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Journal articles on the topic 'Clot retraction'
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1
Kovalenko, Tatiana A., Marie-Noelle Giraud, Anita Eckly, Anne-Sophie Ribba, Fabienne Proamer, Sandrine Fraboulet, Nadezhda A. Podoplelova, et al. "Asymmetrical Forces Dictate the Distribution and Morphology of Platelets in Blood Clots." Cells 10, no. 3 (March 6, 2021): 584. http://dx.doi.org/10.3390/cells10030584.
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Primary hemostasis consists in the activation of platelets, which spread on the exposed extracellular matrix at the injured vessel surface. Secondary hemostasis, the coagulation cascade, generates a fibrin clot in which activated platelets and other blood cells get trapped. Active platelet-dependent clot retraction reduces the clot volume by extruding the serum. Thus, the clot architecture changes with time of contraction, which may have an important impact on the healing process and the dissolution of the clot, but the precise physiological role of clot retraction is still not completely understood. Since platelets are the only actors to develop force for the retraction of the clot, their distribution within the clot should influence the final clot architecture. We analyzed platelet distributions in intracoronary thrombi and observed that platelets and fibrin co-accumulate in the periphery of retracting clots in vivo. A computational mechanical model suggests that asymmetric forces are responsible for a different contractile behavior of platelets in the periphery versus the clot center, which in turn leads to an uneven distribution of platelets and fibrin fibers within the clot. We developed an in vitro clot retraction assay that reproduces the in vivo observations and follows the prediction of the computational model. Our findings suggest a new active role of platelet contraction in forming a tight fibrin- and platelet-rich boundary layer on the free surface of fibrin clots.
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Haling, Jacob R., Susan J. Monkley, David R. Critchley, and Brian G. Petrich. "Talin-dependent integrin activation is required for fibrin clot retraction by platelets." Blood 117, no. 5 (February 3, 2011): 1719–22. http://dx.doi.org/10.1182/blood-2010-09-305433.
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Abstract Talin functions both as a regulator of integrin affinity and as an important mechanical link between integrins and the cytoskeleton. Using genetic deletion of talin, we show for the first time that the capacity of talin to activate integrins is required for fibrin clot retraction by platelets. To further dissect which talin functions are required for this process, we tested clot retraction in platelets expressing a talin1(L325R) mutant that binds to integrins, but exhibits impaired integrin activation ascribable to disruption of the interaction between talin and the membrane-proximal region (MPR) in the β-integrin cytoplasmic domain. Talin-deficient and talin1(L325R) platelets were defective in retracting fibrin clots. However, the defect in clot retraction in talin1(L325R) platelets, but not talin-deficient platelets, was rescued by extrinsically activating integrins with manganese, thereby proving that integrin activation is required and showing that talin1(L325R) can form functional links to the actin cytoskeleton.
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Carr, M. E., and S. L. Zekert. "Abnormal clot retraction, altered fibrin structure, and normal platelet function in multiple myeloma." American Journal of Physiology-Heart and Circulatory Physiology 266, no. 3 (March 1, 1994): H1195—H1201. http://dx.doi.org/10.1152/ajpheart.1994.266.3.h1195.
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Clot retraction, measured by serum expression, is absent in some cases of multiple myeloma. Decreased clot retraction has been attributed to platelet dysfunction. A new instrument allows simultaneous measurement of platelet-mediated force development during clot retraction and of clot elastic modulus. We report 10 patients with immunoglobulin (Ig) G myeloma in whom the abnormalities of fibrin structure were quantitatively defined and platelet-fibrin interactions were assessed. Fiber mass-to-length ratios were calculated from gel turbidity. Platelet force development and clot elastic modula were measured in platelet-rich plasma gels. Fiber mass-to-length ratios for IgG myeloma patients were smaller (means +/- SE) (0.98 +/- 0.19 x 10(13) Da/cm) than for normal controls (1.36 +/- 0.06 x 10(13) Da/cm), indicating thinner fiber formation. Elastic modula of myeloma clots (51,013 +/- 14,660 dyn/cm2) were strikingly larger than modula for normal controls (23,355 +/- 1,887 dyn/cm2), indicating that such clots are mechanically less flexible. Platelet force development 1,200 s after thrombin addition was not diminished in myeloma patients (8,315 +/- 1,155 dyn) vs. controls (6,906 +/- 606 dyn). Abnormal clot retraction in myeloma appears to be primarily due to altered clot structure rather than platelet dysfunction.
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Chen, YP, TE O'Toole, L. Leong, BQ Liu, F. Diaz-Gonzalez, and MH Ginsberg. "Beta 3 integrin-mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta 3 and alpha v beta 3." Blood 86, no. 7 (October 1, 1995): 2606–15. http://dx.doi.org/10.1182/blood.v86.7.2606.2606.
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Abstract Fibrin clot retraction may be important in resolution of thrombi and, in platelets, is mediated by integrin alpha IIb beta 3 (GPIIb-IIIa). Nucleated cells that lack alpha IIb beta 3 can retract fibrin clots, and we now report that integrin alpha v beta 3 can support this process. In addition, we compared the capacities of recombinant beta 3 integrins to mediate clot retraction in Chinese hamster ovary and M21 melanoma cells. We found that alpha v beta 3, but not alpha IIb beta 3, could spontaneously support retraction. Transferring the cytoplasmic domain of alpha v to alpha IIb enabled the resulting chimeric alpha IIb beta 3 to support clot retraction. The capacity of the alpha v cytoplasmic domain to support clot retraction was not caused by activation of the ligand binding function of alpha IIb beta 3 or by enhancement of alpha IIb beta 32s capacity to stimulate the formation of focal adhesions or the tyrosine phosphorylation of pp125FAK. These experiments define requirements for alpha IIb beta 3-mediating clot retraction, establish the capacity of alpha v beta 3 to mediate this process, and suggest differing functional roles of the alpha v and alpha IIb cytoplasmic domains.
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Chen, YP, TE O'Toole, L. Leong, BQ Liu, F. Diaz-Gonzalez, and MH Ginsberg. "Beta 3 integrin-mediated fibrin clot retraction by nucleated cells: differing behavior of alpha IIb beta 3 and alpha v beta 3." Blood 86, no. 7 (October 1, 1995): 2606–15. http://dx.doi.org/10.1182/blood.v86.7.2606.bloodjournal8672606.
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Fibrin clot retraction may be important in resolution of thrombi and, in platelets, is mediated by integrin alpha IIb beta 3 (GPIIb-IIIa). Nucleated cells that lack alpha IIb beta 3 can retract fibrin clots, and we now report that integrin alpha v beta 3 can support this process. In addition, we compared the capacities of recombinant beta 3 integrins to mediate clot retraction in Chinese hamster ovary and M21 melanoma cells. We found that alpha v beta 3, but not alpha IIb beta 3, could spontaneously support retraction. Transferring the cytoplasmic domain of alpha v to alpha IIb enabled the resulting chimeric alpha IIb beta 3 to support clot retraction. The capacity of the alpha v cytoplasmic domain to support clot retraction was not caused by activation of the ligand binding function of alpha IIb beta 3 or by enhancement of alpha IIb beta 32s capacity to stimulate the formation of focal adhesions or the tyrosine phosphorylation of pp125FAK. These experiments define requirements for alpha IIb beta 3-mediating clot retraction, establish the capacity of alpha v beta 3 to mediate this process, and suggest differing functional roles of the alpha v and alpha IIb cytoplasmic domains.
6
Tanaka, Kenichi A., John McNeil, and Ezeldeen Abuelkasem. "Clot Retraction." Anesthesia & Analgesia 128, no. 4 (April 2019): e53-e54. http://dx.doi.org/10.1213/ane.0000000000004030.
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Jansen, Ellen E., and Matthias Hartmann. "Clot Retraction: Cellular Mechanisms and Inhibitors, Measuring Methods, and Clinical Implications." Biomedicines 9, no. 8 (August 21, 2021): 1064. http://dx.doi.org/10.3390/biomedicines9081064.
