-operative bleeding control, pain during pack removal, general satisfaction, willingness to reuse and post-operative adhesion were same for both MerocelR and sponge.
Conclusion
The innovative technique of using a commonly available, commercially prepared sponge which is as good as MerocelR is well supported due to its efficacy in hemostasis, less mucosal trauma and less pain during pack removal. So it may be used in developing countries where cost is a factor for compliance of patients for undergoing surgeries without compromising on quality.
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Yorgun, Hikmet, Uğur Canpolat, Ahmet Hakan Ates, Metin Oksul, Yusuf Ziya Sener, Fatih Akkaya, and Kudret Aytemir. "Comparison of standard vs modified “figure‐of‐eight” suture to achieve femoral venous hemostasis after cryoballoon based atrial fibrillation ablation." Pacing and Clinical Electrophysiology 42, no. 9 (August 5, 2019): 1175–82. http://dx.doi.org/10.1111/pace.13764.
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Toh, Cheng Hock. "APTT Revisited: Detecting Dysfunction in the Hemostatic System Through Waveform Analysis." Thrombosis and Haemostasis 82, no. 08 (1999): 684–87. http://dx.doi.org/10.1055/s-0037-1615897.
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IntroductionDespite extensive investigations in both experimental and clinical disseminated intravascular coagulation (DIC), much remains to be learned about this condition of hemostatic dysfunction. Although the technological armory available to the clinical investigator has expanded enormously since the early 1980s, particularly with regard to immunologically-based testing, the sobering fact is that prognosis has remained dismally unchanged.1 Although some of the new laboratory approaches can identify DIC at an early enough stage to allow for useful clinical intervention, most require a level of stringency in performance that is not attainable in the routine clinical setting.2 The pace of acute DIC precludes many of these specific tests, and reliance is still based on traditional screening assays, such as the prothrombin time (PT), activated partial thromboplastin time (APTT) and platelet count (Plt). These tests lack specificity on an individual basis and are only useful in DIC if there is further determination of fibrinogen (Fgn) and fibrin breakdown products (fdp)/D-Dimers.3 However, changes in these battery of tests seldom occur together. As a result, the realization of DIC is often only made after serial testing, with the inevitable delay and consequent jeopardization of patient critical care.4 The numerous failures of new therapies in sepsis and DIC testifies to this problem of a lack of early definition in the disease process to forewarn the clinician of more timely intervention.5 The gold standard must, therefore, be a single, rather than multiple, assay system that is both sensitive and specific for early DIC and simple and rapid to perform. It is therefore, not too surprising that such a test has remained elusive for such a considerable length of time.In the context of complex scenarios, the simplest of approaches can often be the most rewarding. Such an axiom could fit with the description of APTT transmittance waveform analysis as a useful test in DIC that could also potentially lead to an improved understanding of the condition.
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Sari, Eka Afrima, M. Z. Arifin, and Sari Fatimah. "Perbandingan Hematoma Pasca Kateterisasi Jantung Berdasarkan Penekanan Bantal Pasir dan Cold Pack." JURNAL PENDIDIKAN KEPERAWATAN INDONESIA 3, no. 2 (December 31, 2017): 100. http://dx.doi.org/10.17509/jpki.v3i2.9414.
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ABSTRAK Kateterisasi jantung merupakan prosedur untuk mendiagnosis dan/atau mengevaluasi arteri koroner. Pada akhir prosedur, dilakukan pelepasan femoral sheath dan penekanan manual maupun mekanik pada arteri femoralis untuk mengontrol perdarahan hingga tercapai hemostasis. Teknik penekanan yang tidak adekuat pada sisi akses arteri setelah kateterisasi jantung merupakan salah satu penyebab terjadinya hematoma. Ditemukan kejadian hematoma pada pasien pasca kateterisasi jantung yang timbul beberapa waktu setelah penekanan mekanik bantal pasir. Penelitian ini bertujuan untuk membandingkan ukuran hematoma dalam waktu 24 jam pasca kateterisasi jantung berdasarkan penekanan mekanik bantal pasir dan cold pack. Penelitian ini merupakan penelitian komparatif dengan pendekatan after-only non-equivalent control group design. Subjek penelitian adalah 20 orang pasien pasca kateterisasi jantung yang dibagi menjadi kelompok eksperimen dengan penekanan mekanik cold pack selama 20 menit setelah pelepasan femoral sheath dan kelompok kontrol dengan penekanan mekanik bantal pasir 2,5 Kg selama 6 jam setelah pelepasan femoral sheath. Kejadian hematoma dilihat setelah penekanan manual, penekanan mekanik, dan setiap jam selama 24 jam. Perbedaan kejadian hematoma dilihat dengan menggunakan uji Mann-Whitney. Hasil penelitian menunjukkan bahwa tidak terdapat perbedaan bermakna ukuran hematoma setelah penekanan manual, penekanan mekanik dan setiap jam dalam waktu 24 jam pada pasien pasca kateterisasi jantung yang menggunakan bantal pasir maupun cold pack (ρ > 0,05). Pada kelompok eksperimen ukuran hematoma mengecil di akhir jam ke-24 dan pada kelompok kontrol ukuran hematoma membesar di akhir jam ke-24. Penggunaan penekanan mekanik cold pack dapat mengurangi risiko hematoma sebagaimana bantal pasir, sehingga cold pack dapat digunakan sebagai pilihan alat tekan mekanik pada pasien pasca kateterisasi jantung. ABSTRACT Cardiac catheterization is a procedure to diagnose and/or evaluate coronary arteries. At the end of procedure, performed manual and mechanical compression on the femoral artery to control bleeding until homeostasis is achieved. Inadequate compression techniques on the arterial access after cardiac catheterization is one of the causes of hematoma. Found in hematoma incidence in patients after cardiac cathetherization that arise after mechanical compression using sandbags. The purpose of this study was to compare the size of the hematoma within 24 hours after cardiac catheterization using sandbags and cold pack mechanical compression. The study design was a comparative study with after-only non-equivalent control group design. Research subjects included of 20 after cardiac catheterization patients were divided into an experimental group with cold pack mechanical compression for 20 minutes after femoral sheath release and control group with sandbags mechanical compression for 6 hours after femoral sheath release. Incidence of hematoma was observed after manual compression, mechanical compression, and every hour for 24 hours. Mann-Whitney test was used to see the difference the incidence of hematoma of both of groups. The results showed no significant difference in the size of the hematoma after manual compression, mechanical compression, and every hour within 24 hours after cardiac catheterization in both of groups (ρ> 0.05). But the size of the hematoma in the end of observation time was smaller in experiment group, and bigger in the control group. The use ofcold pack mechanical compressions could reduce the risk of hematoma as well as sandbags. Thus, cold pack could be used as an alternative mechanical compression tool in patients with post cardiac catheterization.
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Shrivastava, Deepak, Shubham Negi, Pankaj Gharde, and Ramsewak Verma. "A study of prostatic fossa packing: a modified technique in Freyer’s prostatectomy for achieving hemostasis." International Surgery Journal 5, no. 2 (January 25, 2018): 711. http://dx.doi.org/10.18203/2349-2902.isj20180379.
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Background: Benign prostatic hyperplasia is one of the most common old age related benign tumor of urinary tract of men. Though now a day the gold standard treatment is only TURP, but this facility is still out of reach for majority of rural population of India. The rate of complications has come down heavily but still complete hemostasis remains a major concern for these patients.Methods: This present study was conducted at the center from March 2014 to December 2016, the aim of present study was to see the effectiveness of bladder packing on blood loss, complications and comfort of patient in the cases of Benign prostatic hyperplasia (BPH) admitted at in patient department of surgery.Results: A Total of 90 cases of BPH (Benign Prostatic Hypertrophy) were operated by Freyer’s suprapubic transvesical prostatectomy. All the patients presented with symptoms of BPH. A detailed clinical history and examination of all patients was recorded and AUA (American Urological Association), International Prostate Symptom Score (IPSS) was calculated. On table clear urine was confirmed with naked eyes and no Foley’s traction was given. After 72 hours of the surgery, pack was removed, saline irrigation was continued for 5 days. The patient was discharged on the 8th post-operative day after removal of stitches.Conclusions: The prostatic fossa packing technique without any traction is effective in control of postoperative bleed, it is an acceptable option where transurethral resection of prostate(TURP) is not available.
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Lyde, Randolph B., Li Zhai, Karen Vo, Danuta Jadwiga Jarocha, Spencer Sullivan, Valder R. Arruda, Rodney M. Camire, Deborah French, and Mortimer Poncz. "Towards the Care of Hemophilia a Patients Using Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Expressing Coagulation Factor (F) VIII." Blood 126, no. 23 (December 3, 2015): 2266. http://dx.doi.org/10.1182/blood.v126.23.2266.2266.
