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1
Hansen, Caroline E., Yongzhi Qiu, Owen J. T. McCarty, and Wilbur A. Lam. "Platelet Mechanotransduction." Annual Review of Biomedical Engineering 20, no. 1 (June 4, 2018): 253–75. http://dx.doi.org/10.1146/annurev-bioeng-062117-121215.
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The vasculature is a dynamic environment in which blood platelets constantly survey the endothelium for sites of vessel damage. The formation of a mechanically coherent hemostatic plug to prevent blood loss relies on a coordinated series of ligand–receptor interactions governing the recruitment, activation, and aggregation of platelets. The physical biology of each step is distinct in that the recruitment of platelets depends on the mechanosensing of the platelet receptor glycoprotein Ib for the adhesive protein von Willebrand factor, whereas platelet activation and aggregation are responsive to the mechanical forces sensed at adhesive junctions between platelets and at the platelet–matrix interface. Herein we take a biophysical perspective to discuss the current understanding of platelet mechanotransduction as well as the measurement techniques used to quantify the physical biology of platelets in the context of thrombus formation under flow.
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Diamond, Scott L. "Systems Biology to Predict Platelet Function." Blood 116, no. 21 (November 19, 2010): SCI—38—SCI—38. http://dx.doi.org/10.1182/blood.v116.21.sci-38.sci-38.
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Abstract Abstract SCI-38 Systems Biology seeks to provide patient-specific prediction of dynamic cellular response to multiple stimuli, critical information toward predicting risk, disease progression, or response to therapy. We deployed two distinct approaches, bottom-up and top-down analyses, to gain insight into platelet signaling. The bottom-up approach required a definition of reaction network and kinetic equations (topology), kinetic parameters, and initial concentrations in order to simulate platelet signaling. We developed a computational platelet model – assembled from 24 peer-reviewed platelet studies to yield 132 measured kinetic rate constants – that accurately predicts resting levels of cytosolic calcium, IP3, diacylglycerol, phosphatidic acid, phosphoinositol, PIP, and PIP2. The model accurately predicts the full transient calcium dynamics in response to increasing levels of ADP. In the first full stochastic simulation of single platelet response to ADP, the model provides an extremely accurate prediction of the statistics of the asynchronous [Ca]i spikes observed in single platelets. Specifically, this is the first work to provide a quantitative molecular explanation of the asynchronous calcium spiking observed in ADP-activated human platelets. We show the asynchronous spiking is a result of the fundamentally stochastic nature of signal transduction in cells as small as human platelets. Specific testable predictions have emerged about the requirement of high SERCA/IP3R ratios in functional platelets, limits on the concentration of calcium in the DTS, and relative potencies of PAR peptides and ADP. For functional phenotyping platelets, a top-down approach linking multiple inputs to functional outputs was used to understand how human platelets integrate diverse signals encountered during thrombosis. We developed a high-throughput platform that measures the human platelet calcium mobilization in response to all pairwise combinations of six major agonists. Agonists tested in this study were: convulxin (CVX; GPVI activator), ADP, the thromboxane analog U46619, PAR1 agonist peptide (SFLLRN), PAR4 agonist peptide (AYPGKF), and PGE2 (activator of IP and EP receptor). The calcium responses to single agonists at 0.1, 1, 10′ EC50 and 135 pairwise combinations trained a neural network (NN) model to predict the entire 6-dimensional platelet response space. The NN model successfully predicted responses to sequential additions and 27 ternary combinations of [ADP], [convulxin], and [SFLLRN] (R=0.881). With 4077 NN simulations spanning the 6-dimensional agonist space, 45 combinations of 4–6 agonists (ranging from synergism to antagonism) were selected and confirmed experimentally (R=0.883), revealing a highly synergistic condition of high U46619/PGE2 ratio, consistent with the risk of COX-2 therapy. Furthermore, pairwise agonist scanning (PAS) provided a direct measurement of 135 synergy values, thus allowing a unique phenotypic scoring of 10 human donors. Patient-specific training of NNs represent a compact and robust approach for prediction of cellular integration of multiple signals in a complex disease milieu. Either bottom-up models or top-down NN models are ideal for incorporation into systems biology simulations of thrombotic pathways under flow conditions. Disclosures: No relevant conflicts of interest to declare.
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Berndt, Michael C., and Robert K. Andrews. "Systems biology meets platelet biology." Blood 112, no. 10 (November 15, 2008): 3920–21. http://dx.doi.org/10.1182/blood-2008-07-170225.