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Platelets have important functions in hemostasis. Best investigated is the aggregation of platelets for primary hemostasis and their role as the surface for coagulation leading to fibrin- and clot-formation. Importantly, the function of platelets does not end with clot formation. Instead, platelets are responsible for clot retraction through the concerted action of the activated αIIbβ3 receptors on the surface of filopodia and the platelet’s contractile apparatus binding and pulling at the fibrin strands. Meanwhile, the signal transduction events leading to clot retraction have been investigated thoroughly, and several targets to inhibit clot retraction have been demonstrated. Clot retraction is a physiologically important mechanism allowing: (1) the close contact of platelets in primary hemostasis, easing platelet aggregation and intercellular communication, (2) the reduction of wound size, (3) the compaction of red blood cells to a polyhedrocyte infection-barrier, and (4) reperfusion in case of thrombosis. Several methods have been developed to measure clot retraction that have been based on either the measurement of clot volume or platelet forces. Concerning the importance of clot retraction in inborn diseases, the failure of clot retraction in Glanzmann thrombasthenia is characterized by a bleeding phenotype. Concerning acquired diseases, altered clot retraction has been demonstrated in patients with coronary heart disease, stroke, bronchial asthma, uremia, lupus erythematodes, and other diseases. However, more studies on the diagnostic and prognostic value of clot retraction with methods that have to be standardized are necessary.
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Rooney, Michael M., David H. Farrell, Bettien M. van Hemel, Philip G. de Groot, and Susan T. Lord. "The Contribution of the Three Hypothesized Integrin-Binding Sites in Fibrinogen to Platelet-Mediated Clot Retraction." Blood 92, no. 7 (October 1, 1998): 2374–81. http://dx.doi.org/10.1182/blood.v92.7.2374.
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AbstractFibrinogen is a plasma protein that interacts with integrin αIIbβ3 to mediate a variety of platelet responses including adhesion, aggregation, and clot retraction. Three sites on fibrinogen have been hypothesized to be critical for these interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the C-terminus of the γ chain and two Arg-Gly-Asp (RGD) sequences in the Aα chain. Recent data showed that AGDV is critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide evidence, using engineered recombinant fibrinogen, that no one of these sites is critical for clot retraction; fibrinogen lacking all three sites still sustains a relatively normal, albeit delayed, retraction response. Three fibrinogen variants with the following mutations were examined: a substitution of RGE for RGD at position Aα 95-97, a substitution of RGE for RGD at position Aα 572-574, and a triple substitution of RGE for RGD at both Aα positions and deletion of AGDV from the γ chain. Retraction rates and final clot sizes after a 20-minute incubation were indistinguishable when comparing the Aα D97E fibrinogen or Aα D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recombinant fibrinogen. Similar results were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from platelet α-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites are important for an initial step in clot retraction, but not for a subsequent step. With the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second step of clot retraction.
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Rooney, Michael M., David H. Farrell, Bettien M. van Hemel, Philip G. de Groot, and Susan T. Lord. "The Contribution of the Three Hypothesized Integrin-Binding Sites in Fibrinogen to Platelet-Mediated Clot Retraction." Blood 92, no. 7 (October 1, 1998): 2374–81. http://dx.doi.org/10.1182/blood.v92.7.2374.2374_2374_2381.
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Fibrinogen is a plasma protein that interacts with integrin αIIbβ3 to mediate a variety of platelet responses including adhesion, aggregation, and clot retraction. Three sites on fibrinogen have been hypothesized to be critical for these interactions: the Ala-Gly-Asp-Val (AGDV) sequence at the C-terminus of the γ chain and two Arg-Gly-Asp (RGD) sequences in the Aα chain. Recent data showed that AGDV is critical for platelet adhesion and aggregation, but not retraction, suggesting that either one or both of the RGD sequences are involved in clot retraction. Here we provide evidence, using engineered recombinant fibrinogen, that no one of these sites is critical for clot retraction; fibrinogen lacking all three sites still sustains a relatively normal, albeit delayed, retraction response. Three fibrinogen variants with the following mutations were examined: a substitution of RGE for RGD at position Aα 95-97, a substitution of RGE for RGD at position Aα 572-574, and a triple substitution of RGE for RGD at both Aα positions and deletion of AGDV from the γ chain. Retraction rates and final clot sizes after a 20-minute incubation were indistinguishable when comparing the Aα D97E fibrinogen or Aα D574E fibrinogen with normal recombinant fibrinogen. However, with the triple mutant fibrinogen, clot retraction was delayed compared with normal recombinant fibrinogen. Nevertheless, the final clot size measured after 20 minutes was the same size as a clot formed with normal recombinant fibrinogen. Similar results were observed using platelets isolated from an afibrinogenemic patient, eliminating the possibility that the retraction was dependent on secretion of plasma fibrinogen from platelet α-granules. These findings indicate that clot retraction is a two-step process, such that one or more of the three putative platelet binding sites are important for an initial step in clot retraction, but not for a subsequent step. With the triple mutant fibrinogen, the second step of clot retraction, possibly the development of clot tension, proceeds with a rate similar to that observed with normal recombinant fibrinogen. These results are consistent with a mechanism where a novel site on fibrin is involved in the second step of clot retraction.
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Flevaris, Panagiotis, Zhenyu Li, Guoying Zhang, Yi Zheng, Junling Liu, and Xiaoping Du. "Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway." Blood 113, no. 4 (January 22, 2009): 893–901. http://dx.doi.org/10.1182/blood-2008-05-155978.
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Abstract Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway.
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Tutwiler, Valerie, Alina Peshkova, Dina Khasanova, John W. Weisel, and Rustem I. Litvinov. "Blood Clot Contraction (Retraction) Is Impaired in Acute Ischemic Stroke." Blood 128, no. 22 (December 2, 2016): 4998. http://dx.doi.org/10.1182/blood.v128.22.4998.4998.
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Abstract Obstructive thrombi or thrombotic emboli of cerebral arteries are the pathogenic basis of ischemic stroke, which is a leading cause of death and disability worldwide. Blood clots undergo volume shrinkage due to the contractile forces that are generated by platelets and propagated through the clot volume due to platelet-fibrin interactions and elasticity of the fibrin network. This process is designed clot contraction (retraction) which remains one of the least studied steps of blood clotting. Importantly, this phenomenon has been shown to occur not only in vitro but also in in vivo thrombi. Clot contraction has been shown to be important in the volume reduction of otherwise obstructive thrombi and has the potential to reduce occlusion and restore blood flow past emboli or thrombi. Despite the potential medical significance of clot contraction, it has not been examined systemically in a clinical setting. This aim of this work was to examine the potential pathogenic role of clot contraction in ischemic stroke. Here we employed a novel automated method to quantify the time of initiation, extent and rate of clot contraction in vitro to compare clot contraction in the blood of healthy subjects with patients suffering a recent ischemic stroke (<6 hours from the onset of symptoms) who had not yet received any treatment with anticoagulants, antiplatelet drugs or thrombolytics.. Parameters of clot contraction were correlated with the severity and etiological types of stroke as well as with hematological, coagulation, and biochemical tests to examine the clinical significance of clot contraction. The main finding of this work is that clot contraction in blood from patients with acute ischemic stroke is reduced on average by ~60% (p<0.0001) when compared to that of healthy subjects. The reduction in clot contraction is correlated with a lower platelet count and platelet dysfunction, higher fibrinogen level, higher hematocrit, leukocytosis as well as other changes in the blood composition of patients with ischemic stroke that may alter the properties of the blood clot. We propose that these changes in the composition of the blood contribute to the impaired mechanism of clot contraction, which may exaggerate vessel occlusion and brain infarct. While stroke severity is determined mainly by the diameter and location of the obstructed cerebral artery, the ability of the thrombi to contract more or less may augment or ease the course of brain damage. Clinical correlations with respect to severity and stroke etiology indicate that reduced clot contraction has the potential to be a pathogenic factor in ischemic stroke. Paradoxically, the extent of clot contraction marginally improved in patients with a more severe stroke (NIHSS>15 vs. NIHSS<15, p<0.01), while it was still significantly reduced compared to healthy subjects. This finding can be presumably explained by the fact that in severe brain damage a massive amount of tissue factor is released into the systemic circulation, which can induce the activation of blood coagulation. We propose that this release of tissue factor results in a secondary wave of thrombin generation that causes patients with more severe stroke to have hyperactivity of platelets. In combination with a higher platelet count (p<0.01) this can enhance contraction of obstructive clots or thrombi, which may be a compensatory mechanism resulting in the recanalization of an otherwise occluded blood vessel. In support of this hypothetical scenario, it was also found that patients with atherothrombotic strokes have an increased extent of clot contraction compared to patients with cardioembolic stroke (p<0.05), and atherothrombotic patients are reported to have increased tissue factor as a consequence of atherosclerotic lesions. In summary, the clinical pathophysiological importance of clot contraction in a thrombotic state has been examined for the first time and the modulation of the ability of clots or thrombi to shrink in volume may be a novel and unappreciated mechanism that aggravates or alleviates the course and outcomes of thrombosis, such as ischemic stroke. The clinical importance of clot or thrombus remodeling in vivo as well as the diagnostic and prognostic value of this blood test for clot contraction needs further exploration. Disclosures Weisel: Bayer: Research Funding.