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Abstract We and others have shown that FVIII expressed ectopically in platelets (pFVIII) is stored in α-granules, released at sites of vascular injury and restores hemostasis in FVIIInull mice, even in the presence of neutralizing antibodies to FVIII. These studies support the concept that unlike therapeutic interventions that correct plasma FVIII, pFVIII may be a useful therapy in hemophilia A with intractable inhibitors and significant bleeds. We have also demonstrated this approach has several limitations that may make pFVIII gene therapy bone marrow transplantation (BMT) strategies problematic: 1) pFVIII is not equivalent to plasma FVIII and its efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors, and 2) pFVIII expressed during megakaryopoiesis can cause injury to the Mks, potentially exacerbating post-BMT thrombocytopenia. We propose an alternative strategy: interval prophylactic infusions of FVIII-containing platelets generated from patient-specific iMks expressing either human B-domain-deleted (BDD) FVIII or variants of this FVIII that have greater stability and longer half-lives; making them especially efficacious as pFVIII as we previously demonstrated. iPSCs are a renewable source of cells that can be pre-screened prior to clinical usage for lines that express optimal levels of pFVIII and also release optimal numbers of platelets after differentiation into iMks. Such iPSCs were transfected with a self-inactivating lentivirus containing cDNA for one of three FVIII variants: wildtype BDD FVIII (WT FVIII), R1645H PACE/furin cleavage site FVIII (FVIIIR1645H), and amino acid 1645 to 1648 deletion FVIII (FVIIIΔ). FVIIIR1645H and FVIIIΔ show greater stability and consequently greater specific activity with no increase in injuring Mks. All FVIII variants were expressed using the MK-specific Cxcl4 promoter and were shown to be effective in several bleeding models in FVIIInull mice. Differentiated and transduced iMKs were analyzed for RNA and protein expression. All of the FVIII variant iMKs expressed at least forty-fold higher levels of mRNA compared to the non-transduced control (n=6) and protein was expressed at >550 pg/106 CD42b+ iMKs (n=6). Transduced MKs released FVIII into the supernatant when activated by thrombin showing the pFVIII was likely stored in α-granules. Annexin staining was the same between FVIII-expressing iMKs and control iMks suggesting that the level of pFVIII did not cause the iMks to become apoptotic. To test the ability of FVIII-expressing iMKs to correct the coagulopathy in hemophilia A, 5x105 iMKs were added to FVIIInull murine whole blood and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each FVIII variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (n=4). These data show that iMKs expressing FVIII variants can improve hemostasis in a whole blood clotting assay. Our next goal is to generate sufficient platelets from these iMKs to test for correction of the bleeding diathesis in immunodeficient FVIIInull mice and to determine their efficacy in improving hemostasis in a number of clinically relevant hemostatic models. Disclosures Arruda: Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Patents & Royalties. Camire:Pfizer: Consultancy, Patents & Royalties, Research Funding; NovoNordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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Abrams, CS, N. Ellison, AZ Budzynski, and SJ Shattil. "Direct detection of activated platelets and platelet-derived microparticles in humans." Blood 75, no. 1 (January 1, 1990): 128–38. http://dx.doi.org/10.1182/blood.v75.1.128.128.
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Abstract Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.
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Abrams, CS, N. Ellison, AZ Budzynski, and SJ Shattil. "Direct detection of activated platelets and platelet-derived microparticles in humans." Blood 75, no. 1 (January 1, 1990): 128–38. http://dx.doi.org/10.1182/blood.v75.1.128.bloodjournal751128.
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Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.
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Nguyen, Giang N., Joshua I. Siner, Robert J. Davidson, Valder R. Arruda, Rodney M. Camire, and Denise E. Sabatino. "Human Factor VIII Variants with Diminished PACE-Furin Cleavage Exhibit Increased Biological Activity and Therapeutic Efficacy." Blood 124, no. 21 (December 6, 2014): 104. http://dx.doi.org/10.1182/blood.v124.21.104.104.
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Abstract During intracellular processing, factor VIII (FVIII) undergoes proteolysis at multiple sites with the most predominant cleavage at a Paired basic Amino acid Cleaving Enzyme (PACE)-furin cleavage site at the carboxy-terminus of the B-domain at residue 1648 to give rise to two polypeptide chains, the heavy chain and the light chain. Through a metal ion dependent association, these chains form a heterodimer that is the secreted form of the protein. Previously, we observed that canine B-domain deleted FVIII (cFVIII-BDD) is secreted primarily as a single chain (SC) molecule (170kD) while human FVIII-BDD (hFVIII-BDD) is secreted predominantly as a heterodimer. In addition, cFVIII-BDD is more stable and has higher biological activity. Amino acid sequence analysis revealed a single amino acid difference between cFVIII (1645-HHQR-1648) and hFVIII (1645-RHQR-1648) at the PACE-furin cleavage recognition site. Characterization of hFVIII-R1645H demonstrated that this residue is responsible for the secretion of cFVIII predominantly as a single polypeptide chain and its inherent increased stability and specific activity. Furthermore, hFVIII-R1645H has enhanced hemostatic effects upon vascular injury and in the setting of AAV delivery was associated with increased circulating levels compared to wild type hFVIII-BDD. These data suggest that there is suboptimal cleavage of cFVIII and hFVIII-R1645H by PACE-furin. Here we tested whether deletion of part or all of the PACE-furin recognition sequence may further decrease the efficiency of cleavage at the site resulting in a larger portion of single SC molecules and thus may confer increased stability compared to hFVIII-R1645H. A series of hFVIII-BDD deletion variants (n=5) of the PACE-furin cleavage recognition site (1645-1648) were generated: del1645, del1645-46, del1645-47, del1645-48 and del1648. Stable BHK cell lines were established for each variant and protein was purified using ion exchange chromatography. On an SDS-PAGE gel under reducing conditions the deletion variants migrate in a similar manner, however, the proportion of the hFVIII in the single chain form compared to hFVIII-BDD (15% SC) was 3.5-fold higher for del1645-46 (57% SC), del1645-47 (53% SC) and del1645-48 (48% SC) which is similar to R1645H (52% SC). The single deletion variants on SDS-PAGE were 2-fold higher (del1645, 34% SC) or identical (del1648, 15% SC) to hFVIII-BDD. In a one-stage aPTT assay, all variants had activity similar to hFVIII-BDD. While in the two-stage aPTT assay, three variants (del1645-46, del1645-47, del1645-48) had 2-fold higher activity than hFVIII-BDD. Variants del1645 and del1648 had similar activity to hFVIII-BDD. Using an A2 domain dissociation clotting-based assay, we found that del1645-46, del1645-47, del1645-48 and del1648 were more stable following thrombin (IIa) activation compared to hFVIII-BDD suggesting that the A2 domain of these variants may dissociate more slowly than the wild type human A2 domain following IIa cleavage. These findings explain, at least in part, the superior clotting activity determined in multiple assays. The hFVIII PACE-furin variants were also introduced into an adeno-associated viral vector serotype 8 (AAV8-hAAT-hFVIII-BDD) for liver targeted delivery. Each AAV8-hAAT-hFVIII-P/F variant was delivered to hemophilia A mice (5x1011vector genomes/mouse)(n=6/group). At 4 weeks post vector administration, the hFVIII expression of del1645-47 was 3-fold higher than hFVIII-BDD followed by del1645, del1645-48, del1645-46 while del1648 was similar to hFVIII-BDD. In summary, these in vitro data demonstrate that deletion of residues 1645-46, 1645-47 or 1645-48 of the PACE-furin cleavage recognition site confers 2-3-fold higher biological activity than wild type hFVIII-BDD. In vivo these variants result in 2-3 fold higher levels of hFVIII expression after AAV delivery compared to wild type hFVIII-BDD. Together these data suggest that the hFVIII-P/F deletion variants have enhanced hemostatic function and are superior to wild type hFVIII. In the setting of gene-based therapeutics for hemophilia A, these novel variants provide a unique strategy for increasing factor VIII expression which will allow the use of a lower vector dose which is a critical improvement for translation of this approach. Disclosures Nguyen: Pfizer, Inc.: Research Funding. Camire:Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding; Pfizer, Inc.: Research Funding.
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Kholmukhamedov, Andaleb, Yumiko Sakurai, Elaissa L. Hardy, Wilbur A. Lam, and Shawn M. Jobe. "Mitochondrial Uncoupling Prevents Procoagulant Platelet Formation Resulting in Decreased Thrombus Stability." Blood 132, Supplement 1 (November 29, 2018): 2421. http://dx.doi.org/10.1182/blood-2018-99-117004.
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Abstract Secondary hemostasis is the cascade of coagulation reactions that has two pathways, both of which cultivate in the formation of insoluble fibrin to stabilize the primary platelet clot. Although some biochemical reactions of secondary hemostasis can occur in solution their amplification, to the extent capable of supporting active hemostasis, occurs only in the presence of the membranes provided by cells and particles of blood and vasculature. Procoagulant platelets, a subpopulation of activated platelets, are believed to be the key contributor of the phosphatidylserine surface for the membrane dependent reactions. This subpopulation of activated platelets is generated upon the onset of mitochondrial permeability transition (MPT) triggered by mitochondrial calcium overload or intramitochondrial reactive oxygen species (ROS) formation. MPT leads to phosphatidyl serine (PSer) exposure by yet unknown mechanisms. Prevention of MPT however, completely disrupts transition of platelets to a procoagulant phenotype. It has been debated for more than a decade whether procoagulant platelets get formed during thrombogenesis in vivo. Numerous recent studies suggest that higher procoagulant platelet formation potential correlates with pathologic thromboses. However, the role of procoagulant platelet phenotype in hemostasis has been poorly investigated. Here we demonstrate that procoagulant platelets are essential in secondary hemostasis. Washed human platelets were stimulated with thrombin (THR), convulxin (CVX) or both. PSer exposure, granule release and integrin activation were measured by annexin V (AnnV), P-selectin and PAC1, respectively. Aggregation was done by standard aggregometry. Thrombin generation was measured using tissue factor initiated thrombinoscopy. Hemostasis experiments were performed as described previously (Sakurai Y, Hardy ET, Ahn B, et al. A microengineered vascularized bleeding model that integrates the principal components of hemostasis. Nature communications. 2018;9(1):509). Isolated platelets were treated with vehicle (DMSO) or DNP, washed, resuspended in plasma, mixed with red and white blood cell mass (so called "red layer") at a ratio of 1 to 1. Whole blood then was perfused over the bleeding channels at a shear rate of 500 s-1. Mitochondrial transmembrane potential is the primary driving force for mitochondrial calcium uptake, as well as the generation of ROS upon platelet stimulation. Mitochondrial uncouplers dissipate the proton gradient across the inner mitochondrial membrane leading to mitochondrial depolarization. Dissipation of membrane potential, by uncouplers carbonyl cyanide-p-trifluoromethoxy-phenylhydrazone and 2,4-dinitrophenol (FCCP and DNP, respectively), decreased formation of procoagulant platelets in a dose dependent manner as measured by AnnV binding. DNP decreased CVX stimulated THR generation to the baseline. Although mitochondrial uncoupling alleviated the spatio-temporal transition of proaggregatory platelets to procoagulant phenotype, it did not affect THR nor CVX stimulated platelet aggregation. Mitochondrial uncoupling did not affect granule release, whereas it did increase platelet accretion by ~10-fold. On hemostasis model there was no statistical difference between experimental groups in platelet accumulation as measured by CD41 fluorescence, whereas fibrin (59D8 fluorescence) in DMSO group was ~2-fold higher than in DNP. More striking finding is that bleeding time after DMSO treatment averaged 10.5 minutes, whereas hemostasis in DNP treatment group was never achieved. Together, these results identify a key role of procoagulant platelets in the formation of a stable clot; hence procoagulant platelets are essential in secondary hemostasis. Manipulations targeted to affect percentage of procoagulant subpopulation may provide a novel pharmacologic strategy for the treatment of procoagulant related hematopathologies. Disclosures Jobe: CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.