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Franco, Aime T., Adam Corken, and Jerry Ware. "Platelets at the interface of thrombosis, inflammation, and cancer." Blood 126, no. 5 (July 30, 2015): 582–88. http://dx.doi.org/10.1182/blood-2014-08-531582.
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Abstract Although once primarily recognized for its roles in hemostasis and thrombosis, the platelet has been increasingly recognized as a multipurpose cell. Indeed, circulating platelets have the ability to influence a wide range of seemingly unrelated pathophysiologic events. Here, we highlight some of the notable observations that link platelets to inflammation, reinforcing the platelet’s origin from a lower vertebrate cell type with both hemostatic and immunologic roles. In addition, we consider the relevance of platelets in cancer biology by focusing on the hallmarks of cancer and the ways platelets can influence multistep development of tumors. Beyond its traditional role in hemostasis and thrombosis, the platelet’s involvement in the interplay between hemostasis, thrombosis, inflammation, and cancer is likely complex, yet extremely important in each disease process. The existence of animal models of platelet dysfunction and currently used antiplatelet therapies provide a framework for understanding mechanistic insights into a wide range of pathophysiologic events. Thus, the basic scientist studying platelet function can think beyond the traditional hemostasis and thrombosis paradigms, while the practicing hematologist must appreciate platelet relevance in a wide range of disease processes.
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Masselli, Elena, Giulia Pozzi, Mauro Vaccarezza, Prisco Mirandola, Daniela Galli, Marco Vitale, Cecilia Carubbi, and Giuliana Gobbi. "ROS in Platelet Biology: Functional Aspects and Methodological Insights." International Journal of Molecular Sciences 21, no. 14 (July 9, 2020): 4866. http://dx.doi.org/10.3390/ijms21144866.
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Reactive oxygen species (ROS) and mitochondria play a pivotal role in regulating platelet functions. Platelet activation determines a drastic change in redox balance and in platelet metabolism. Indeed, several signaling pathways have been demonstrated to induce ROS production by NAPDH oxidase (NOX) and mitochondria, upon platelet activation. Platelet-derived ROS, in turn, boost further ROS production and consequent platelet activation, adhesion and recruitment in an auto-amplifying loop. This vicious circle results in a platelet procoagulant phenotype and apoptosis, both accounting for the high thrombotic risk in oxidative stress-related diseases. This review sought to elucidate molecular mechanisms underlying ROS production upon platelet activation and the effects of an altered redox balance on platelet function, focusing on the main advances that have been made in platelet redox biology. Furthermore, given the increasing interest in this field, we also describe the up-to-date methods for detecting platelets, ROS and the platelet bioenergetic profile, which have been proposed as potential disease biomarkers.
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Hundelshausen, Philipp, Frank Petersen, and Ernst Brandt. "Platelet-derived chemokines in vascular biology." Thrombosis and Haemostasis 97, no. 05 (2007): 704–13. http://dx.doi.org/10.1160/th07-01-0066.
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SummaryUndoubtedly, platelets are key elements in the regulation of thrombosis and haemostasis. Along with their primary task to prevent blood loss from injured vessels, platelets have emerged as regulators of a variety of processes in the vasculature. Multiple challenges, from the contact and adhesion to subendothelial matrix after injury of the vessel wall, to interactions with blood cells in inflammatory conditions, result in platelet activation with concomitant shape change and release of numerous substances. Among these, chemokines have been found to modulate several processes in the vasculature, such as atherosclerosis and angiogenesis. In particular, the chemokines connective tissue activating protein III (CTAP-III) and its precursors, or truncation products (CXCL7), platelet factor 4, (PF4, CXCL4) and its variant PF4alt (CXCL4L1) or regulated upon activation and normal T cell expressed and secreted (RANTES, CCL5), have been investigated thoroughly. Defined common properties as their aptitude to bind glycosaminoglycans or their predisposition to associate and form homooligomers are prerequisites for their role in the vasculature and function in vivo. The current review summarizes the development of these single chemokines, and their cooperative effects that may in part be dependent on their physical interactions.
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Littleton-Kearney, Marguerite T., Patricia D. Hurn, Thomas S. Kickler, and Richard J. Traystman. "Incomplete global cerebral ischemia alters platelet biology in neonatal and adult sheep." American Journal of Physiology-Heart and Circulatory Physiology 274, no. 4 (April 1, 1998): H1293—H1300. http://dx.doi.org/10.1152/ajpheart.1998.274.4.h1293.