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Mondoro, Traci Heath, Melanie McCabe White, and Lisa K. Jennings. "Active GPIIb-IIIa conformations that link ligand interaction with cytoskeletal reorganization." Blood 96, no. 7 (October 1, 2000): 2487–95. http://dx.doi.org/10.1182/blood.v96.7.2487.
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Abstract Glycoprotein (GP) IIb-IIIa plays a critical role in platelet aggregation and platelet-mediated clot retraction. This study examined the intramolecular relationship between GPIIb-IIIa activation and fibrinogen binding, platelet aggregation, and platelet-mediated clot retraction. To distinguish between different high-affinity activation states of GPIIb-IIIa, the properties of an antibody (D3) specific for GPIIIa that induces GPIIb-IIIa binding to adhesive protein molecules and yet completely inhibits clot retraction were used. Clot retraction inhibition by D3 was not due to altered platelet-fibrin interaction; however, combination treatments of D3 and adenosine diphosphate (ADP) inhibited full-scale aggregation and decreased the amounts of GPIIb-IIIa and talin incorporated into the core cytoskeletons. Morphologic evaluation of the D3/ADP aggregates showed platelets that were activated but to a lesser extent when compared to ADP only. ADP addition to platelets caused an increase in the number of D3 binding sites indicating that ligand had bound to the GPIIb-IIIa receptor. These data suggest that high-affinity GPIIb-IIIa– mediated ligand binding can be separated mechanistically from GPIIb-IIIa–mediated clot retraction and that clot retraction requires additional signaling through GPIIb-IIIa after ligand binding. The conformation recognized by D3 represents the expression of a GPIIb-IIIa activation state that participates in full-scale platelet aggregation, cytoskeletal reorganization, and clot retraction.
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Mondoro, Traci Heath, Melanie McCabe White, and Lisa K. Jennings. "Active GPIIb-IIIa conformations that link ligand interaction with cytoskeletal reorganization." Blood 96, no. 7 (October 1, 2000): 2487–95. http://dx.doi.org/10.1182/blood.v96.7.2487.h8002487_2487_2495.
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Glycoprotein (GP) IIb-IIIa plays a critical role in platelet aggregation and platelet-mediated clot retraction. This study examined the intramolecular relationship between GPIIb-IIIa activation and fibrinogen binding, platelet aggregation, and platelet-mediated clot retraction. To distinguish between different high-affinity activation states of GPIIb-IIIa, the properties of an antibody (D3) specific for GPIIIa that induces GPIIb-IIIa binding to adhesive protein molecules and yet completely inhibits clot retraction were used. Clot retraction inhibition by D3 was not due to altered platelet-fibrin interaction; however, combination treatments of D3 and adenosine diphosphate (ADP) inhibited full-scale aggregation and decreased the amounts of GPIIb-IIIa and talin incorporated into the core cytoskeletons. Morphologic evaluation of the D3/ADP aggregates showed platelets that were activated but to a lesser extent when compared to ADP only. ADP addition to platelets caused an increase in the number of D3 binding sites indicating that ligand had bound to the GPIIb-IIIa receptor. These data suggest that high-affinity GPIIb-IIIa– mediated ligand binding can be separated mechanistically from GPIIb-IIIa–mediated clot retraction and that clot retraction requires additional signaling through GPIIb-IIIa after ligand binding. The conformation recognized by D3 represents the expression of a GPIIb-IIIa activation state that participates in full-scale platelet aggregation, cytoskeletal reorganization, and clot retraction.
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Egot, Marion, Alexandre Kauskot, Dominique Lasne, Pascale Gaussem, and Christilla Bachelot-Loza. "Biphasic myosin II light chain activation during clot retraction." Thrombosis and Haemostasis 110, no. 12 (2013): 1215–22. http://dx.doi.org/10.1160/th13-04-0335.
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SummaryClot retraction is an essential step during primary haemostasis, thereby promoting thrombus stability and wound healing. Integrin αIIbβ3 plays a critical role in clot retraction, by inducing acto-myosin interactions that allow platelet cytoskeleton reorganisation. However, the signalling pathways that lead to clot retraction are still misunderstood. In this study, we report the first data on the kinetics of myosin II light chain (MLC) phosphorylation during clot retraction. We found an early phosphorylation peak followed by a second peak. By using specific inhibitors of kinases and small G proteins, we showed that MLC kinase (MLCK), RhoA/ROCK, and Rac-1 were involved in clot retraction and in the early MLC phosphorylation peak. Only Rac-1 and actin polymerisation, controlled by outside-in signalling, were crucial to the second MLC phosphorylation peak.
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Peshkova, A. D., A. P. Lozhkin, L. S. Fathullina, D. V. Malyasev, R. A. Bredikhin, and R. I. Litvinov. "Dependence of clot contraction (retraction) on the molecular and cellular blood composition." Kazan medical journal 97, no. 1 (February 15, 2016): 70–77. http://dx.doi.org/10.17750/kmj2016-70.
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Aim. To study the effect of the blood components on clot contraction dynamics in vitro.Methods. The original method based on the optical detection of changes in the blood clot volume over time was used. Whole blood, as well as reconstructed samples using washed platelets, erythrocytes, purified fibrinogen, platelet-poor and platelet-rich plasma were studied.Results. Blood clot contraction has a non-linear kinetics, reflecting the complexity of the underlying mechanisms. Platelets increase the blood clot contraction, while the red blood cells have an inhibitory effect. Blocking the fibrin and platelets interaction using the RGDS peptide, an integrin αIIbβ3 antagonist, reduces the extent and rate of clot contraction. The exogenous Ca2+ is not required for contraction, but its addition stabilizes clots by inhibiting the erythrocytes. Thrombin has a dose-dependent effect and increases the rate and extent of contraction. In blood samples of patients taking warfarin, blood clot contraction was delayed.Conclusion. The blood clot contraction is a process which depends on many factors, including the blood cell composition, amount of fibrinogen, the endogenous thrombin activity and platelets interaction with fibrin; understanding the mechanisms of the blood clot contraction could form the basis for the development of novel approaches to the hemostatic disorders treatment.
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Kunitada, S., GA FitzGerald, and DJ Fitzgerald. "Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction." Blood 79, no. 6 (March 15, 1992): 1420–27. http://dx.doi.org/10.1182/blood.v79.6.1420.1420.
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Abstract Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t- PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet- induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.
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Kunitada, S., GA FitzGerald, and DJ Fitzgerald. "Inhibition of clot lysis and decreased binding of tissue-type plasminogen activator as a consequence of clot retraction." Blood 79, no. 6 (March 15, 1992): 1420–27. http://dx.doi.org/10.1182/blood.v79.6.1420.bloodjournal7961420.
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Tissue-type plasminogen activator (t-PA) is less active in vivo and in vitro against clots that are enriched in platelets, even at therapeutic concentrations. The release of radioactivity from 125I-fibrin-labeled clots was decreased by 47% 6 hours after the addition of t-PA 400 U/mL when formed in platelet-rich versus platelet-poor plasma. This difference was not due to the release of plasminogen activator inhibitor-1 (PAI-1) by platelets. Thus, the fibrinolytic activity of t- PA in the supernatant was similar in the two preparations and fibrin autography demonstrated only a minor degree of t-PA-PAI-1 complex formation. Furthermore, a similar platelet-dependent reduction in clot lysis was seen with a t-PA mutant resistant to inhibition by PAI-1. The reduction in t-PA activity correlated with a decrease in t-PA binding to platelet-enriched clot (60% +/- 3% v platelet-poor clot, n = 5). This reduction in binding was also shown using t-PA treated with the chloromethylketone, D-Phe-Pro-Arg-CH2Cl (PPACK) (36% +/- 13%, n = 3), and with S478A, a mutant t-PA in which the active site serine at position 478 has been substituted by alanine (43% +/- 6%, n = 3). In contrast, fixed platelets and platelet supernatants had no effect on the binding or lytic activity of t-PA. Pretreatment with cytochalasin D 1 mumol/L, which inhibits clot retraction, also abolished the platelet- induced inhibition of lysis and t-PA binding by platelets. These data suggest that platelets inhibit clot lysis at therapeutic concentrations of t-PA as a consequence of clot retraction and decreased access of fibrinolytic proteins.