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Greene, Teshell K., Li Zhai, Beatrice Razzo, Karen Vo, Denise E. Sabatino, Rodney M. Camire, Valder R. Arruda, and Mortimer Poncz. "Platelet Factor VIII-Induced Megakaryocyte Apoptosis: Implications for Hemophilia A Gene Therapy." Blood 120, no. 21 (November 16, 2012): 2051. http://dx.doi.org/10.1182/blood.v120.21.2051.2051.
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Abstract Abstract 2051 Hemophilia A is an X-chromosome-linked bleeding disorder due to a deficiency in clotting factor (F) VIII, affecting ∼1:5000 males. Inhibitor development to circulating FVIII is not an uncommon complication for patients receiving protein replacement therapy. We had proposed that the ectopic expression of FVIII in platelet alpha-granules will be targeted to sites of vascular injury, and be protected from circulating FVIII inhibitors. We and others have confirmed that this platelet (p)FVIII is in fact effective in a number of hemostatic models and is protected from circulating inhibitors. However, pFVIII has different temporal and spatial availability from plasma FVIII that may underlie the limited efficacy of pFVIII observed in some hemostatic models using FVIIInull mice. No group has been able to achieve levels greater than the equivalent of ∼200 mU/ml of whole blood pFVIII levels using standard human (h) B-domainless FVIII (hBFVIII). We therefore sought to improve pFVIII efficacy by expressing higher levels and/or by using pFVIII species with increased activity. We tested canine (c) BFVIII because of its reported higher efficacy in vitro and in mice, and because it is secreted at 1.5–2 fold HIGHER levels than hBFVIII in cell lines. We found that the level of pcBFVIII protein in Megs was actually ∼3-fold LOWER than phBFVIII. In spite of this lower level, pcBFVIII corrected the bleeding diathesis in FVIIInull mice more effectively than phBFVIII. Using Megs from transgenic FVIIInull mice that expressed pcBFVIII or phBFVIII, we found that mRNA levels of the FVIIIs were comparable. However, compared to littermate pFVIIInull mice, FVIIInull mice that expressed either pFVIII had lower ploidy Megs and these Megs showed higher levels of apoptosis. This apoptosis was enhanced by increasing the pFVIII level in the Megs by exposure to sodium butyrate. These effects were more marked in the pcBFVIII- than phBFVIII-expressing Megs. We then tested whether this phenomenon was limited to murine Megs, but found that lentiviral expression of pBFVIII increased Meg apoptosis whether the Megs were derived from mice, canine or human marrow, and that in each, levels of pcBFVIII were ∼3-fold lower than phBFVIII. In vivo studies support these in vitro apoptotic effects of pFVIII on Megs: In transgenic mice, steady-state platelet counts were normal in pFVIII mice, but TPO levels were 4-times higher in the phBFVIII and 8-times higher in the pcBFVIII compared to littermate controls (p<0.02 and p<0.0002 for phBFVIII/FVIIInull and pcBFVIII/FVIIInull vs. FVIIInull, respectively). In FVIII lentiviral transfected marrow/bone marrow transplant (lenti/BMT) studies, platelet counts did not differ in recovering recipient mice to negative controls, but both pBFVIII-expressing recipient lenti/BMT mice showed a peak in recipient platelets contributing to the total platelet mass at 4 weeks that decreased over the following month. This was especially true for the pcBFVIII-expressing lenti/BMT mice wherein >12% of the platelets were recipient-derived at 4 weeks compared to <2% in mice receiving a control lentivirus-transfected marrow (p<0.002). Thus, these studies suggest that there is a limit as to how much pFVIII can be stored in platelets, so efforts should be focused on increasing pFVIII activity. As an example of this approach, we had identified an R1645H Furin/PACE cleavage site difference between hBFVIII and cBFVIII that increases the level of single-chain to two-chain FVIII released from cell lines. The more single-chain FVIII, the slower the loss of FVIII activity, and the more overall activity was seen. We introduced this R1645H substitution into phBFVIII Megs and show antigen levels of this phBFVIIIR1645H comparable to phBFVIII in vitro and in lenti/BMT-reconstituted FVIIInull mice. Excitingly, these phBFVIIIR1645H lenti/BMT FVIIInull mice demonstrated normalization of hemostasis in several hemostatic models. Since pFVIII tends to be released deep inside a growing thrombus from degranulating platelets with limited washout, a more stable version of FVIII such as FVIIIR1645H may be particularly efficacious as pFVIII, and we intend to pursue this hFVIII variant in larger animal studies as it may be especially useful for clinical gene therapy. Disclosures: No relevant conflicts of interest to declare.
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Lucas, Charles E. "The Evolution of Liver Injury Diagnosis and Treatment in the Past 50 Years." Panamerican Journal of Trauma, Critical Care & Emergency Surgery 3, no. 3 (2014): 124–31. http://dx.doi.org/10.5005/jp-journals-10030-1103.
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ABSTRACT Background During the past 50 years many changes have occurred in treating liver injury. Nonoperative therapy (NOT) for blunt penetrating injuries is now common. Study design This report highlights the current therapy of liver injury. Results Complications of NOT include early rebleeding requiring prompt operative intervention; intrahepatic hematoma which becomes infected necessitating drainage; bile peritoneum requiring exploration and drainage; and hemobilia requiring embolization, hepatotomy with ligation, or resection. Operative exposure through a midline incision which can be extended as a median sternotomy is preferred. Prehepatic and intrahepatic packs are helpful. Full mobilization of the right and left triangular ligaments augments exposure. Hemostasis for both blunt and penetrating, usually, is obtained by hepatorrhaphy using the 2’ blunt tipped needle swedged onto a 2-O chromic suture. Through-and-through injuries may require hepatotomy with intrahepatic ligation of cross-linking vessels. Locally destructive wounds may require nonanatomic debridement to the point of healthy liver tissue which is then sutured. Formal segmentectomy, or lobectomy, is seldom needed. Hepatic artery ligation controls deep arterial not involving the portal venous supply. The retrohepatic caval atrial shunt will facilitate hemostasis from central liver injuries involving the hepatic veins or retrohepatic cava. Debridement of emacerated liver tissue should be extended to good liver parenchyma where deep liver sutures help with approximate the edges. Drainage is not used for minor injuries. Closed suctions are best for larger wounds. Common duct drainage should be avoided. Conclusion Most liver injuries are treated by NOT. Operative therapy involves hemostasis, debridement when necessary, and selective drainage. How to cite this article Lucas CE. The Evolution of Liver Injury Diagnosis and Treatment in the Past 50 Years. Panam J Trauma Crit Care Emerg Surg 2014;3(3):124-131.
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Siner, Joshua I., Julie M. Crudele, Courtney T. Connolly, Shangzhen Zhou, Elizabeth P. Merricks, Timothy C. Nichols, Rodney M. Camire, and Valder R. Arruda. "Unexpected Role of PACE/Furin Cleavage Site in FVIII Biology: Implications for Hemophilia a Therapy." Blood 124, no. 21 (December 6, 2014): 105. http://dx.doi.org/10.1182/blood.v124.21.105.105.
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Abstract The paired basic amino acid cleaving enzyme (PACE)/Furin is a protein convertase system that plays a vital role in several biological processes, including coagulation. The propeptide processing of human FIX by PACE/Furin is a critical posttranslational modification, so cells co-expressing PACE/Furin and FIX are used for production of clinical recombinant protein. In the development of recombinant B-domain deleted (BDD) FVIII for hemophilia A (HA), a 14 amino acid B-domain sequence containing a putative cleavage site for PACE/Furin was retained because it was believed to be critical for intracellular processing and secretion. In contrast to FIX, we report here a surprising detrimental effect of PACE/Furin in FVIII activity and intracellular processing and secretion. We engineered a human FVIII variant where the PACE/Furin site at residues 1645-1648 was deleted from FVIII-BDD (FVIII-ΔP/F). Notably, FVIII-ΔP/F exhibits a 3-fold increased activity over FVIII-BDD (p=0.0004) in a 2-stage APTT assay. Moreover, the A2-domain dissociation of activated FVIII-ΔP/F was also 3-fold longer compared to FVIII-BDD, suggesting a more stable activated FVIII molecule. The amount of FVIII secreted from stably transduced BHK cells was about 3-fold higher for FVIII-ΔP/F than for FVIII-BDD. Conversely, the amount of intracellular FVIII antigen was lower for FVIII-ΔP/F than for FVIII-BDD. To confirm that PACE/Furin was implicated in the underlying mechanisms for the observed differences in FVIII secretion, we inhibited PACE/Furin in FVIII-BDD producing BHKs by transducing them with vectors expressing an engineered α1-antitrypsin variant (haat-PDX) that specifically inhibits PACE/Furin. This resulted in a 40% increase in FVIII secretion (p=0.017) and a decrease in intracellular FVIII-BDD, whereas transduction with the haat-wild type control, which does not inhibit PACE/Furin, did not significantly change the amount of FVIII secreted (p=0.32). Importantly, the secretion and intracellular levels of FVIII-ΔP/F were not affected by the inhibition of PACE/Furin by haat-PDX, indicating that the secretion of this FVIII variant does not benefit from further inhibition of PACE/Furin cleavage. Together these data suggest that the increased secretion of FVIII-ΔP/F compared to FVIII-BDD is due to the former circumventing PACE/Furin. Furin is ubiquitously expressed in mammal tissues. In a stringent cellular model, we used LoVo, a unique human cell line that lacks functional Furin to determine whether expression of FVIII-ΔP/F and FVIII-BDD would differ, as we have observed in cells expressing Furin. Interestingly, the secretion of FVIII-ΔP/F and FVIII-BDD were comparable. This result confirms that FVIII-BDD is secreted better in the absence of Furin. In summary, our novel variant FVIII-ΔP/F exhibits enhanced secretion primarily by bypassing PACE/Furin cleavage; inhibiting this cellular process also enhances the secretion of FVIII. Futhermore in vivo experiments also demonstrated a beneficial effect of FVIII-ΔP/F: HA mice (n=4-7/dose) given adeno-associated viral 8 (AAV8) vectors for liver gene expression of FVIII-ΔP/F resulted in a 3-fold higher circulating FVIII levels than FVIII-BDD-expressing mice (p=0.025). These exciting results from human FVIII-ΔP/F prompt us to test this variant HA canine model. First we found that recombinant canine FVIII with the entire PACE/Furin site deleted (cFVIII-ΔP/F) had increased activity in a 2-stage aPTT assay compared to wild-type cFVIII-BDD. Injection of cFVIII-ΔP/F effectively corrects the hemophilia coagulopathy in two HA dogs. Further, AAV8 liver gene therapy with cFVIII-ΔP/F in additional two HA dogs at doses of ~6 x 1012 vg/kg, a log lower than previously used for canine FVIII-BDD AAV8 gene therapy, resulted in therapeutic levels of cFVIII and shortening of clotting times. Preliminary data on injection of cFVIII-BDD protein was well tolerated in cFVIII-ΔP/F-expressing dogs. In conclusion, these data suggest that PACE/Furin cleavage of FVIII hampers protein biological activity. FVIII variants lacking PACE/Furin recognition sequences are secreted more efficiently and exhibit improved hemostatic effects in both protein- and gene-based strategies. Inhibition of PACE/Furin in manufacturing systems for recombinant human FVIII may increase the yields of protein production. Thus these strategies have a strong rationale for translation to HA therapy. Disclosures No relevant conflicts of interest to declare.