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Platelets are implicated as etiologic agents in cerebral ischemia and as modulators of neural injury following an ischemic insult. We examined the effects of severe, transient global ischemia on platelet aggregation during 45-min ischemia and 30-, 60-, and 120-min reperfusion in adult and neonatal lambs. We also examined postischemic platelet deposition in brain and other tissues (120-min reperfusion) using indium-111-labeled platelets. Ischemic cerebral blood flow fell to 5 ± 1 and 5 ± 2 ml ⋅ min−1⋅ 100 g−1in lambs and sheep, respectively. During ischemia, platelet counts fell to 47.5 ± 5.1% of control ( P < 0.05) in lambs and 59 ± 4.9% of control in sheep ( P < 0.05). Ischemia depressed platelet aggregation response ( P < 0.01) to 4 μg collagen in lambs and sheep (20.4 ± 29.2 and 26 ± 44.7% of control, respectively). Marked platelet deposition occurred in brain and spleen in sheep, whereas significant platelet entrapment occurred only in brain in lambs. Our findings suggest that ischemia causes platelet activation and deposition in brain and noncerebral tissues.
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Gianazza, Erica, Maura Brioschi, Roberta Baetta, Alice Mallia, Cristina Banfi, and Elena Tremoli. "Platelets in Healthy and Disease States: From Biomarkers Discovery to Drug Targets Identification by Proteomics." International Journal of Molecular Sciences 21, no. 12 (June 25, 2020): 4541. http://dx.doi.org/10.3390/ijms21124541.
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Platelets are a heterogeneous small anucleate blood cell population with a central role both in physiological haemostasis and in pathological states, spanning from thrombosis to inflammation, and cancer. Recent advances in proteomic studies provided additional important information concerning the platelet biology and the response of platelets to several pathophysiological pathways. Platelets circulate systemically and can be easily isolated from human samples, making proteomic application very interesting for characterizing the complexity of platelet functions in health and disease as well as for identifying and quantifying potential platelet proteins as biomarkers and novel antiplatelet therapeutic targets. To date, the highly dynamic protein content of platelets has been studied in resting and activated platelets, and several subproteomes have been characterized including platelet-derived microparticles, platelet granules, platelet releasates, platelet membrane proteins, and specific platelet post-translational modifications. In this review, a critical overview is provided on principal platelet proteomic studies focused on platelet biology from signaling to granules content, platelet proteome changes in several diseases, and the impact of drugs on platelet functions. Moreover, recent advances in quantitative platelet proteomics are discussed, emphasizing the importance of targeted quantification methods for more precise, robust and accurate quantification of selected proteins, which might be used as biomarkers for disease diagnosis, prognosis and therapy, and their strong clinical impact in the near future.
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Andrade, Sheila Siqueira, Alessandra Valéria de Sousa Faria, Manoel João Batista C. Girão, Gwenny M. Fuhler, Maikel P. Peppelenbosch, and Carmen V. Ferreira-Halder. "Biotech-Educated Platelets: Beyond Tissue Regeneration 2.0." International Journal of Molecular Sciences 21, no. 17 (August 23, 2020): 6061. http://dx.doi.org/10.3390/ijms21176061.
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The increasing discoveries regarding the biology and functions of platelets in the last decade undoubtedly show that these cells are one of the most biotechnological human cells. This review summarizes new advances in platelet biology, functions, and new concepts of biotech-educated platelets that connect advanced biomimetic science to platelet-based additive manufacturing for tissue regeneration. As highly responsive and secretory cells, platelets could be explored to develop solutions that alter injured microenvironments through platelet-based synthetic biomaterials with instructive extracellular cues for morphogenesis in tissue engineering beyond tissue regeneration 2.0.
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De Kock, Lore, and Kathleen Freson. "The (Patho)Biology of SRC Kinase in Platelets and Megakaryocytes." Medicina 56, no. 12 (November 24, 2020): 633. http://dx.doi.org/10.3390/medicina56120633.