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Li, Zida, Xiang Li, Brendan McCracken, Yue Shao, Kevin Ward, and Jianping Fu. "Clot Retraction: A Miniaturized Hemoretractometer for Blood Clot Retraction Testing (Small 29/2016)." Small 12, no. 29 (August 2016): 3925. http://dx.doi.org/10.1002/smll.201670144.
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Nair, Prajeeda M., Michael Adam Meledeo, Xiaowu Wu, Grantham Peltier, Bin Liu, Kristin M. Reddoch-Cardenas, James Bynum, and Andrew P. Cap. "Effect of Extended Platelet Storage on Platelet Metabolism and Clot Retraction." Blood 134, Supplement_1 (November 13, 2019): 2450. http://dx.doi.org/10.1182/blood-2019-131116.
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Background: Platelet (PLT) hemostatic responses are heavily dependent on PLT bioenergetics which drives the generation of PLT contractile forces and contribute to mechanical hemostasis. Although effective clot retraction (CR) requires PLT adhesion, aggregation and formation of strong clots, CR remains an understudied PLT function. In this work, we hypothesize that well-conserved PLT bioenergetics during storage is crucial for highly energy-dependent PLT functions including CR. To further investigate this correlation, we characterized the effect of extended PLT storage for up to 15 days on PLT bioenergetics and CR at room temperature (22°C, RT) and refrigeration (4°C, 4C). Methods: Apheresis PLT concentrates in plasma were collected from 5 healthy donors and stored for 21 days at RT with agitation, or stored stationary in the cold (4C). Fresh PLTs were treated with metabolic inhibitors for 1 hour at 37°C, recalcified with 20mM calcium chloride, and incubated at 37°C to initiate CR. The clot weights were measured post retraction, and the clots were prepared for immunohistochemistry to assess clot structure. Similarly, extent of retraction and mitochondrial respiration levels were estimated in PLTs stored for 5, 10 and 15 days. Additionally, Platelet additive solution (PAS) stored PLTs were tested as a viable alternative to plasma storage to improve metabolic exhaustion during storage. Results: 4C-stored PLTs in plasma retracted similar to fresh PLTs for up to 15 days. In comparison, RT-stored PLTs in plasma did not retract after 10 days, whereas PLTs stored in PAS at RT or 4C seemed to retain their contractile function for up to 15 days (Fig 1A). 4C-stored and fresh PLTs yielded heavier clots with a highly organized structure compared to the disorganized structure of clots from RT-stored PLTs. Mitochondrial respiration was significantly reduced over 10 days in RT- plasma stored PLTs, which was mitigated by PAS storage. Similarly, 4C storage in PAS mitigated the reduction in respiration seen in 15 days 4C- stored PLTs in plasma (Fig 1B). Conclusion: Retraction properties of 4C-stored PLTs were similar to those of fresh PLTs, together with superior clot structure and function compared to RT- stored PLTs. This suggests that 4C storage could be a suitable alternative for extended storage. In RT-stored PLTs, reduction in mitochondrial respiration did not show any correlation with the reduced ability of PLTs to retract, suggesting that preserved glycolysis in PAS- stored RT-stored PLTs enabled CR. Thus, storing PLTs in PAS at RT or 4C could potentially mitigate mitochondrial dysfunction during extended storage. Disclosures No relevant conflicts of interest to declare.
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Samson, Andre L., Imala Alwis, Jessica A. A. Maclean, Pramith Priyananda, Brian Hawkett, Simone M. Schoenwaelder, and Shaun P. Jackson. "Endogenous fibrinolysis facilitates clot retraction in vivo." Blood 130, no. 23 (December 7, 2017): 2453–62. http://dx.doi.org/10.1182/blood-2017-06-789032.
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Key Points Localized vascular injury with thrombin microinjection produces a fibrin network that undergoes myosin IIa–dependent retraction in vivo. Using this model, we demonstrate that endogenous fibrinolysis promotes fibrin clot retraction.
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Sabovic, M., H. R. Lijnen, D. Keber, and D. Collen. "Effect of Retraction on the Lysis of Human Clots with Fibrin Specific and Non-Fibrin Specific Plasminogen Activators." Thrombosis and Haemostasis 62, no. 04 (1989): 1083–87. http://dx.doi.org/10.1055/s-0038-1647122.
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SummaryThe effect of the serum content of human clots on their sensitivity to lysis with plasminogen activators was studied in a system composed of 125I-fibrin labeled clots immersed in buffer or in citrated plasma. The effect was studied with plasma clots before or after mechanical compression and with whole blood clots before or after retraction, using either the fibrin specific plasminogen activators recombinant tissue-type plasminogen activator (rt-PA) or recombinant single chain urokinase-type plasminogen activator (rscu-PA), and the non-fibrin specific activators recombinant two chain urokinase-type plasminogen activator (rtcu-PA), or streptokinase (SK).In a buffer milieu, all plasminogen activators had a similar fibrinolytic potency towards serum-rich clots (non-compressed plasma clots or non-retracted blood clots): 50% clot lysis in 4 h required 50 to 85 ng plasminogen activator per ml. Serum-poor clots (compressed plasma clots or retracted blood clots) were resistant to lysis in a buffer milieu but became sensitive to lysis following preincubation in plasma for 48 h. These findings indicate that plasma proteins entrapped in clots contribute significantly to their sensitivity to lysis and suggest that the amount of bound or entrapped plasminogen may be a limiting factor. In a plasma milieu, all plasminogen activators lysed serum- rich plasma or blood clots, albeit at higher concentrations (3 to 40 times higher than in the buffer milieu) and with different efficiencies: 50% clot lysis in 4 h required approximately 600 ng/ ml of rtcu-PA but 1,500 to 2,000 ng/ml of rscu-PA. These findings suggest that components of plasma are responsible for increased resistance of clots towards lysis and that the effect is variable for different plasminogen activators. Serum-poor plasma or blood clots were very resistant to lysis with non-fibrin specific agents, but became more sensitive after preincubation in plasma. However, serum-poor plasma or blood clots were sensitive to lysis with fibrin specific plasminogen activators, suggesting that during clot lysis with fibrin specific agents, plasminogen recruited from surrounding plasma may contribute significantly to clot lysis. The concentration of plasminogen activator required to obtain 50% clot lysis in a plasma milieu of compressed plasma clots or retracted blood clots was 390 and 1,600 ng/ml respectively for rt-PA and 1,100 and 3,200 ng/ml respectively for rscu-PA. These data suggest that in a plasma milieu retracted blood clots are more sensitive to lysis with fibrin specific plasminogen activators than with non-fibrin specific agents.
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Hudák, Renáta, János Vincze, László Csernoch, Ildikó Beke Debreceni, Tamás Oláh, Ferenc Erdődi, Kenneth J. Clemetson, and János Kappelmayer. "The Phosphatase Inhibitor Calyculin-A Impairs Clot Retraction, Platelet Activation, and Thrombin Generation." BioMed Research International 2017 (2017): 1–10. http://dx.doi.org/10.1155/2017/9795271.
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The aim of this study was to investigate the effect of the serine/threonine protein phosphatase inhibitor, calyculin-A (CLA), on clot formation and on the procoagulant activity of human platelets. Platelet-rich plasma (PRP) samples were preincubated with buffer or CLA and subsequently platelets were activated by the protease-activated receptor 1 (PAR-1) activator, thrombin receptor activating peptide (TRAP). Clot retraction was detected by observing clot morphology up to 1 hour, phosphatidylserine- (PS-) expression was studied by flow cytometry, and thrombin generation was measured by a fluorimetric assay. For the intracellular Ca2+assay, platelets were loaded with calcium-indicator dyes and the measurements were carried out using a ratiometric method with real-time confocal microscopy. CLA preincubation inhibited clot retraction, PS-expression, and thrombin formation. TRAP activation elicited Ca2+response and PS-expression in a subset of platelets. The activated PRP displayed significantly faster and enhanced thrombin generation compared to nonactivated samples. CLA pretreatment abrogated PS-exposure and clot retraction also in TRAP-activated samples. As a consequence of the inhibitory effect on calcium elevation and PS-expression, CLA significantly downregulated thrombin generation in PRP. Our results show that CLA pretreatment may be a useful tool to investigate platelet activation mechanisms that contribute to clot formation and thrombin generation.