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Fernandez-Bello, Ihosvany, Raul Justo Sanz, Elena Monzón Manzano, Francisco García Río, Carolina Cubillos, Cristina Balbás-García, Teresa Álvarez-Roman, et al. "Platelet Dysfunction and Cellular Microparticles May be Involved in the Hipercoagulable State Observed in Obstructive Sleep Apnea Syndrome." Blood 132, Supplement 1 (November 29, 2018): 5048. http://dx.doi.org/10.1182/blood-2018-99-116018.
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Abstract Introduction: Obstructive sleep apnea syndrome (OSA) is a common disorder characterized by repetitive episodes of nocturnal breathing cessation due to upper airway collapse that cause intermittent hypoxia. Because of this, there is an increase in oxidative stress, endothelial damage and mortality associated with the incidence of thrombotic events. The evaluation of platelet function by flow cytometry (FCM) and hemostatic capacity through global hemostasis tests have been successfully used in the study of the prothrombotic state associated to numerous diseases. We anticipate that these techniques may help to clarify the mechanism involved in the prothrombotic state of OSA. Materials and Methods: We included 16 OSA patients diagnosed in the Pneumology Unit of the University Hospital La Paz and 59 healthy controls recruited in the Donor Unit of the same Hospital. Platelet surface receptors were analysed by FCM (FACScan, BD Biosciences) with specific monoclonal antibodies (mAbs) against CD41/αIIb and CD61/β3, CD42a and CD42b. Platelet activation was assessed by FCM through PAC1-FITC binding (mAbs that recognizes activated conformation of fibrinogen receptor), P-selectin and CD63 exposure in baseline condition and after stimulation with 100 µM TRAP (agonist of the PAR-1 receptor) and 10 µM ADP. Platelet apoptosis was analysed by FCM through FITC-annexin V (BD Pharmingen) binding to phosphatidylserine (PS) exposed on platelet membrane under basal conditions and by determination of activity of caspases -3/7, -8, and -9 (BD Millipore). The overall hemostatic capacity was determined by the evaluation of thrombin generation using the Calibrated Automatic Thrombinography (CAT) with fresh platelet-poor plasma (obtained after double centrifugation of whole blood at 2500g, 20 minutes and 24ºC) and by the assessment of kinetics of clot formation using Rotational Thromboelastometry (ROTEM® Gamma, Tem Innovations GmbH, Spain) using whole blood and activation by recalcification (naTEM® test, Tem Innovations GmbH, Spain). The statistical analysis was performed using SPSS 9.0 software (SPSS Inc., Chicago, Illinois, USA). Statistical significance was established at p <0.05. Results: No significant differences in age or sex were found between groups. Patients presented anxiety-depressive disorder (15%), ischemic cardiomyopathy (8%), dyslipidemias (38%), arterial hypertension (69%) and lower minimum oxygen pressures during sleep (mean±SD; patients: 76±8 % vs controls: 89±1 %; p< 0.001) indicating hypoxia. In basal conditions, an increase of CD42a subunit of the von Willebrand factor receptor was observed in patients with OSA but no differences were found in the exposure of fibrinogen receptor either at basal condition or after activation with TRAP or ADP (Table 1 and Figure 1). Platelet PS exposure before and after TRAP stimulation was higher in the patient group (Figure 1) but no differences between groups were observed in caspase activities (Table 2). Clotting time (CT), maximum clot firmness (MCF) and MP-associated thrombin generation were higher in patients with OSA (Figure 2). Conclusion: Mechanisms related to the mobilization of P-selectin to platelet surface and with the maintenance of phospholipids distribution in platelet membrane might be altered in OSA. The increment of P-selectin exposure may favor the interaction between white cells and platelets through PSGL-1 ligand from lymphocytes which in turn may produce endothelial damage explaining part of OSA's pathophysiology. A lower CT and higher MCF reflected a hypercoagulable state in patients, perhaps related to their higher MP-associated thrombin generation and, at least in part, due to the enhanced platelet-PS exposure. Our results also suggest that both ROTEM and CAT are useful tools for characterizing prothrombotic states in OSA patients. Work supported by grant from FIS-FEDER PI15/01457. NB holds a Miguel Servet II (FIS-FEDER CP14/00024). Disclosures Álvarez-Roman: Shire: Consultancy; NovoNordisk: Consultancy; SOBI: Consultancy. Jimenez-Yuste:Grifols: Consultancy, Research Funding; Octapharma: Consultancy, Research Funding; CSL Behring: Consultancy; Bayer: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Shire: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; NovoNordisk: Consultancy, Research Funding; Sobi: Consultancy, Research Funding. Butta:FIS-Fondos FEDER: Research Funding.
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Thahir, Hasanuddin, Arni I. Djais, Shek Wendy, Muhammad H. Achmad, and Fuad H. Akbar. "Management of maxillary labial frenum with comparison of conventional and incision below the clamp techniques: a case report." Journal of Dentomaxillofacial Science 3, no. 1 (April 1, 2018): 61. http://dx.doi.org/10.15562/jdmfs.v3i1.634.
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Objective: Describes and compare the procedure of superior labial frenum frenectomies with conventional technique and incision below the clamp technique.Methods: Two female patient came to Departement of periodonsia, Unhas Dental Hospital to have frenectomies. The first patient was 28 year old with labialis superior frenulum reached attached gingiva, gingival recession 1-2 mm with calculus deposits, and referred to do frenectomy with Incision below the Clamp. While the second patient was 15 year old with labialis superior frenulum extend up to palatine papilla, central diastema and referred to do frenectomy with conventional technique.Results: The conventional techniques is done by engaged the frenum by a haemostat that inserted into the depth of the vestibule, and incision were placed on the upper and the under surface of haemostat, then followed by suturing the wound and periodontal pack. Insision below the clamp technique is done by placing a hemostat in position adjacent and parallel to the lip mucosa, and incision carried out below the clamp, then followed by suturing at the mucolabial fold and periodontal pack.Conclusions: Patients were very satisfied with the results that achieved. Technique Incision below the Clamp is an alternative treatment with good aesthetic and less bleeding during frenectomies by using a scalpel.
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Wang, Lili, Yang Yang, Camilo Ayala Breton, John White, Jia Zhang, Yan Che, Alexei Saveliev, et al. "CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX–knockout mice." Blood 133, no. 26 (June 27, 2019): 2745–52. http://dx.doi.org/10.1182/blood.2019000790.
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Abstract Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.
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Escobar, Miguel A., Kenneth Chong, and Keith W. Hoots. "Experience with the “Off Label” Use of Recombinant Factor VIIa in a University Hospital." Blood 104, no. 11 (November 16, 2004): 4017. http://dx.doi.org/10.1182/blood.v104.11.4017.4017.
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Abstract Recombinant factor VIIa (rFVIIa), NovoSeven@ (Denmark), is a bypass agent approved in the United States for patients with hemophilia and inhibitors. The off-label use of this medication (non approved use) is becoming common in large institutions. Its main use seems to be for excessive bleeding due to trauma and surgery. Here we report our 2 year experience with the off-label use of this product and describe a protocol that is followed at our teaching hospital. Administration of rFVIIa was based on the described protocol. The medication is controlled by the hospital pharmacy and authorization for its use has to be given by the hematologists in collaboration with the clinical pharmacist. To be eligible for the use of rFVIIa the patient has to have received at least 10 units of red pack cells and fresh frozen plasma. In addition, the patient should have fibrinogen level greater than 100 mg/dl, platelet count greater than 70,000/mm3 and fibrin split products less than 80 mcg/ml. A total of 25 patients where treated off-label for excessive bleeding and 4 patients where given rFVIIa preoperative for prevention of bleeding. The doses ranged from 35 to 120 micrograms/kg of body weight with an average of 2 doses given per patient. The use of Factor VIIa was more common in the neurosurgical (NS) setting (7), followed by cardiovascular (CV) (6), gastrointestinal (GI) surgery (6), trauma (6) and others (4). Overall 62% of the patients had adequate hemostasis after the administration of rFVIIa. Successful hemostasis with rFVIIa by diagnosis was 100% for preoperative prophylaxis and GI, for NS 57%, for trauma 50% and for CV 17%. All the patients in the CV group where admitted with acute dissecting aortic aneurysms. No evidence of thromboembolic events where described. The “off-label” use of rFVIIa continues to be widely used with variable results. Until further randomized or controlled, double blind, clinical trials are done, no definitive recommendations can be given. Although this report includes a small cohort of patients, we will consider revising our protocol for bleeding following cardiovascular surgery and trauma.
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Kibrik, Pavel, Justin Eisenberg, Marc A. Bjurlin, Natalie Marks, Anil Hingorani, and Enrico Ascher. "Endoureteral coil embolization of an ureteral arterial fistula." Vascular 25, no. 5 (April 24, 2017): 557–60. http://dx.doi.org/10.1177/1708538117704522.