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Proto-oncogene tyrosine-protein kinase SRC (SRC), as other members of the SRC family kinases (SFK), plays an important role in regulating signal transduction by different cell surface receptors after changes in the cellular environment. Here, we reviewed the role of SRC in platelets and megakaryocytes (MK). In platelets, inactive closed SRC is coupled to the β subunit of integrin αIIbβ3 while upon fibrinogen binding during platelet activation, αIIbβ3-mediated outside-in signaling is initiated by activation of SRC. Active open SRC now further stimulates many downstream effectors via tyrosine phosphorylation of enzymes, adaptors, and especially cytoskeletal components. Functional platelet studies using SRC knockout mice or broad spectrum SFK inhibitors pointed out that SRC mediates their spreading on fibrinogen. On the other hand, an activating pathological SRC missense variant E527K in humans that causes bleeding inhibits collagen-induced platelet activation while stimulating platelet spreading. The role of SRC in megakaryopoiesis is much less studied. SRC knockout mice have a normal platelet count though studies with SFK inhibitors point out that SRC could interfere with MK polyploidization and proplatelet formation but these inhibitors are not specific. Patients with the SRC E527K variant have thrombocytopenia due to hyperactive SRC that inhibits proplatelet formation after increased spreading of MK on fibrinogen and enhanced formation of podosomes. Studies in humans have contributed significantly to our understanding of SRC signaling in platelets and MK.
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Morrell, Craig N. "Immunomodulatory Mediators in Platelet Transfusion Reactions." Hematology 2011, no. 1 (December 10, 2011): 470–74. http://dx.doi.org/10.1182/asheducation-2011.1.470.
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Abstract Our appreciation of the roles that platelets play in vascular biology is constantly expanding. One of the major roles of platelets is in initiating and accelerating immune responses. Platelet transfusion may be associated with adverse inflammatory outcomes manifested as fever, discomfort, tachycardia, and respiratory issues. This may in part be due to immune mediators either expressed by activated platelets or released into the platelet media during platelet storage. This review will highlight some more recent knowledge gained regarding the platelet storage lesion and potential mediators of platelet transfusion reactions.
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Flaumenhaft, Robert. "Bioengineering in Platelet Biology." Thrombosis Research 133, no. 4 (April 2014): 523–24. http://dx.doi.org/10.1016/j.thromres.2013.12.036.
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KICKLER, THOMAS S. "Platelet biology ? an overview." Transfusion Alternatives in Transfusion Medicine 8, no. 2 (June 2006): 79–85. http://dx.doi.org/10.1111/j.1778-428x.2006.00013.x.
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Noh, Ji-Yoon. "Megakaryopoiesis and Platelet Biology: Roles of Transcription Factors and Emerging Clinical Implications." International Journal of Molecular Sciences 22, no. 17 (September 5, 2021): 9615. http://dx.doi.org/10.3390/ijms22179615.
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Platelets play a critical role in hemostasis and thrombus formation. Platelets are small, anucleate, and short-lived blood cells that are produced by the large, polyploid, and hematopoietic stem cell (HSC)-derived megakaryocytes in bone marrow. Approximately 3000 platelets are released from one megakaryocyte, and thus, it is important to understand the physiologically relevant mechanism of development of mature megakaryocytes. Many genes, including several key transcription factors, have been shown to be crucial for platelet biogenesis. Mutations in these genes can perturb megakaryopoiesis or thrombopoiesis, resulting in thrombocytopenia. Metabolic changes owing to inflammation, ageing, or diseases such as cancer, in which platelets play crucial roles in disease development, can also affect platelet biogenesis. In this review, I describe the characteristics of platelets and megakaryocytes in terms of their differentiation processes. The role of several critical transcription factors have been discussed to better understand the changes in platelet biogenesis that occur during disease or ageing.
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Stolla, Moritz, Renata Grozovsky, Melissa M. Lee-Sundlov, Herve Falet, and Karin M. Hoffmeister. "Effects of Platelet Circulatory Age on Platelet Function." Blood 128, no. 22 (December 2, 2016): 413. http://dx.doi.org/10.1182/blood.v128.22.413.413.