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White, James G. "Platelet secretion during clot retraction." Platelets 11, no. 6 (January 2000): 331–43. http://dx.doi.org/10.1080/09537100050144759.
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Weyrich, Andrew S., Melvin M. Denis, Hansjorg Schwertz, Neal D. Tolley, Jason Foulks, Eliott Spencer, Larry W. Kraiss, Kurt H. Albertine, Thomas M. McIntyre, and Guy A. Zimmerman. "mTOR-dependent synthesis of Bcl-3 controls the retraction of fibrin clots by activated human platelets." Blood 109, no. 5 (November 16, 2006): 1975–83. http://dx.doi.org/10.1182/blood-2006-08-042192.
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Abstract New activities of human platelets continue to emerge. One unexpected response is new synthesis of proteins from previously transcribed RNAs in response to activating signals. We previously reported that activated human platelets synthesize B-cell lymphoma-3 (Bcl-3) under translational control by mammalian target of rapamycin (mTOR). Characterization of the ontogeny and distribution of the mTOR signaling pathway in CD34+ stem cell–derived megakaryocytes now demonstrates that they transfer this regulatory system to developing proplatelets. We also found that Bcl-3 is required for condensation of fibrin by activated platelets, demonstrating functional significance for mTOR-regulated synthesis of the protein. Inhibition of mTOR by rapamycin blocks clot retraction by human platelets. Platelets from wild-type mice synthesize Bcl-3 in response to activation, as do human platelets, and platelets from mice with targeted deletion of Bcl-3 have defective retraction of fibrin in platelet-fibrin clots mimicking treatment of human platelets with rapamycin. In contrast, overexpression of Bcl-3 in a surrogate cell line enhanced clot retraction. These studies identify new features of post-transcriptional gene regulation and signal-dependant protein synthesis in activated platelets that may contribute to thrombus and wound remodeling and suggest that posttranscriptional pathways are targets for molecular intervention in thrombotic disorders.
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Berndt, Michael C., and Robert K. Andrews. "Full clot retraction: running on mTOR." Blood 109, no. 5 (March 1, 2007): 1791–92. http://dx.doi.org/10.1182/blood-2006-12-062000.
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Rooney, Michael M., Leslie V. Parise, and Susan T. Lord. "Dissecting Clot Retraction and Platelet Aggregation." Journal of Biological Chemistry 271, no. 15 (April 12, 1996): 8553–55. http://dx.doi.org/10.1074/jbc.271.15.8553.
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Lu, Jun, Peng Hu, Guangyu Wei, Qi Luo, Jianlin Qiao, and Deqin Geng. "Effect of alteplase on platelet function and receptor expression." Journal of International Medical Research 47, no. 4 (February 24, 2019): 1731–39. http://dx.doi.org/10.1177/0300060519829991.
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Objective To investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function. Methods Human platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined. Results This study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction. Conclusions Alteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.
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Kuchay, Shafi M., Nayoung Kim, Elizabeth A. Grunz, William P. Fay, and Athar H. Chishti. "Double Knockouts Reveal that Protein Tyrosine Phosphatase 1B Is a Physiological Target of Calpain-1 in Platelets." Molecular and Cellular Biology 27, no. 17 (June 18, 2007): 6038–52. http://dx.doi.org/10.1128/mcb.00522-07.
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ABSTRACT Calpains are ubiquitous calcium-regulated cysteine proteases that have been implicated in cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. Gene targeting was used to evaluate the physiological function of mouse calpain-1 and establish that its inactivation results in reduced platelet aggregation and clot retraction potentially by causing dephosphorylation of platelet proteins. Here, we report that calpain-1 null (Capn1 −/−) platelets accumulate protein tyrosine phosphatase 1B (PTP1B), which correlates with enhanced tyrosine phosphatase activity and dephosphorylation of multiple substrates. Treatment of Capn1 −/− platelets with bis(N,N-dimethylhydroxamido)hydroxooxovanadate, an inhibitor of tyrosine phosphatases, corrected the aggregation defect and recovered impaired clot retraction. More importantly, platelet aggregation, clot retraction, and tyrosine dephosphorylation defects were rescued in the double knockout mice lacking both calpain-1 and PTP1B. Further evaluation of mutant mice by the ferric chloride-induced arterial injury model suggests that the Capn1 −/− mice are relatively resistant to thrombosis in vivo. Together, our results demonstrate that PTP1B is a physiological target of calpain-1 and suggest that a similar mechanism may regulate calpain-1-mediated tyrosine dephosphorylation in other cells.
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Faes, Camille, Anton Ilich, Erica M. Sparkenbaugh, Alisa S. Wolberg, Brian C. Cooley, Kenneth I. Ataga, Nigel S. Key, and Rafal Pawlinski. "Alteration of the Structure and Dynamics of Venous Clot Formation in Human and Murine Sickle Cell Disease." Blood 128, no. 22 (December 2, 2016): 2478. http://dx.doi.org/10.1182/blood.v128.22.2478.2478.
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Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation and an increased risk of venous thromboembolism. Traditionally, it is believed that during venous thrombosis, red blood cells (RBC) are simply trapped within fibrin-rich thrombi and do not actively affect thrombosis. However, a study from our group showed that factor XIII (FXIII) activity is critical for the retention of RBC within clots and directly affects thrombus size. Others reported that during clot contraction polyhedral shaped RBC formed a densely packed core and that SCD alters the formation of polyhedrocytes which may affect clot stability (Strauss et al, 2015, ASH abstract). We further investigated if SCD affects the structure and the dynamics of clot formation. Ex vivo clot retraction was performed using blood from sickle cell patients and Townes mice (a model of SCD). Citrated blood was added to siliconized wells of 96-well plates containing CaCl2 (10mM) and tissue factor (1pM) and incubated for 2 hours at 37°C. The number of RBC extruded from the clot was counted in serum by Hemavet™ and expressed as a percentage of initial RBC number in the anticoagulated blood. Morphology of the clots was evaluated using scanning and transmission electron microscopy (EM). Thrombosis in Townes SS (sickle) and AA (control) mice was studied using femoral vein thrombosis induced by electrolytic injury and inferior vena cava stenosis models. The number of mouse SS RBC in the serum extruded during clot contraction was dramatically reduced compared to the number of AA RBC (0.8±0.8% vs. 19.4±0.8%, n=3, p<0.0001, Fig. A). A similar result was observed for human RBC. Since SS mice and sickle patients have a lower hematocrit (HCT), we investigated if the number of RBC affects the extrusion of these cells during clot retraction. Indeed, lowering HCT in AA mouse blood reduced RBC extrusion from the clots. However, increasing HCT in SS mouse blood to that of AA blood did not increase the number of SS RBC extruded from the clot. Furthermore, inhibition of FXIIIa activity with T101 (10µM) increased the release of AA (by 64%, n=6, p<0.05) but not SS RBC from mouse clots. These data indicate that the entrapment of SS RBCs within the clot is not simply caused by lower RBC number, and is FXIII-independent. Mixing the platelet poor plasma (PPP) and cellular fraction of AA and SS mouse blood revealed that the entrapment of SS RBC is not mediated by PPP (AA blood recombined = 6.9±3.6%; SS blood recombined = 0.4±0.4%; AA cells/SS PPP = 6.7±6.4%; SS cells/AA PPP = 0.1±0.2%, n=3 per group). Clots formed ex vivo from AA blood had a gel-like, soft structure, whereas SS clots were more firm and stiff. EM demonstrated that RBC within AA clots had polyhedral shapes and were tightly packed in the central part of the clot. In contrast, most of the mouse SS RBC did not have polyhedral shapes, underwent sickling and were not compacted within the clot. They also formed long "spicule-like" processes that intertwined with fibrin fibers (Fig. C). Similar results were observed in blood of sickle patients; however, the sickled RBC phenotype was less prominent. Importantly, sickling of RBC was observed in clots formed in the inferior vena cava of SS mice, 2 hours after vessel stenosis (Fig. D). We also performed a tPA challenge assay on clots formed ex vivo from human blood and showed that SS clots challenged with low tPA concentration (0.6nM) were more resistant to fibrinolysis compared to AA clots (clot lysis time, 714±6 vs. 388.3±120.7 minutes, n=6, p=0.08). The electrolytic injury model of venous thrombosis was used to investigate the dynamics of clot formation in SS mice in vivo. Mice were infused with fluorescently labeled antibodies for fibrin (green) and platelets (red). Electrolytic injury was applied to the femoral vein; a relative intensity of fibrin and platelet accumulation was assessed by fluorescence microscopy for one hour at 10-minute intervals. SS mice have increased platelet and fibrin accumulation compared to AA mice (~ 2 fold, n=5-7, p<0.05 for 40, 50 and 60 minute time points). Interestingly, in the AA clots, platelets were mostly localized on the surface, in contrast to their widespread distribution throughout the clot in SS mice (Fig. E, yellow color). Our data demonstrated that SCD alters the structure and dynamics of venous clot formation. Experiments investigating the consequence of these observations in mouse models of stroke and pulmonary embolism are currently ongoing. Figure Figure. Disclosures No relevant conflicts of interest to declare.