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Background Ureteral arterial fistulas are rare but potentially life threatening. We present a female who developed a ureteral arterial fistula following a right robotic nephrectomy. After several endovascular interventions to control the bleeding had failed, we approached the fistula through the right ureteral stump with coil embolization. Methods Coil embolization of the right ureteral stump was performed. We utilized a 6Fr × 45 cm sheath inserted through one of the cystoscope channels to cannulate the right ureteral orifice. We then performed a retrograde ureterogram. After, we were able to visualize full length of the ureter, ahd we began placing several 10–12 mm Nester coils to pack the ureter and tamponade the fistula for hemostasis. After the ureter was packed, we injected 1 g of Vancomycin into the ureter. The sheath and cytoscope were removed and the patient did well and was sent to the recovery room. Results Postoperatively, the patient had no complaints of hematuria and her hemoglobin level remained unchanged. She was observed for a few days prior to being discharged to home. The patient’s follow-up at six months revealed resolution of her hematuria. Conclusion Ureteral arterial fistula is a potentially life-threatening condition. Endovascular stenting has provided a safe, reliable alternative to open surgery. However, when endovascular options are not satisfactory, coil embolization of the ureteral stump may serve as a safe and effective alternative treatment for these cases.
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Cruceru, Adelina Maria, Ionut Negoi, Sorin Paun, Sorin Hostiuc, Ruxandra Irina Negoi, and Mircea Beuran. "Complex Perineal Trauma with Anorectal Avulsion." Case Reports in Surgery 2016 (2016): 1–5. http://dx.doi.org/10.1155/2016/4830712.
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Introduction. The objective of this case report is to illustrate a severe perineal impalement injury, associated with anorectal avulsion and hemorrhagic shock.Results. A 32-year-old male patient was referred to our hospital for an impalement perineal trauma, associated with complex pelvic fracture and massive perineal soft tissue destruction and anorectal avulsion. On arrival, the systolic blood pressure was 85 mm Hg and the hemoglobin was 7.1 g/dL. The patient was transported to the operating room, and perineal lavage, hemostasis, and repacking were performed. After 12 hours in the Intensive Care Unit, the abdominal ultrasonography revealed free peritoneal fluid. We decided emergency laparotomy, and massive hemoperitoneum due to intraperitoneal rupture of pelvic hematoma was confirmed. Pelvic packing controlled the ongoing diffuse bleeding. After 48 hours, the relaparotomy with packs removal and loop sigmoid colostomy was performed. The postoperative course was progressive favorable, with discharge after 70 days and colostomy closure after four months, with no long-term complications.Conclusions. Severe perineal injuries are associated with significant morbidity and mortality. Their management in high volume centers, with experience in colorectal and trauma surgery, allocating significant human and material resources, decreases the early mortality and long-term complications, offering the best quality of life for patients.
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Liu, Xuqiang, Jing Dong, Qiongxin Liang, Hui-min David Wang, Zhenhua Liu, Ruian Xu, and Wenyi Kang. "Coagulant Effects and Mechanism of Schefflera heptaphylla (L.) Frodin." Molecules 24, no. 24 (December 12, 2019): 4547. http://dx.doi.org/10.3390/molecules24244547.
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Schefflera heptaphylla (L.) Frodin, are commonly used in anti-inflammatory, analgesic, traumatic bleeding and hemostasisas. In this paper, the coagulation effect of the ethanol extract (Set), ethyl acetate phase (Sea) and n-butanol phase (Sbu) was evaluated by prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen content (FIB) assays in vitro. Then, Three main lupanine triterpenes (compounds A–C) were isolated and identified from Sea and Sbu by a bioassay-guided method and their structure were identified as 3α-Hydroxy-lup-20(29)-ene-23, 28-dioic acid, betulinic acid 3-O-sulfate and 3α-Hydroxy-lup-20(29)-ene-23, 28-dioic acid 28-O-(α-l-rhamnopyranosyl(1→4)-O-β-d-glucopyranosyl(1→6))-β-d-glucopyranoside) by spectroscopic data analysis. Among of them, compound B was confirmed to have significant coagulant effect in vitro. Furthermore, the pro-coagulation mechanism of S. heptaphylla extracts and compound B were investigated by measuring whole blood viscosity (WBV), plasma viscosity (PV), erythrocyte sedimentetion rate (ESR), pack cell volume (PCV), APTT, PT, TT, and FIB in vivo. Meanwhile, the levels of thromboxane B2 (TXB2), 6-keto prostaglandin F1α (6-keto-PGF1α), endothelial nitric oxide synthase (eNOS) and (endothelin-1) ET-1 were detected. The bleeding time (BT) was tested by tail bleeding method, which proved the traumatic bleeding and hemostasis activities of S. heptaphylla. The pharmacology experiments showed that the Set, Sea, Sbu and compound B has significant pro-coagulation effect. In addition, compound B might be the main constituent of pro-coagulation in S. heptaphylla These results could support the fact that S. heptaphylla could be used traditionally to cure traumatic bleeding, and the pro-coagulation effects were associated with the regulation of vascular endothelium active substance and hemorheology parameters.
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Casonato, Alessandra, Francesca Sartorello, Maria Grazia Cattini, Elena Pontara, Carmen Soldera, Antonella Bertomoro, and Antonio Girolami. "An Arg760Cys mutation in the consensus sequence of the von Willebrand factor propeptide cleavage site is responsible for a new von Willebrand disease variant." Blood 101, no. 1 (January 1, 2003): 151–56. http://dx.doi.org/10.1182/blood-2002-04-1046.
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Abstract We describe a von Willebrand disease (VWD) variant characterized by the persistence of von Willebrand factor (VWF) propeptide as a result of a C>T transition at nucleotide 2527 in exon 17 of the VWF gene. This mutation, which was present in the proband and his father, predicts the substitution of Cys for Arg at position 760 of pre–pro-VWF, 4 residues before the propeptide cleavage site belonging to a consensus sequence for substrate recognition by the processing enzyme paired dibasic amino acid–cleaving enzyme (PACE)/furin. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) documented the presence of both processed and unprocessed VWF in the patient's plasma, with unprocessed VWF relatively less represented. The patient's hemostatic phenotype was characterized by a mild decrease in plasma factor VIII (FVIII) and VWF, a decrease in plasma VWF multimers, and a mild reduction in the FVIII binding capacity of VWF. The FVIII binding defect was more pronounced in the proband than in the father because he also inherited the type 2N Arg91Gln mutation from his mother. The persistence of VWF propeptide did not impair VWF synthesis because platelet VWF content was normal, nor did it compromise VWF storage in endothelial cells, because of the normal post–1-deamino-8-D-arginine vasopressin (DDAVP) increase in plasma VWF. Coexpression of wild-type and Arg760Cys VWF into a Furin-producing BHK cell line resulted in decreased VWF secretion and a defect in the FVIII binding capacity of VWF, together with the persistence of VWF propeptide. These findings confirm that a normal consensus sequence for VWF propeptide cleavage and efficient cleavage are required in vivo for normal FVIII binding capacity of VWF.
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Lyde, Randolph B., Hyunsook Ahn, Karen K. Vo, Danuta Jadwiga Jarocha, Li Zhai, Spencer K. Sullivan, Valder R. Arruda, et al. "Infusion of Coagulation Factor VIII-Containing Induced Pluripotent Stem Cell (iPSC)-Derived Megakaryocytes (iMks) Shows Potential As Hemophilia a Treatment." Blood 128, no. 22 (December 2, 2016): 2559. http://dx.doi.org/10.1182/blood.v128.22.2559.2559.
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Abstract Ectopically expressed factor VIII (FVIII) in megakaryocytes (MKs) and platelets (pFVIII) is stored in a-granules and released at sites of vascular injury by activated platelets (Plts), restoring hemostasis in FVIIInull mice, even in the presence of neutralizing inhibitors. These studies support the idea that unlike therapies that correct plasma levels of FVIII, pFVIII may be a useful therapy in patients with hemophilia A who have intractable inhibitors and significant bleeds. Expressing FVIII in Plts, however, has limitations that make pFVIII gene therapy through bone marrow transplantation (BMT) problematic: 1) pFVIII expressed during megakaryopoiesis can injure the Mks, potentially exacerbating post-BMT thrombocytopenia, and 2) pFVIII's efficacy in joint and intracranial bleeds has yet to be shown, especially in the presence of inhibitors. Due to these limitations we propose an alternative strategy: infusing patient-specific iMks derived from personalized iPSCs and expressing either human B-domain-deleted (BDD) FVIII or variants of FVIII that have greater stability and longer half-lives. Our group has shown that infusing in vitro-grown Mks into mice releases functional Plts in the recipient animals. iPSCs are a renewable source of stem cells that can be pre-screened to select clones that both express high levels of pFVIII and also release high numbers of Plts after differentiation into iMks. As proof-of-principle, iMks were transfected with a self-inactivating lentivirus containing cDNA for 1 of 3 FVIII variants: wildtype BDD FVIII (WT FVIII), a PACE/furin cleavage site FVIII (FVIIIR1645H) variant, and an amino acid 1645 to 1648 deletion FVIII (FVIIIΔ) variant that removes the entire PACE/furin cleavage site. FVIIIR1645H and FVIIIΔ showed greater stability and consequently greater specific activity with no increase in injuring Mks. We previously published that hemophilia A mice expressing pFVIIIR1645H were more hemostatically corrected than comparable mice expressing WT pFVIII. All of the FVIII variant iMks expressed at least a 40-fold higher level of mRNA compared to the non-transduced control (N=6) and integration levels show the same number of viral copies between the groups (N=6). All variants expressed >550 pg FVIII/106 CD42b+ iMKs (N=6). Upon activation with thrombin, transduced Mks released the FVIII into the supernatant. To examine whether this pFVIII injured the developing Mks, baseline PAC-1 binding for Mk activation in culture (N=3), TUNEL staining and Annexin-5 binding for apoptosis (N=4) were analyzed with no differences observed with WT Mks not expressing pFVIII. To test the ability of FVIII-expressing iMks to correct the coagulopathy in hemophilia A, 5x105 iMks were added to FVIIInull murine whole blood (0.11 ml) and evaluated for clot formation using rotational thromboelastometry (ROTEM). Each pFVIII iMk variant showed a decrease in clotting time, clot formation time, and an increase in maximum clot firmness when compared to the non-transduced control (p<0.007 for each, N=4). These FVIII expressing iMks were also tested in vivo in a FeCl3 carotid artery injury murine model. 24 hours prior to infusion, recipient hemophilia A mice were treated with clodronate liposomes to eliminate circulating monocytes and to improve the survival of infused human iMks and their released Plts. Immediately post iMks (5x106)infusion, a 20% FeCl3 solution was applied to the carotid artery for 3 mins and flow rate through the injured vessel was measured for 30 mins. Both WT FVIII and FVIIIR1645H showed a significant decrease in blood flow through the injured vessel from 1.2 ml/min seen in FVIIInull mice receiving control iMks to 0.4 ml/min (p<0.05, N=10). Wild-type mice had a flow rate of 0.13 ml/min. These data indicate that pFVIII within iMKs or their derived Plts expressing FVIII can improve hemostasis in vitro and in vivo. These studies provide the groundwork to examine whether infused iMks pFVIII can improve hemostasis in the setting of inhibitors. Disclosures Arruda: Pfizer: Patents & Royalties, Research Funding. Sabatino:Spark Therapeutics: Research Funding. Camire:Bayer: Consultancy; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding.