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Abstract The human body produces and removes 1011 platelets daily to maintain a normal steady-state platelet count. However, the regulatory mechanisms remain elusive. We have shown that platelets lacking sialic acid (desialylated platelets) are removed by the hepatic Ashwell-Morell receptor (AMR or asialoglycoprotein receptor type 2), thereby regulating platelet survival and hepatic TPO levels. Platelet counts and lifetime were increased in Asgr2-/- mice (AMR-null mice), compared to wild type (WT) mice. Platelet volume and immature platelet fraction (IPF) are decreased in AMR-null mice, consistent with the notion that platelets in AMR-null mice (AMR-null platelets) circulate longer and are older. By contrast, deficiency of the sialyltransferase St3gal4 gene induces a marked thrombocytopenia (St3gal4-null platelets), due to rapid platelet clearance by the hepatic AMR. Consistent with the rapid platelet clearance, platelet volume and IPF were increased in St3gal4-/- mice, reflecting high platelet turnover and younger platelets. While both AMR-null and St3gal4-null platelets are desialylated, they differ substantially in their time, i.e. age, in circulation. Here we investigated the effects of in vivo and in vitro aging on platelet function. Freshly isolated St3gal4-null platelets showed significantly increased integrin activation when stimulated with convulxin and thrombin, while AMR-null platelets showed a significantly lower response compared to WT platelets. Secretion of α-granule was significantly increased in St3gal4-null platelets. By contrast no significant difference was measured between WT and AMR-null platelets. Despite increased platelet counts, the tail-bleeding time was significantly prolonged in AMR-null mice, compared to WT mice, suggesting that increased circulatory time (age) negatively affects platelet function in vivo. We next performed in vitro storage for up to 72 hours at room temperature to stress platelet aging. Stored St3gal4-deficient platelets had increased integrin activation, α-granule secretion in response to convulxin and thrombin and showed increased phosphatidyl serine exposure as detected by Annexin V binding in response to calcium ionophore, compared to stored control platelets. By contrast, stored AMR-null platelets had significantly impaired integrin activation, α-granule secretion and Annexin V binding compared to controls. To further evaluate the propensity to undergo apoptosis, we tested caspase-3 activation and mitochondrial membrane potential. Surprisingly, we found that both St3gal4-null (young) and AMR-null (old) platelets showed a significantly lower caspase-3 activation in response to calcium ionophore and ABT-737 compared to WT platelets. Furthermore, the mitochondrial membrane potential was lower in both St3gal4-deficient and AMR-null platelets compared to WT platelets, indicating functionally impaired mitochondria. Taken together, our data indicate that younger St3gal4-null platelets have an increased baseline function while by contrast older AMR-null platelets have decreased function in vitro and in vivo. Interestingly, younger and older (longer circulating platelets) had reduced propensity to undergo apoptosis and impaired mitochondrial function. Overall, younger platelets represent a highly favorable profile during storage. Identification of donors with a larger fraction of younger platelets could result in safer and more efficacious platelet transfusions. Disclosures No relevant conflicts of interest to declare.
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Battinelli, Elisabeth M., Rajesh Kulenthirarajan, Joseph E. Italiano, and Kelly Johnson. "Tamoxifen Directly Disrupts Platelet Angiogenic Potential and Inhibits Platelet-Mediated Metastasis." Blood 124, no. 21 (December 6, 2014): 4169. http://dx.doi.org/10.1182/blood.v124.21.4169.4169.
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Abstract Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in metastasis. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown directly impact platelet function; however, the reported effects have been varied (Vitseva et al,. 2005, Nayak et al., 2011). Because platelets are critical for metastatic spread, we posited that tamoxifen may exert anti-tumor effects by directly altering platelet function. To explore this, we first examined the effect of tamoxifen on platelet activation. P-selectin expression was determined by flow cytometry for platelets pretreated with or without tamoxifen and then activated with ADP or MCF-7 cells. Both ADP and exposure to MCF-7 cells resulted in platelet activation and treatment with tamoxifen lead to a partial inhibition of activation. Platelets are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. We have previously shown that we can manipulate the angiogenic potential of the platelet releasate through physiological (platelet agonists) and pathological activation (MCF-7 tumor cells) (Battinelli et al., 2011). We hypothesized that tamoxifen may impact malignancy by altering the release of angiogenesis regulatory proteins from platelets. To explore the impact of tamoxifen on the angiogenic potential of platelets, we analyzed the releasates from platelets exposed to tamoxifen alone or activated with ADP or MCF-7 cells in conjunction with tamoxifen. Our data reveals that platelets exposed to tamoxifen release significantly decreased amounts of VEGF in response to activation by either the platelet agonist ADP or interaction with tumor cells (MCF-7 cells). Next, in vitro angiogenesis assays were performed to further examine the effect of tamoxifen on the angiogenic potential of platelets. We observed dramatically diminished capillary tube branch point formation and decreased migration in endothelial cell cultures exposed to releasates generated from tamoxifen treated platelets compared to control releasates, demonstrating that tamoxifen inhibits the ability of activated platelets to promote angiogenesis. Platelets play a critical role in aiding the intravasation and extravasation of tumor cells in the circulation and therefore we postulated that tamoxifen could alter the ability of platelets to aid tumor cells in crossing the vascular endothelium. We performed transendothelial migration assays in which platelets were pretreated with tamoxifen or vehicle control, washed and mixed with MCF-7 tumor cells in endothelialized transwells. Platelets significantly increased the number of tumor cells that crossed the endothelial barrier; however this increase was lost when platelets were pretreated with tamoxifen. Overall our results demonstrate that tamoxifen directly alters platelet function, leading to decreased angiogenic and metastatic potential. These studies highlight a previously unknown mechanism of action for tamoxifen and may shed light on the efficacy of tamoxifen in estrogen-receptor negative cancers. Furthermore our work stresses the importance of crosstalk between platelets and cells within the tumor microenvironment for successful angiogenesis and metastatic spread and, ultimately, support the idea of utilizing targeted platelet therapies to inhibit the platelet’s role in malignancy. Disclosures No relevant conflicts of interest to declare.