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Randriamboavonjy, Voahanginirina, Johann Isaak, Timo Frömel, Benoit Viollet, Beate Fisslthaler, Klaus T. Preissner, and Ingrid Fleming. "AMPK α2 subunit is involved in platelet signaling, clot retraction, and thrombus stability." Blood 116, no. 12 (September 23, 2010): 2134–40. http://dx.doi.org/10.1182/blood-2010-04-279612.
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Abstract The adenosine monophosphate (AMP)–activated protein kinase (AMPK) is a regulator of energy balance at the cellular and whole-body levels, but little is known about the role of AMPK in platelet activation. We report that both the α1 and α2 AMPK isoforms are expressed by human and murine platelets and that thrombin elicits the phosphorylation of AMPKα as well as the upstream kinase, liver kinase B1 (LKB1). In human platelets, the kinase inhibitors iodotubercidin and compound C significantly inhibited thrombin-induced platelet aggregation and clot retraction without affecting the initial increase in [Ca2+]i. Clot retraction was also impaired in platelets from AMPKα2−/− mice but not from wild-type littermates or AMPKα1−/− mice. Moreover, rebleeding was more frequent in AMPKα2−/− mice, and the FeCl3-induced thrombi formed in AMPKα2−/− mice were unstable. Mechanistically, AMPKα2 was found to phosphorylate in vitro the Src-family kinase, Fyn, and isoform deletion resulted in the attenuated threonine phosphorylation of Fyn as well as the subsequent tyrosine phosphorylation of its substrate, β3 integrin. These data indicate that AMPKα2—by affecting Fyn phosphorylation and activity—plays a key role in platelet αIIbβ3 integrin signaling, leading to clot retraction and thrombus stability.
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Khatlani, Tanvir, Lavanya Kailasam, Nawaf Alrehani, Subhashree Pradhan, and Vinod K. Vijayan. "Distinct roles for the α, β and γ1 isoforms of protein phosphatase 1 in the outside-in αIIbβ3 integrin signalling-dependent functions." Thrombosis and Haemostasis 109, no. 01 (2013): 118–29. http://dx.doi.org/10.1160/th12-04-0237.
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SummaryAlthough protein kinases and phosphatases participate in integrin αIIbβ3 signalling, whether integrin functions are regulated by the catalytic subunit of protein phosphatase 1 (PP1c) isoforms are unclear. We show that siRNA mediated knockdown of all PP1c isoforms (α, β and γ1) in 293 αIIbβ3 cells decreased adhesion to immobilised fibrinogen and fibrin clot retraction. Selective knockdown of only PP1cγ1 did not alter adhesion or clot retraction, while depletion of PP1cβ decreased both functions. Unexpectedly, knockdown of PP1cα enhanced αIIbβ3 adhesion to fibrinogen and clot retraction. Protein interaction studies revealed that all PP1c isoforms can interact with the integrin αIIb subunit. Phospho-profiling studies revealed an enhanced activation of mitogen- activated protein kinase (MAPK) p38 in the PP1cα depleted cells. Enhanced adhesive phenotype displayed by the PP1cα-depleted 293 αIIbβ3 cells was blocked by pharmacological inhibition of p38. Conversely, the decreased adhesion of PP1cα overexpressing cells was rescued by the expression of constitutively active p38α or p38γ. Thus, PP1c isoforms have distinct contribution to the outside-in αIIbβ3 signalling- dependent functions in 293 αIIbβ3 cells. Moreover, PP1cα negatively regulates integrin function by suppressing the p38 pathway.
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Naik, Meghna Ulhas, Timothy J. Stalker, Lawrence F. Brass, and Ulhas P. Naik. "Platelet Junctional Adhesion Molecule-A Regulates Thrombosis by Negatively Regulating Outside-in Signaling through Integrin αIIbβ3." Blood 114, no. 22 (November 20, 2009): 155. http://dx.doi.org/10.1182/blood.v114.22.155.155.
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Abstract Abstract 155 Platelet aggregation plays an important role in physiological hemostasis and pathological thrombosis. Platelet agonists induce a series of signaling events called inside-out signaling that leads to the activation of integrin αIIbβ3. Upon ligand binding to integrin αIIbβ3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How these signaling events are regulated is not well understood. We have previously identified JAM-A, a junctional adhesion molecule, on the platelet surface. Genetic ablation of Jam-A significantly (P<0.00001) shortened tail bleeding times (mean 54 seconds) compared to wild-type littermates (mean 98 seconds), suggesting an enhanced clot formation. FeCl3-induced carotid artery thrombosis and laser-induced cremaster arteriole thrombosis, two well recognized in vivo thrombosis model, both showed greatly shortened time of vessel occlusion and increased thrombus formation compared to wild-type (WT). Since JAM-A is expressed both on the endothelium and on platelets, the observed defect could arise from the lack of JAM-A in either cell type. To identify the involvement of platelet derived JAM-A, we performed a collagen-epinephrine-induced pulmonary thromboembolism assay. In this assay, pulmonary vessel occlusion occurs primarily through platelet thrombus without injury to the endothelium. We found that a significantly increased number of Jam-A−/− mice died within two minutes compared to WT mice. When analyzed for the extent of pulmonary vascular occlusion by Evans blue exclusion as well as histochemical analysis, we found a significantly greater extent of thromoembolism in Jam-A−/− mice compared to WT mice. Consistent with this finding, agonist-induced platelet aggregation, but not secretion, was significantly enhanced in Jam-A−/− platelets. Interestingly, however, the expression or activation of integrin αIIbβ;3 was not affected by Jam-A deficiency. When we analyzed the rate of clot retraction, we found that Jam-A−/− platelets showed 85% clot retraction within 90 minutes compared to only a 20% clot retraction in WT platelets, indicating that the absence of Jam-A significantly increases the rate of clot retraction (P<0.0001). JAM-A was found to associate with integrin αIIbβ3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation assay suggesting that JAM-A may suppress signaling through the integrin. To delineate the molecular mechanism influenced by JAM-A, we analyzed the known signaling cascade involved in clot retraction. We found that outside-in signaling-induced activation of ERK1 and p38 MAP kinase was significantly enhanced in Jam-A−/− platelets. However, activation of focal adhesion kinase was unaffected in Jam-A−/− platelets compared to WT. Furthermore, outside-in signaling-induced phosphorylation of the myosin light chain was increased in Jam-A−/− platelets. These in vivo and in vitro results clearly show that JAM-A negatively regulates outside-in signaling through integrin αIIbβ;3, thus protecting from thrombosis. Disclosures: No relevant conflicts of interest to declare.
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Jen, C. J., and L. V. McIntire. "Platelet Microtubules in Clot Structure Formation and Contractile Force Generation: Investigation of a Controversy." Thrombosis and Haemostasis 56, no. 01 (1986): 023–27. http://dx.doi.org/10.1055/s-0038-1661596.
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SummaryWhether platelet microtubules are involved in clot retraction/ contraction has been controversial. To address this question we have simultaneously measured two clotting parameters, clot structural rigidity and isometric contractile force, using a rheological technique. For recalcified PRP clots these two parameters began rising together at about 15 min after CaCl2 addition. In the concentration range affecting microtubule organization in platelets, colchicine, vinca alkaloids and taxol demonstrated insignificant effects on both clotting parameters of a recalcified PRP clot. For PRP clots induced by adding small amounts of exogenous thrombin, the kinetic curves of clot rigidity were biphasic and without a lag time. The first phase corresponded to a platelet-independent network forming process, while the second phase corresponded to a platelet-dependent process. These PRP clots began generating contractile force at the onset of the second phase. For both rigidity and force parameters, only the second phase of clotting kinetics was retarded by microtubule affecting reagents. When PRP samples were clotted by adding a mixture of CaCl2 and thrombin, the second phase clotting was accelerated and became superimposed on the first phase. The inhibitory effects of micro tubule affecting reagents became less pronounced. Thrombin clotting of a two-component system (washed platelets/ purified fibrinogen) was also biphasic, with the second phase being microtubule-dependent. In conclusion, platelet microtubules are important in PRP clotted with low concentrations of thrombin, during which fibrin network formation precedes platelet-fibrin interactions. On the other hand they are unimportant if a PRP clot is induced by recalcification, during which the fibrin network is constructed in the presence of platelet-fibrin interactions. The latter is likely to be more analogous to physiological processes in vivo.