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Quinlan, Mark R., Damien M. Bolton, and Rowan G. Casey. "The management of rectal bleeding following transrectal prostate biopsy: A review of the current literature." Canadian Urological Association Journal 12, no. 3 (December 16, 2017): E146–53. http://dx.doi.org/10.5489/cuaj.4660.
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Introduction: Since the advent of prostate-specific antigen (PSA)-based testing, transrectal ultrasound (TRUS)-guided prostate biopsy has become a standard part of the diagnostic pathway for prostate cancer (PCa). Rectal bleeding is one of the common side effects of this transrectal route. While rectal bleeding is usually mild and self-limiting, it can be life-threatening. In this article, we examine rectal bleeding post TRUS-guided prostate biopsy and explore the literature to evaluate techniques and strategies aimed at preventing and managing this common and important complication.Methods: A PubMed literature search was carried out using the keywords “transrectal-prostate-biopsy-bleed.” A search of the bibliography of reviewed studies was also conducted. Additionally, papers in non-PubMed-listed journals of which the authors were aware were appraised.Results: Numerous modifiable risk factors for this bleeding complication exist, particularly anticoagulants/antiplatelets and the number of core biopsies taken. Successfully described corrective measures for such rectal bleeding include tamponade (digital/packs/catheter/tampon/condom), endoscopic sclerotherapy/banding/clipping, radiological embolization, and surgical intervention.Conclusions: We advocate early consultation with the colorectal/gastroenterology and interventional radiology services and a progressive, stepwise approach to the management of post-biopsy rectal bleeding, starting with resuscitation and conservative tamponade measures, moving to endoscopic hemostasis ± radiological embolization ± transanal surgical methods. Given the infrequent but serious nature of major rectal bleeding after TRUS biopsy, we recommend the establishment of centralized databases or registries forthwith to prospectively capture such data. To the best of our knowledge, this is the first comprehensive look specifically at the management of post-TRUS biopsy rectal bleeding.
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Lambert, Michele P. "Improving interpretation of genetic testing for hereditary hemorrhagic, thrombotic, and platelet disorders." Hematology 2020, no. 1 (December 4, 2020): 76–81. http://dx.doi.org/10.1182/hematology.2020000091.
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Abstract The last 10 years have seen an explosion in the amount of data available through next-generation sequencing. These data are advancing quickly, and this pace makes it difficult for most practitioners to easily keep up with all of the new information. Complicating this understanding is sometimes conflicting information about variant pathogenicity or even about the role of some genes in the pathogenesis of disease. The more widespread clinical use of sequencing has expanded phenotypes, including the identification of mild phenotypes associated with previously serious disease, such as with some variants in RUNX1, MYH9, ITG2A, and others. Several organizations have taken up the task of cataloging and systematically evaluating genes and variants using a standardized approach and making the data publicly available so that others can benefit from their gene/variant curation. The efforts in testing for hereditary hemorrhagic, thrombotic, and platelet disorders have been led by the International Society on Thrombosis and Haemostasis Scientific Standardization Committee on Genomics in Thrombosis and Hemostasis, the American Society of Hematology, and the National Institutes of Health National Human Genome Research Institute Clinical Genome Resource. This article outlines current efforts to improve the interpretation of genetic testing and the role of standardizing and disseminating information. By assessing the strength of gene–disease associations, standardizing variant curation guidelines, sharing genomic data among expert members, and incorporating data from existing disease databases, the number of variants of uncertain significance will decrease, thereby improving the value of genetic testing as a diagnostic tool.
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Small, Juliana, Shannon Zintner, Lynn E. Dankner, and Paris Margaritis. "Characterization of the Interaction of Activated FVII with the Endothelial Protein C Receptor (EPCR) in the Rat." Blood 132, Supplement 1 (November 29, 2018): 2455. http://dx.doi.org/10.1182/blood-2018-99-116892.
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Abstract Activated FVII (FVIIa) has been shown to interact with EPCR resulting in long-term extravascular tissue presence in mice, well beyond its short circulating half-life. In addition, data in hemophilia patients on short term FVIIa prophylaxis indicate that clinical improvements can persist even in the post-prophylaxis period. Taken together, these data suggest that EPCR may sequester administered FVIIa in the extravascular space that retains activity, potentially explaining the hemostatic improvements that persist long after its infusion has ceased. As such, the EPCR-FVIIa interaction may have mechanistic and translational ramifications in the treatment of bleeds in hemophilia which needs to be further investigated. Unfortunately, hemophilic mice do not model the human disorder in terms of bleeding diathesis. In contrast, the novel rat model of hemophilia A (Nielsen LN et al, 2014) exhibits spontaneous bleeds that, if untreated, can be fatal. Therefore, this model can be utilized to better understand the FVIIa-EPCR interaction in the on-demand and prophylactic action of FVIIa administration in hemophilia. As an initial step towards that goal, here we characterize for the first time the rat FVIIa-EPCR interaction in vitro. We generated rat FVIIa by introduction of a PACE/Furin intracellular cleavage site (RKRRKR) between the light and heavy chains of rat FVII, an approach we have previously shown to result in secretion of two-chain activated FVII protease of various species (human, mouse and canine) in vitro. Recombinant rat FVIIa was purified in a two-chain form using conditioned medium from a HEK-293 stable cell line. The affinity of rat FVIIa to rat EPCR was assessed by incubation of the protease on engineered CHO-K1 cells that stably expressed full length rat EPCR. We found that rat FVIIa exhibited minimal binding to rat EPCR; in contrast human FVIIa bound rat EPCR with a Kd of ~400 nM. The lack of EPCR binding of rat FVIIa, similar to what we previously observed with mouse FVIIa (Pavani G et al., 2014 and 2016), complicates studies aimed at deciphering the hemostatic effects of the FVIIa-EPCR interaction in hemophilia using the rat. Therefore, we decided to engineer a rat FVIIa molecule with EPCR binding capacity so that it can be compared to rat FVIIa (non-EPCR binder) in subsequent in vivo experiments. For this, a rat FVIIa chimeric protease was generated using the first 11 amino acids of the Gla domain of rat protein C (rat FVIIa-FVRAG, with substitutions L4F, L8V, W9R, S10A, S11G). Using CHO-K1 expressing rat EPCR, we found that purified recombinant rat FVIIa-FVRAG bound rat EPCR with a Kd of ~500 nM, comparable to human FVIIa. Considering that the Gla domain, where the modifications are located, is essential for protease function, we also determined the procoagulant activity of rat FVIIa-FVRAG vs. FVIIa. We performed a thrombin generation assay in which each protease was added at increasing concentration (0-500 nM) and we measured the lag time, peak thrombin, endogenous thrombin potential. We found that rat FVIIa, rat FVIIa-FVRAG and human FVIIa (control) behaved similarly in all measured parameters for each concentration tested. In conclusion, we have defined the lack of EPCR binding of rat FVIIa and characterized a recombinant rat FVIIa molecule that can bind rat EPCR in vitro as a gain of function, but is otherwise similar to rat FVIIa. This molecule can now be used to probe the FVIIa-EPCR interaction in the novel and relevant hemophilia A rat model. Disclosures Margaritis: Bayer Hemophilia Awards: Research Funding; Bristol Myers Squibb: Other: Salary (spouse); Novo Nordisk A/S: Research Funding.
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Greene, Teshell K., Denise E. Sabatino, Nicholas P. Iacobelli, Li Zhai, Steven W. Pipe, M. Anna Kowalska, Rodney M. Camire, and Valder Arruda. "Understanding Ectopically Expressed Factor VIII (F8) In Megakaryocytes: Implications for Optimum Platelet-Delivered F8 Activity for Gene Therapy." Blood 116, no. 21 (November 19, 2010): 2205. http://dx.doi.org/10.1182/blood.v116.21.2205.2205.