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Battinelli, Elisabeth M., Kelly Elizabeth Johnson, Jodi A. Forward, Mason Tippy, Rajesh Kulenthirarajan, Erica L. Mayer, and Joseph E. Italiano. "Tamoxifen Directly Inhibits Platelet Activation, Angiogenic Potential and Platelet-Mediated Metastasis." Blood 128, no. 22 (December 2, 2016): 3723. http://dx.doi.org/10.1182/blood.v128.22.3723.3723.
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Abstract Objective - Platelets, which are mainly known for their role in hemostasis, are now known to play a crucial role in tumor metastasis and neovascularization. Metastatic disease is the cause of roughly 90% of all cancer-related deaths and understanding the mechanisms leading to dissemination of tumor cells to distant sites remains one of the main challenges of cancer research. Platelets carry a plethora of potent angiogenic and metastatic mediators within their alpha-granules and exposure to breast tumor cells induces platelet activation, leading to release of these mediators. Tamoxifen is a selective estrogen receptor modulator that is widely used for the treatment and prevention of breast cancer; its use is associated with a 50% reduction in the risk of invasive and noninvasive breast cancer in women who utilized the drug for at least 5 years. Interestingly, tamoxifen has demonstrated anti-cancer efficacy in estrogen negative breast cancers suggesting that this drug has additional mechanisms of action. Previously tamoxifen and its metabolites have been shown to directly impact platelet function. Because platelets are critical for metastatic spread, we posited that tamoxifen or its metabolites may exert anti-tumor effects through direct platelet inhibition. To test this hypothesis, we examined the impact of tamoxifen on platelet activation, angiogenic potential and metastasis both ex vivoand in breast cancer patients utilizing tamoxifen therapy. Approach and Results - We found that ex vivo pretreatment with tamoxifen or its metabolite 4-hydroxytamoxifen (4-OH) lead to a significant inhibition of platelet activation in response to TRAP, ADP or the breast tumor cell lines MDA-MB-231 and MCF-7 in platelets from healthy human donors. Platelets, known to promote tumor angiogenesis, are a reservoir for angiogenic proteins that are secreted in a differentially regulated process. Activated platelets exposed to tamoxifen or 4-OH released significantly lower amounts of the pro-angiogenic regulator VEGF in while anti-angiogenic endostatin release is unaffected, thus shifting the balance of angiogenic regulators that are released. To examine the impact of this alteration in angiogenic protein release, we performed functional angiogenesis assays using releasates generated from tamoxifen or 4-OH treated platelets. These in vitroangiogenesis assays confirmed that tamoxifen pretreatment led to dramatically diminished capillary tube formation and decreased endothelial migration. Tamoxifen also significantly inhibited the ability of platelets to promote metastasis in vitro, causing a dramatic decrease in breast tumor cell invasion and transendothelial migration. Next, we utilized membrane-based cytokine arrays to further interrogate the effect of tamoxifen on the release of stored platelet factors. Using this method, we identified several key proteins known to be associated with metastasis that were lower in releasate from tamoxifen treated platelets including TGF-b, IL-6 and IGF-1 while anti-angiogenic angiopoietin-1 was elevated. Platelets isolated from patients on tamoxifen maintenance therapy were also found to have decreased activation responses, diminished VEGF release and lower angiogenic potential. Conclusions - Overall we demonstrate that tamoxifen directly influences the release of specific stored platelet factors leading to decreased tumor cell support. The mechanism is directly linked to tamoxifen's inhibitory role in platelet activation, causing altered of release of key platelet-derived angiogenic and metastatic factors including VEGF, angiopoietin-1, TGF-b, IL-6 and IGF-1 during tumor cell and platelet crosstalk. Furthermore, translational studies confirmed that platelet activation and angiogenic potential are significantly suppressed in breast cancer patients utilizing tamoxifen therapy. Our work stresses the importance of platelets for successful angiogenesis and metastatic spread and, ultimately, supports the idea of utilizing targeted platelet therapies to inhibit the platelet's role in malignancy. Disclosures No relevant conflicts of interest to declare.