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Evtugina, N. G., S. S. Sannikova, A. D. Peshkova, S. I. Safiullina, I. A. Andrianova, G. R. Tarasova, R. R. Khismatullin, Sh M. Abdullaeva, and R. I. Litvinov. "Quantitative and qualitative changes in blood cells associated with COVID-19." Kazan medical journal 102, no. 2 (April 6, 2021): 141–55. http://dx.doi.org/10.17816/kmj2021-141.
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Aim. To establish the relationship of hematological disorders with the pathogenesis, course and outcomes of COVID-19.
Methods. We examined 235 hospitalized patients with moderate and severe forms of acute COVID-19 receiving anticoagulants and immunosuppressive drugs. We studied the full blood cell counts and morphology along with the platelet function by flow cytometry in comparison with the clinical features and synthesis of inflammatory markers. To assess platelet contractility, blood clot contraction (retraction) kinetics was used in combination with scanning electron microscopy of platelets and blood clots.
Results. Hemolytic anemia, neutrophilia and lymphopenia were associated with immature erythrocytes and leukocytes, indicating activation of hematopoiesis. Contraction of blood clots in COVID-19 was impaired, especially in severe and lethal cases, as well as in the presence of comorbidities, including myeloproliferative and coronary heart diseases and acute cerebrovascular disease. In male patients, the changes in clot contraction were more pronounced. Suppression of clot contraction correlated directly with anemia and coagulopathy, including a high D-dimer level, which confirms the pathogenetic significance of blood clot contraction in COVID-19. A decrease in platelet contractility was due to moderate thrombocytopenia in combination with chronic platelet activation and secondary platelet dysfunction. The structure and cellular composition of blood clots depended on the extent of contraction; clots with impaired contraction were porous, had a low content of deformed polyhedral erythrocytes (polyhedrocytes) and an even distribution of fibrin.
Conclusion. Blood cells undergoing both quantitative and qualitative changes are involved in the pathogenesis of COVID-19; the suppressed platelet-driven contraction of intravital blood clots may be a part of the prothrombotic mechanisms.
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Colucci, Mario, Silvia Scopece, Antonio V. Gelato, Donato Dimonte, and Nicola Semeraro. "In Vitro Clot Lysis as a Potential Indicator of Thrombus Resistance to Fibrinolysis – Study in Healthy Subjects and Correlation with Blood Fibrinolytic Parameters." Thrombosis and Haemostasis 77, no. 04 (1997): 725–29. http://dx.doi.org/10.1055/s-0038-1656041.
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SummaryUsing an in vitro model of clot lysis, the individual response to a pharmacological concentration of recombinant tissue plasminogen activator (rt-PA) and the influence on this response of the physiological variations of blood parameters known to interfere with the fibrinolytic/thrombolytic process were investigated in 103 healthy donors. 125I-fibrin labelled blood clots were submersed in autologous plasma, supplemented with 500 ng/ml of rt-PA or solvent, and the degree of lysis was determined after 3 h of incubation at 37° C. Baseline plasma levels of t-PA, plasminogen activator inhibitor 1 (PAI-1), plasminogen, α2-anti-plasmin, fibrinogen, lipoprotein (a), thrombomodulin and von Willebrand factor as well as platelet and leukocyte count and clot retraction were also determined in each donor. rt-PA-induced clot lysis varied over a wide range (28-75%) and was significantly related to endogenous t-PA, PAI-1, plasminogen (p <0.001) and age (p <0.01). Multivariate analysis indicated that both PAI-1 antigen and plasminogen independently predicted low response to rt-PA. Surprisingly, however, not only PAI-1 but also plasminogen was negatively correlated with rt-PA-ginduced clot lysis. The observation that neutralization of PAI-1 by specific antibodies, both in plasma and within the clot, did not potentiate clot lysis indicates that the inhibitor, including the platelet-derived form, is insufficient to attenuate the thrombolytic activity of a pharmacological concentration of rt-PA and that its elevation, similarly to the elevation of plasminogen, is not the cause of clot resistance but rather a coincident finding. It is concluded that the in vitro response of blood clots to rt-PA is poorly influenced by the physiological variations of the examined parameters and that factors other than those evaluated in this study interfere with clot dissolution by rt-PA. In vitro clot lysis test might help to identify patients who may be resistant to thrombolytic therapy.
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LUNDSTEEN, ERLING. "On the Clot-retraction of the blood." Acta Medica Scandinavica 112, no. 3-4 (April 24, 2009): 302–13. http://dx.doi.org/10.1111/j.0954-6820.1942.tb13096.x.
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Bottecchia, Domenico, Gianpaolo Fantin, Paolo Gruppo, Gino Nassuato, and Pietro Zatti. "Reptilase Clot Retraction Induced by Electrical Stimulation." Scandinavian Journal of Haematology 16, no. 1 (April 24, 2009): 75–80. http://dx.doi.org/10.1111/j.1600-0609.1976.tb01120.x.
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KUBISZ, PETER, JANA SURANOVÁ, and STIG CRONBERG. "Mechanism of Calcium-Induced Reptilase Clot Retraction." Scandinavian Journal of Haematology 16, no. 4 (April 24, 2009): 263–65. http://dx.doi.org/10.1111/j.1600-0609.1976.tb01148.x.
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Vaiyapuri, Sakthivel, Tanya Sage, Rekha H. Rana, Michael P. Schenk, Marfoua S. Ali, Amanda J. Unsworth, Chris I. Jones, et al. "EphB2 regulates contact-dependent and contact-independent signaling to control platelet function." Blood 125, no. 4 (January 22, 2015): 720–30. http://dx.doi.org/10.1182/blood-2014-06-585083.
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Banerjee, Meenakshi, Smita Joshi, Jinchao Zhang, Carole L. Moncman, Shilpi Yadav, Beth A. Bouchard, Brian Storrie, and Sidney W. Whiteheart. "Cellubrevin/vesicle-associated membrane protein-3–mediated endocytosis and trafficking regulate platelet functions." Blood 130, no. 26 (December 28, 2017): 2872–83. http://dx.doi.org/10.1182/blood-2017-02-768176.
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Le Minh, Giang, Alina D. Peshkova, Izabella A. Andrianova, Timur B. Sibgatullin, Adelia N. Maksudova, John W. Weisel, and Rustem I. Litvinov. "Impaired contraction of blood clots as a novel prothrombotic mechanism in systemic lupus erythematosus." Clinical Science 132, no. 2 (January 19, 2018): 243–54. http://dx.doi.org/10.1042/cs20171510.
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The aim of this work was to examine a possible role of clot contraction/retraction in thrombotic complications of systemic lupus erythematosus (SLE). Using a novel automated method, we investigated kinetics of clot contraction in the blood of 51 SLE patients and 60 healthy donors. The functionality of platelets in the SLE patients was assessed using flow cytometry by expression of P-selectin and fibrinogen-binding capacity. The rate and degree of clot contraction were significantly reduced in SLE patients compared with healthy subjects, especially in the patients with higher blood levels of anti-dsDNA antibodies. The reduced platelet contractility correlated with partial refractoriness of platelets isolated from the blood of SLE patients to stimulation induced by the thrombin receptor activating peptide. To test if the anti-dsDNA autoantibodies cause continuous platelet activation, followed by exhaustion and dysfunction of the cells, we added purified exogenous anti-dsDNA autoantibodies from SLE patients to normal blood before clotting. In support of this hypothesis, the antibodies first enhanced clot contraction and then suppressed it in a time-dependent manner. Importantly, a direct correlation of clot contraction parameters with the disease severity suggests that the reduced compactness of intravascular clots and thrombi could be a pathogenic factor in SLE that may exaggerate the impaired blood flow at the site of thrombosis. In conclusion, autoantibodies in SLE can affect platelet contractility, resulting in reduced ability of clots and thrombi to shrink in volume, which increases vessel obstruction and may aggravate the course and outcomes of thrombotic complications in SLE.