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Abstract Abstract 2205 F8 ectopically expressed during megakaryopoiesis is stored in platelet (p) a-granules and released at sites of injury. This F8 is effective in hemostasis even in the presence of circulating inhibitors so that pF8 represents a novel strategy for therapy for patients with hemophilia A with inhibitors. However, cremaster laser injury studies showed that pF8 release from a-granules has a distinct temporal and spatial availability that leads to clot instability. We proposed that F8s, such as canine (c) B-domainless (B) F8, with greater specific activity than human (h) BF8, would prevent pF8 clot instability. We confirmed our thesis; however at the same time, pcBF8 levels were only ∼30% of phBF8 whether studied in transgenic mice models or by lentiviral/bone marrow transplantation (lenti/BMT) into F8null mice. This was surprising because in baby hamster kidney cells, cBF8 was secreted into the media at three times greater than hBF8. This lower level in murine megakaryocytes (Megs) was not due to mRNA levels as shown by qRT-PCR analysis. Using cultured marrow from transgenic mice expressing hBF8 or cBF8, there was a marked decrease in relative number of Megs in both pF8s compared to wildtype (WT) Megs, with the pcBF8 cells showing a greater decrease (49 ± 5% for cBF8 Megs vs. 36 ± 8% for phBF8, p < 0.02). Cultured Megs from pcBF8 and phBF8 mice both showed increased numbers of small, low ploidy Megs, and this was again higher for pcBF8 Megs (58 ± 9% for cBF8 Megs vs. 32 ± 6% for phBF8, p < 0.02). TUNEL studies as an indication of apoptosis showed that Megs expressing either F8s showed significant increased apoptosis than WT Megs with pcBF8 showing 37 ± 22% vs. 19 ± 11% in phBF8. The above data show that pcBF8 is deleterious to Meg development and was supported by a retrospective analysis of lenti/BMT pF8 platelet counts where recipient mice platelets made up a higher % of recovered platelet counts (17% ± 3% for pcBF8 vs. 4 ± 1% for phBF8, p < 0.05). We then tested whether we can separate the greater specific activity affect of pcBF8 from its deleterious affect on megakaryopoiesis. Our group has previously shown that cBF8 may have increased stability because it is predominantly expressed as a single chain, likely involving an R1645H (RH) substitution at a conserved PACE/furin site in most F8 species. Lenti/BMT pF8 studies expressing phBF8RH showed that this pF8 was expressed at the same level in reconstituted mice as phBF8, but was more efficacious in several bleeding models, including near-normal hemostasis in the cremaster laser injury model in F8null recipient mice, thus becoming the first lenti/BMT pF8-expressing F8null mouse with near-normal hemostasis in a F8null setting. Preliminary Meg count and apoptosis studies show that phBF8RH is no more deleterious to Megs than phBF8. Thus, our studies point out that F8 is deleterious to Megs with some species being more deleterious than others. This apoptosis limits F8 levels in Megs, and this deleterious effect can influence post-BMT outcome. We also present a model of how one can take advantage of a F8 variant that had a high specific activity while avoiding its low expression levels in Megs. Thus our studies provide important new insights into the biology of pF8 that may be important in developing this platelet-delivery strategy for the treatment of hemophilia A. Disclosures: Pipe: Baxter BioScience: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; Inspiration Biopharmaceuticals: Honoraria, Research Funding; CSL Behring: Honoraria.
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Siner, Joshua I., Nicholas P. Iacobelli, Lacramioara Ivanciu, Denise E. Sabatino, Mortimer Poncz, Rodney M. Camire, and Valder R. Arruda. "Bioengineering Factor VIII B-Domain Sequences Improves Function and Efficacy in Hemophilia A Models." Blood 120, no. 21 (November 16, 2012): 2208. http://dx.doi.org/10.1182/blood.v120.21.2208.2208.
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Abstract Abstract 2208 We previously reported that recombinant canine B-domain deleted factor VIII (FVIII) was expressed predominantly (>75%) as a single-chain protein and, upon activation with thrombin, yielded an active cofactor species with increased stability due to delayed A2 domain dissociation. We hypothesized that this could be in part due to a unique recognition sequence (HHQR) for intracellular PACE/Furin protease present in the canine FVIII, but absent in other mammals including human, porcine and murine FVIII where the sequence is RHQR. Here, we changed position 1645 from R to H in the B-domain deleted human FVIII (hFVIII-RH) and found that much more of the protein was expressed in a single chain form (3-fold) compared to hFVIII-wild type (hFVIII-WT). Importantly, the variant hFVIII-RH was biologically active exhibiting a 2-fold higher activity measured in a 2-stage activated partial thromboplastin time (p<0.05) likely due to slower dissociation of the A2-domain upon thrombin activation compared to hFVIII-WT (t½ 3.48 vs. 1.48 minutes, respectively). We next sought to determine the potential in vivo efficacy and safety of circulating hFVIII-RH in murine models of hemophilia A (HA) by hepatocyte-restricted transgene expression using adeno-associated viral (AAV serotype 8) vector and by exogenous administration of the recombinant protein. HA mice received varying doses (8×1012-4×1013 vg/kg) of AAV-hFVIII-WT or AAV-hFVIII-RH and exhibited a corresponding dose-dependent response of FVIII with plateau antigen levels 2–3 fold higher in hFVIII-RH than hFVIII-WT expressing mice (n = 5/group, p < 0.05 at all doses). At the low dose cohort, circulating FVIII antigen levels were 79±6.6 and 50±9 ng/ml for the FVIII-RH and FVIII-WT, respectively. In the mid dose cohort, FVIII levels were 165±54 and 84±9 ng/ml and in the high dose 274±39 and 120±35 ng/ml. We monitored the blood loss following tail-clip assay in groups of mice stratified into groups determined by expression to be low (28–60 ng/mL), mid (60–100 ng/mL), or high (above 100 ng/mL) for analysis (n= 3–9/group). Expression of either hFVIII variant was capable of decreasing the blood loss of mice in the low group compared to untreated HA mice, but did not reach that of hemostatically normal mice. Notably, at the mid expression group only hFVIII-RH expressing mice had corrected blood loss to normal hemostasis (hFVIII-RH vs hFVIII-WT p < 0.02). In the high dose all mice (FVIII-RH and FVIII-WT) exhibited blood loss similar to that of hemostatically normal mice. The increased effect of hFVIII-RH is a result of more stable clot as seen in the FeCl3- carotid artery injury model. At comparable FVIII levels, 15/16 mice expressing hFVIII-RH form stable occlusive thrombi whereas only 14/21 in hFVIII-WT group. Laser-induced injury at microcirculation resulted in increased fibrin deposition by 2–3 fold in HA mice expressing FVIII-RH (n=4 mice, 20 injuries) compared to those expressing FVIII-WT (n=3, 10 injuries). Similar findings were obtained upon injection of recombinant protein to achieve ∼40% of normal for both FVIII-RH (n=3, 13 injuries) and FVIII-WT (n=3, 18 injuries). Moreover, we assessed the immunogenicity of hFVIII-RH by administering AAV vectors to transgenic HA mice tolerant to B-domain deleted hFVIII-WT. At 4 weeks post AAV administration all mice exhibited stable hFVIII-WT/hFVIIl-RH levels (n=5/group) with no evidence of inhibitor by Bethesda assay or by mouse specific-hFVIII-IgG titers. In summary, FVIII-RH is superior to FVIII-WT in terms of expression levels and total hemostastic function without evidence of increased immunogenicity. Therefore FVIII-RH is an attractive molecule for protein replacement and gene/cell-based strategies for HA. Disclosures: No relevant conflicts of interest to declare.
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Yasuoka, Hidekata, Komei Sakata, Keiko Yoshimoto, and Tsutomu Takeuchi. "Phenotype of Platelets Are Altered and Activated in Circulation of Patients with Systemic Sclerosis." Blood 132, Supplement 1 (November 29, 2018): 3732. http://dx.doi.org/10.1182/blood-2018-99-117833.
Full textAbstract:
Abstract Systemic sclerosis (SSc), a systemic, connective tissue disease, is characterized by excessive fibrosis of skin and multiple internal organs associated with microvascular injury and autoantibody production. However, the disease process is still under investigation. Among circulating blood cells, the roles of lymphocytes and monocytes were well-examined, however, other cellular components are not well-focused. Platelets play a significant role in hemostasis physiologically. However, recent studies have revealed that platelets contain humoral factors such as cytokines, chemokines and growth factors and can distribute systemically through circulation. We hypothesized that platelets could contribute to the pathogenesis through the release of these factors. To elucidate the role of platelets in SSc, activation status and phenotype of circulating platelets in patients and association with clinical characteristics were examined. Twenty-one patients with SSc who fulfilled 2013 classification criteria of American College of Rheumatology and European League Against Rheumatism and 16 healthy controls were involved. Platelets or platelet-derived microparticles (MPs) were defined as vesicles in platelet-rich plasma which is more or less than 1mm in diameter by forward and side scatter, respectively, and positive staining with anti-CD41 antibody using flow cytometry. Activation status of platelets was examined by the expression of activation markers on platelets such as P-selectin (CD62P) or activated glycoprotein IIb/IIIa (PAC1). Production of microparticles (MPs) is defined as ratio of proportion of MP to that of platelets. Association or correlation between proportion of activated platelets and clinical characteristics or parameters of patients with SSc was also examined. For the further phenotypic analysis of platelets, expression of selected surface markers on platelets such as membrane-bound transforming growth factor (TGF)-beta, CD147 (emmprin), CD142 (tissue factor), CD31 (platelet endothelial cell adhesion molecule (PECAM)-1) was examined using flow cytometry. Furthermore, release reaction of platelets was evaluated by release of platelet factor 4 (PF4) in culture supernatant of coculture with skin fibroblasts using enzyme-linked immunosorbent assay (ELISA). As for the characteristics of 21 patients, male was 14 %, proportion of diffuse cutaneous SSc was 24 %, mean age was 63 ± 13 years, and mean disease duration was 16 ± 13 years. In SSc, both proportion of CD62P+ or PAC1+ activated platelets (P < 0.05, P < 0.05, respectively) and production of MP were higher in SSc (P < 0.05) compared to those in healthy controls. Of these, proportion of CD62P+ platelets and MP production were correlated each other (r = 0.88, P < 0.05). When activation status of platelets was compared to clinical parameters, only proportion of CD62P+ platelets was higher in diffuse cutaneous SSc and correlated with modified Rodnan skin score (mRSS) (P < 0.05). Phenotype of platelets was also altered based on the upregulation of expression of selected surface markers on platelets, such as membrane-bound TGF-beta and CD147. Interestingly, proportion of membrane-bound TGF-beta+ platelets was positively correlated with that of CD62P+ activated platelets and furthermore, proportion of membrane-bound TGF-beta+ platelets was correlated with mRSS (P < 0.05). Functionally, release reaction of platelet was enhanced in platelets from patient with SSc compared to healthy controls (P < 0.05). In conclusion, circulating platelets in SSc were activated and their phenotype is altered in circulation. Furthermore, activation status of platelets is associated with skin fibrotic phenotype, suggesting that circulating platelets reflects and can contribute to the disease process of SSc. Also, phenotype of platelets can be a novel biomarker of the disease. Disclosures No relevant conflicts of interest to declare.
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Kashiwagi, Hirokazu, Masamichi Shiraga, Hisashi Kato, Tsuyoshi Kamae, Naoko Yamamoto, Seiji Tadokoro, Yoshiyuki Kurata, Yoshiaki Tomiyama, and Yuzuru Kanakura. "Negative Regulation of Platelet Function by a Secreted Cell Repulsive Protein, Semaphorin 3A." Blood 104, no. 11 (November 16, 2004): 1546. http://dx.doi.org/10.1182/blood.v104.11.1546.1546.