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Morrell, Craig N., Angela A. Aggrey, Lesley M. Chapman, and Kristina L. Modjeski. "Emerging roles for platelets as immune and inflammatory cells." Blood 123, no. 18 (May 1, 2014): 2759–67. http://dx.doi.org/10.1182/blood-2013-11-462432.
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AbstractDespite their small size and anucleate status, platelets have diverse roles in vascular biology. Not only are platelets the cellular mediator of thrombosis, but platelets are also immune cells that initiate and accelerate many vascular inflammatory conditions. Platelets are linked to the pathogenesis of inflammatory diseases such as atherosclerosis, malaria infection, transplant rejection, and rheumatoid arthritis. In some contexts, platelet immune functions are protective, whereas in others platelets contribute to adverse inflammatory outcomes. In this review, we will discuss platelet and platelet-derived mediator interactions with the innate and acquired arms of the immune system and platelet-vessel wall interactions that drive inflammatory disease. There have been many recent publications indicating both important protective and adverse roles for platelets in infectious disease. Because of this new accumulating data, and the fact that infectious disease continues to be a leading cause of death globally, we will also focus on new and emerging concepts related to platelet immune and inflammatory functions in the context of infectious disease.
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Nagalla, Srikanth, Chad Shaw, Xianguo Kong, Altaf A. Kondkar, Leonard C. Edelstein, Lin Ma, Junmei Chen, et al. "Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity." Blood 117, no. 19 (May 12, 2011): 5189–97. http://dx.doi.org/10.1182/blood-2010-09-299719.
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Abstract MicroRNAs (miRNAs) regulate cell physiology by altering protein expression, but the biology of platelet miRNAs is largely unexplored. We tested whether platelet miRNA levels were associated with platelet reactivity by genome-wide profiling using platelet RNA from 19 healthy subjects. We found that human platelets express 284 miRNAs. Unsupervised hierarchical clustering of miRNA profiles resulted in 2 groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed (DE) between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. Using whole genome mRNA expression data on these same subjects, we computationally generated a high-priority list of miRNA-mRNA pairs in which the DE platelet miRNAs had binding sites in 3′-untranslated regions of DE mRNAs, and the levels were negatively correlated. Three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5, and miR-107:CLOCK) were selected from this list, and all 3 miRNAs knocked down protein expression from the target mRNA. Reduced activation from platelets lacking PRKAR2B supported these findings. In summary, (1) platelet miRNAs are able to repress expression of platelet proteins, (2) miRNA profiles are associated with and may predict platelet reactivity, and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets.
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Smyth, Susan S. "Platelets Unplugged: An ATVB Special Series Focused on Platelet Biology." Arteriosclerosis, Thrombosis, and Vascular Biology 30, no. 12 (December 2010): 2339–40. http://dx.doi.org/10.1161/atvbaha.110.217992.
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DeHelian, Daniel, Shuchi Gupta, Jie Wu, Chelsea Thorsheim, Brian Estevez, Matthew Cooper, Kelly Litts, et al. "RGS10 and RGS18 differentially limit platelet activation, promote platelet production, and prolong platelet survival." Blood 136, no. 15 (October 8, 2020): 1773–82. http://dx.doi.org/10.1182/blood.2019003251.
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Abstract G protein–coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor–activating peptide, an increased maximum response to adenosine 5′-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10−/− platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18−/− mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18−/− and RGS10−/−18−/− mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.
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Josefsson, Emma C., Simone Schoenwaelder, Michael White, Matthew Goschnick, Andrew W. Roberts, Kylie D. Mason, Shaun Jackson, David C. Huang, and Benjamin T. Kile. "Platelet Death." Blood 112, no. 11 (November 16, 2008): sci—40—sci—40. http://dx.doi.org/10.1182/blood.v112.11.sci-40.sci-40.