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Peshkova, Alina, Dmitry Malyasyov, Roman Bredikhin, Giang Le Minh, Izabella Andrianova, Valerie Tutwiler, Chandrasekaran Nagaswami, John Weisel, and Rustem Litvinov. "Reduced Contraction of Blood Clots in Venous Thromboembolism Is a Potential Thrombogenic and Embologenic Mechanism." TH Open 02, no. 01 (January 2018): e104-e115. http://dx.doi.org/10.1055/s-0038-1635572.
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AbstractContraction (retraction) of the blood clot is a part of the clotting process driven by activated platelets attached to fibrin that can potentially modulate the obstructiveness and integrity of thrombi. The aim of this work was to reveal the pathogenic importance of contraction of clots and thrombi in venous thromboembolism (VTE). We investigated the kinetics of clot contraction in the blood of 55 patients with VTE. In addition, we studied the ultrastructure of ex vivo venous thrombi as well as the morphology and functionality of isolated platelets. Thrombi from VTE patients contained compressed polyhedral erythrocytes, a marker for clot contraction in vivo. The extent and rate of contraction were reduced by twofold in clots from the blood of VTE patients compared with healthy controls. The contraction of clots from the blood of patients with pulmonary embolism was significantly impaired compared with that of those with isolated venous thrombosis, suggesting that less compacted thrombi are prone to embolization. The reduced ability of clots to contract correlated with continuous platelet activation followed by their partial refractoriness. Morphologically, 75% of platelets from VTE patients were spontaneously activated (with filopodia) compared with only 21% from healthy controls. At the same time, platelets from VTE patients showed a 1.4-fold reduction in activation markers expressed in response to chemical activation when compared with healthy individuals. The results obtained suggest that the impaired contraction of thrombi is an underappreciated pathogenic mechanism in VTE that may regulate the obstructiveness and embologenicity of venous thrombi.
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Buitrago, Lorena, Wallace Y. Langdon, Archana Sanjay, and Satya P. Kunapuli. "Tyrosine phosphorylated c-Cbl regulates platelet functional responses mediated by outside-in signaling." Blood 118, no. 20 (November 17, 2011): 5631–40. http://dx.doi.org/10.1182/blood-2011-01-328807.
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Abstract c-Cbl protein functions as an E3 ligase and scaffolding protein, where 3 residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses after integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet-spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet-spreading, suggesting a differential role of these tyrosine residues. The physiologic role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-CblYF/YF knock-in mice. c-Cbl KO and c-CblYF/YF platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-CblYF/YF platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet-spreading and clot retraction.
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Kasahara, Kohji, Mizuho Kaneda, Toshiaki Miki, Kazuko Iida, Naoko Sekino-Suzuki, Ikuo Kawashima, Hidenori Suzuki, et al. "Clot retraction is mediated by factor XIII-dependent fibrin-αIIbβ3-myosin axis in platelet sphingomyelin-rich membrane rafts." Blood 122, no. 19 (November 7, 2013): 3340–48. http://dx.doi.org/10.1182/blood-2013-04-491290.
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Key Points Clot retraction of sphingomyelin-rich raft-depleted platelets from sphingomyelin synthase knockout mouse is delayed. Translocation of fibrin to sphingomyelin-rich rafts in platelet membrane is induced by thrombin in the presence of FXIII crosslinking activity.
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Rao, K. Murali Krishna, and Thomas F. Newcomb. "Clot Retraction in a Factor XIII Free System." Scandinavian Journal of Haematology 24, no. 2 (April 24, 2009): 142–48. http://dx.doi.org/10.1111/j.1600-0609.1980.tb02358.x.
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HORÁNYI, M. "Studies on the Retraction of the Blood Clot." Acta Medica Scandinavica 140, no. 2 (April 24, 2009): 140–48. http://dx.doi.org/10.1111/j.0954-6820.1951.tb10164.x.
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Li, Zida, Xiang Li, Brendan McCracken, Yue Shao, Kevin Ward, and Jianping Fu. "A Miniaturized Hemoretractometer for Blood Clot Retraction Testing." Small 12, no. 29 (June 1, 2016): 3926–34. http://dx.doi.org/10.1002/smll.201600274.
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Parfirieva, E. M., A. O. Ivchenko, O. A. Ivchenko, E. V. Gavrilin, A. I. Chirev, I. O. Savel'ev, I. O. Savel'ev, and V. E. Gunther. "3-D Incubator Principle in Nikelid-Titanium Porous Plates Hemostasis." KnE Materials Science 2, no. 1 (July 17, 2017): 347. http://dx.doi.org/10.18502/kms.v2i1.818.
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Biological liquids (blood particularly) imbibe 3-D construction of nikelid-titan porous plates easily. Clot formation takes place in large porous volume, its retraction by fibrin fibers forming and then maturation of connective tissue all together additionally fix the implant in bleeding zone.
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Carr, Marcus E., Sheryl L. Carr, and Philip E. Greilich. "Heparin Ablates Force Development during Platelet Mediated Clot Retraction." Thrombosis and Haemostasis 75, no. 04 (1996): 674–78. http://dx.doi.org/10.1055/s-0038-1650341.
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SummaryAlthough snake venom enzymes such as reptilase do not cause viscous metamorphosis, platelet secretion or clot retraction; when batroxobin and calcium are added to citrated blood significant platelet force development occurs. When this batroxobin-calcium system was applied to the study of platelet function during cardiopulmonary bypass (CPB), force development was found to be completely inhibited. After heparin reversal by protamine sulfate, significant recovery of force occurred. The present investigation was performed to evaluate the role of heparin in reducing force development during CPB. At concentrations above 0.10 U/ml, heparin totally suppressed force development in normal plasma. Addition of protamine sulfate to heparinized plasma caused complete recovery of force development. These concentrations of heparin had little effect on platelet aggregation by ADP or collagen. Possible direct effects of heparin on fibrin assembly and structure were studied by adding varying amounts of heparin to plasma and then inducing clot formation with batroxobin. At 1 U/ml, heparin reduced the size of fibrin fibers by 33%. Higher heparin concentrations had no additional effect. These results indicate that heparin may be responsible for a significant component of the decreased platelet force noted during cardiopulmonary bypass. To test whether heparin’s effect could be due to suppression of thrombin activity, the effects of the antithrombin hirudin on force development were measured. Hirudin also inhibited force development in a concentration dependent manner. Thus, heparin’s reduction of platelet force development may be due, at least in part, to suppression of thrombin activity.
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Ward, Christopher M., Anita S. Kestin, and Peter J. Newman. "A Leu262Pro mutation in the integrin β3 subunit results in an αIIb-β3 complex that binds fibrin but not fibrinogen." Blood 96, no. 1 (July 1, 2000): 161–69. http://dx.doi.org/10.1182/blood.v96.1.161.
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Abstract Platelet retraction of a fibrin clot is mediated by the platelet fibrinogen receptor, IIbβ3. In certain forms of the inherited platelet disorder, Glanzmann thrombasthenia (GT), mutant IIbβ3 may interact normally with fibrin yet fail to support fibrinogen-dependent aggregation. We describe a patient (LD) with such a form of GT. Platelets from LD supported normal clot retraction but failed to bind fibrinogen. Platelet analysis using flow cytometry and immunoblotting showed reduced but clearly detectable IIbβ3, findings consistent with type II GT. Genotyping of LD revealed 2 novel β3 mutations: a deletion of nucleotides 867 to 868, resulting in a premature stop codon at amino acid residue 267, and a T883C missense mutation, resulting in a leucine (Leu) 262-to-proline (Pro) substitution. Leu262 is highly conserved among β integrin subunits and lies within an intrachain loop implicated in subunit association. Leu262Proβ3 cotransfected with wild-type IIb into COS-7 cells showed delayed intracellular maturation and reduced surface expression of easily dissociable complexes. In human embryonic kidney 293 cells, Leu262Proβ3 formed a complex with endogenous av and retracted fibrin clots similarly to wild-type β3. The same cells, however, were unable to bind immobilized fibrinogen. The molecular requirements for IIbβ3 to interact with fibrin compared with fibrinogen, therefore, appear to differ. The region surrounding β3 Leu262 may maintain β3 in a fibrinogen-binding, competent form, but it appears not to be required for receptor interactions with fibrin.