Full textAbstract:
Abstract Semaphorin (Sema) 3A is a secreted disulfide-bound homodimeric molecule that induces growth cone collapse and repulsion of axon growth in the nervous system. Recently, it has been demonstrated that Sema3A is produced by endothelial cells and it regulates vascular morphogenesis by inhibiting integrin function in an autocrine fashion. Since platelet interaction with endothelial cells is critical for thrombus formation as well as hemostasis, we investigated effects of Sema3A on platelet function. We used two distinct Sema3A fusion proteins in this study: human Sema3A fused to Fc (Sema3A/Fc) and human Sema3A fused to placental alkaline phosphatase (Sema3A/AP). We detected dose-dependent and saturable binding of Sema3A fusion proteins to platelets. Flow cytometric analysis with PAC1 or soluble fibrinogen showed that Sema3A/Fc and Sema3A/AP inhibited activation of αIIbβ3 by all agonists examined, including ADP, thrombin, U46619, convulxin, and PMA. Sema3A also inhibited aggregation induced by thrombin or collagen. Moreover, Sema3A inhibited CD62P and CD63 expression after agonist stimulation, indicating that Sema3A inhibits granular secretion as well as activation of αIIbβ3. However, Sema3A did not inhibited thrombin-induced rapid intracellular calcium increase. Sema3A inhibited platelet adhesion to immobilized fibrinogen partially, and it severely impaired spreading of adhered platelets and even shrinking of spread platelets was observed after addition of Sema3A, suggesting that Sema3A impairs rearrangement of platelet cytoskeleton profoundly. Activation of platelets by thrombin or PAR1 peptide increases F-actin contents, and Sema3A inhibited the agonist-induced elevation of F-actin contents. Sema3A treatment also decreased phosphorylation of cofilin in both resting and thrombin-stimulated platelets, suggesting that Sema3A may keep cofilin in the activated state leading to increase severing of F-actin. Since phosphorylation of cofilin was regulated by LIM-kinase, an effecter of Rac-PAK signaling pathway, we next examined effects of Sema3A on Rac1 activation after thrombin stimulation. PAR1 peptide induced rapid activation of Rac1 in platelets, and Sema3A almost completely inhibited the activation of Rac1. These results suggest that Sema3A inhibits agonist-induced actin rearrangement via Rac1-dependent pathway including phosphorylation of cofilin. In conclusion, we demonstrated that Sema3A binds to platelets and inhibits platelet functions extensively. The inhibition of platelet functions by Sema3A appeared to be mediated at least in part through impairment of agonist-induced Rac1-dependent actin rearrangement.
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Wang, Qizhao, Jenni Firrman, Katie A. Pokiniewski, Wenjing Cao, Hongying Wei, Roland Herzog, and Weidong Xiao. "The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity." Blood 126, no. 23 (December 3, 2015): 2038. http://dx.doi.org/10.1182/blood.v126.23.2038.2038.
Full textAbstract:
Abstract Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies. In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors. Disclosures No relevant conflicts of interest to declare.
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Kamae, Tsuyoshi, Masamichi Shiraga, Hirokazu Kashiwagi, Hisashi Kato, Seiji Tadokoro, Yoshiyuki Kurata, Yoshiaki Tomiyama, and Yuzuru Kanakura. "Critical Role of Endogenous ADP Via P2Y12 Receptor in Maintenance of αIibβ3 Activation." Blood 106, no. 11 (November 16, 2005): 1655. http://dx.doi.org/10.1182/blood.v106.11.1655.1655.
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Abstract Platelet αIIbβ3 (GPIIb-IIIa), a noncovalently associated heterodimer, is a prototypic integrin that functions as a physiologic receptor for fibrinogen and von Willebrand factor. αIIbβ3 plays a crucial role in platelet aggregation, a key event of hemostatic plug formation and pathologic thrombus formation. During thrombogenesis, the affinity of αIIbβ3 for macromolecular ligands is dynamically regulated. After exposure to subendothelial matrix, several mediators such as ADP and thromboxane A2, or shear stress, platelets becomes activated and inside-out signaling that induces a high-affinity state of αIIbβ3 for soluble ligands (αIIbβ3 activation) are generated. Previous studies revealed that activation of αIIbβ3 is a reversible process. When platelets are stimulated with weak agonists such as adenosine diphosphate (ADP) and epinephrine in the absence of fibrinogen, αIIbβ3 gradually looses its binding capacity. In contrast, thrombin can induce long-lasting αIIbβ3 activation in the absence of fibrinogen. Although much attention has been directed to the nature of inside-out signaling, the mechanisms by which αIIbβ3 keep in the high-affinity state still remains elusive. In this study, we have demonstrated a critical role of endogenous ADP via its P2Y12 receptor in the maintenance of αIIbβ3 activation. Washed platelets adjusted to 50 x 106/ml were stimulated with 0.2 U/ml thrombin or 5 mM U46619 under static conditions. After the 15-min stimulation, 1 mM AR-C69931MX (a P2Y12 antagonist), 1 mM A3P5P (a P2Y1 antagonist) or buffer alone was added to the suspensions for additional 5 min. The platelet suspensions were then incubated with FITC-PAC1 and PE-anti-CD62P for 30 min and analyzed in a flow cytometer. AR-C69931MX and A3P5P attenuated the number of activated αIIbβ3 about 95% and 45%, respectively on the already activated platelets with thrombin- or U46619 without inhibiting CD62P expression. In an another set of experiments, platelets stimulated with thrombin or U46619 for 15min were then diluted to 1:100 with buffer containing the same agonist concentration (0.5 x 106/ml). In these conditions the number of activated αIIbβ3 was also markedly decreased (~85% reduction). Furthermore, the reduction in activated αIIbβ3 by the dilution was reversed by the addition of exogenous ADP in a dose-dependent fashion. HPLC analysis revealed that the amounts of ADP released from thrombin- and U46619-stimulated platelets were 2.6 and 0.75 mmol/1011platelets, respectively, and these values were comparable with ADP doses required for sustained αIIbβ3 activation in the diluted platelet suspension. Thus, released endogenous ADP plays a critical role in the maintenance of αIIbβ3 activation, and certain platelet concentrations are needed for this action. We also examined Rap 1b activation during the maintenance of αIIbβ3 activation. Thrombin induced sustained Rap 1b activation in the absence of ligand. However, AR-C69931MX disrupted the sustained Rap 1b activation. Thus, there was a close relationship between the maintenance αIIbβ3 activation and Rap 1b activation. Our data provide that the continuous interaction between released ADP and P2Y12 receptor is critical for the maintenance of αIIbβ3 activation.
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Lehmann, Marcus, Annie K. Burton, Savannah G. Szemethy, Jorge Valdez, and Jonathan N. Thon. "In Vitro Functionality of hiPSC Derived PLT+: The Utility of Microfluidic Assays in Determining Potency." Blood 134, Supplement_1 (November 13, 2019): 1168. http://dx.doi.org/10.1182/blood-2019-131896.
Full textAbstract:
Introduction: There is a growing shortage of platelets, the principal blood cells responsible for clot formation and blood vessel repair at sites of active bleeding. Platelet BioGenesis (PBG) is developing a commercial-scale, donor-free platelet (PLT+) production process using a cGMP-grade human induced pluripotent stem cell line (hiPSC) to address this issue. The PLT+ activity is being characterized extensively through multiple approaches that measure hemostatic function both in vitro and in vivo, with the ultimate goal of determining a clinical dose in humans. Alongside tests such as the current gold standard thrombin generation assay (TGA), we have developed an in-house microfluidic model to measure the ability of PLT+ to adhere to extracellular proteins under flow. The assay captures the surface receptor dynamics, as well as the ability of PLT+ to initiate coagulation. Materials and Methods: The thrombin generation assays are performed according to manufacturer's instructions. For the in-house microfluidic assay (MFA), fibrillar collagen, fibrinogen, or von Willebrand Factor is patterned in a commercial microfluidic device (Ibidi Slide VI 0.1). PLT+ (DNA-, calcein +, CD61+, CD42a+ cells from our bioreactor) are concentrated in normal pooled plasma, platelet additive solution, or platelet wash buffer to a normalized concentration of 1e8/mL, and are labeled with the mitochondrial dye DiOC6. Calcium chloride (7.5 mM) is added and the solution is perfused with a syringe pump at 50 1/s over the bioactive surface for 10 min. Images are captured every 10 seconds and quantified with ImageJ. After perfusion, the samples are fixed with 4% paraformaldehyde and can be stained for further characterization. Results and Discussion: PLTs+ are functionally non-inferior to human donor (blood) platelets and appear more active than Day 4/5 stored apheresis unit platelets. The TGA of PLT+ shows a more rapid generation of thrombin but similar total potential to donor platelets (Figure A). In the microfluidic assay, PLT+ (green DiOC6, red PAC1, figure B) adhere readily to the collagen surface under low shear flow, but do not adhere to non-functionalized surfaces, indicating GPVI functionality. As measured by surface coverage, the PLT+ resuspended in PAS adhere faster to the surface than either freshly washed donor platelets or apheresis platelets (p=0.01). When the PLT+ are preincubated in normal pooled plasma prior to perfusion, we observe fibrin formation and the aggregates show clot retraction, which supports the TGA results. Adhesion to surface bound fibrinogen and VWF is also comparable to donor platelets (GP IIb/IIa and GPIb function,). Combined, the TGA and MFA data reflect results of traditional more resource intensive assays, suggesting their viability as a rapid and flexible platform to study platelet function and function as release assays for in vitro generated PLTs+ Conclusion: Current work demonstrates that PLT+ are functional and a future alternative to donor derived platelet units. The MFA is a valuable tool for the characterization of this new therapeutic because of its specific control of surface protein-receptor interactions, shear forces, and flexibility to look at multiple markers. Alongside the TGA, the MFA will be a release assay for the PLT+. Figure Disclosures Lehmann: Platelet Biogenesis: Employment. Burton:Platelet Biogenesis: Employment. Szemethy:Platelet Biogenesis: Employment. Valdez:Platelet BioGenesis: Employment. Thon:Platelet BioGenesis: Employment.