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Abstract Human platelets exhibit a circulating lifespan of ~10 days, mouse platelets ~5 days. This finite existence is circumscribed by members of the Bcl-2 family of proteins, which control the intrinsic apoptosis pathway. Pro-survival Bcl-xL is the critical regulator of platelet lifespan, functioning to keep pro-death Bak and Bax in check, thereby maintaining platelet viability. After 5–10 days in the circulation, platelets not consumed in hemostatic processes initiate a Bak and Bax-dependent cell death program and clearance from the bloodstream. Mutations in Bcl-xL reduce platelet lifespan in a dose-dependent fashion, while deletion of Bak and Bax extend it. Studies with the BH3 mimetic compound ABT-737, which inhibits pro-survival Bcl-xL, have shown that platelets induced to undergo cell death in vitro exhibit many of the hallmarks of apoptosis in nucleated cells, including mitochondrial damage, caspase activation and externalization of membrane phosphatidylserine (PS). Whether any of these features occur during physiological platelet clearance remains unclear. Certainly, mitochondrial damage can reduce the recovery of transfused platelets, but whether PS – which is known to promote the pro-coagulant activity of agonist-activated platelets – also acts as a clearance signal for dying platelets in vivo is yet to be established. Conversely, Bak and Bax may play a role in mediating PS exposure triggered by activation. Supporting the idea that there may be crosstalk between classical platelet signaling pathways and the intrinsic apoptosis pathway is recent evidence that platelet agonists can also activate caspases. Intriguingly, elements of the intrinsic pathway may also contribute to the generation of platelets by megakaryocytes. Several groups have demonstrated that megakaryocytes contain activated caspases and that their inhibition can block platelet shedding by cultured cells. Preliminary evidence we have generated suggests that Bcl-2 family proteins may be required for platelet production in vivo. Thus, it appears that there is much to be understood about the role of the intrinsic apoptosis pathway in the regulation of platelet biogenesis, function, and death.
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Bix, Gregory, Rex A. Iozzo, Ben Woodall, Michelle Burrows, Angela McQuillan, Shelly Campbell, Gregg B. Fields, and Renato V. Iozzo. "Endorepellin, the C-terminal angiostatic module of perlecan, enhances collagen-platelet responses via the α2β1-integrin receptor." Blood 109, no. 9 (December 29, 2006): 3745–48. http://dx.doi.org/10.1182/blood-2006-08-039925.
Full textAbstract:
Abstract Endorepellin, a C-terminal fragment of the vascular basement membrane proteoglycan perlecan, inhibits angiogenesis via the α2β1-integrin receptor. Because this integrin is also implicated in platelet-collagen responses and because endorepellin or its fragments are generated in response to injury and inflammation, we hypothesized that endorepellin could also affect platelet biology. We discovered that endorepellin supported α2β1-dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of an α2β1-integrin function-blocking antibody. Collectively, these results suggest that endorepellin/α2β1-integrin interaction and effects are specific and dependent on cell type, differ from those emanated by exposure to collagen, and may be due to cellular differences in α2β1-integrin activation/ligand affinity state. These studies also suggest a heretofore unrecognized role for angiostatic basement membrane fragments in platelet biology.
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Flaumenhaft, Robert, James R. Dilks, and Derek S. Sim. "Protein Palmitoylation in Platelet Function: Role in G Protein-Mediated Platelet Activation and Platelet Recruitment into Thrombi." Blood 106, no. 11 (November 16, 2005): 650. http://dx.doi.org/10.1182/blood.v106.11.650.650.
Full textAbstract:
Abstract Protein palmitoylation represents the covalent linkage of a 16-carbon saturated fatty acid to a protein. This reversible post-translational modification directs protein-protein interactions as well as protein association with membranes and lipid rafts. Protein palmitoylation participates in ligand-induced signal transduction in several nucleated cells. Its role in platelet activation, however, has not previously been evaluated. We have found that platelets contain the palmitoyl transfer proteins GODZ and HIP14 as well as the palmitoyltransferase, acyl-protein thioesterase 1 (APT1). Thus, platelets possess the basic machinery for regulated palmitoylation. Studies using [3H]-labeled platelets identified several platelet proteins that were palmitoylated following exposure to the protease-activated receptor 1 (PAR-1) ligand, SFLLRN. To determine whether protein palmitoylation functions in activation-induced platelet functions, we infused recombinant APT1 into permeabilized platelets prior to activation with SFLLRN. Infusion of APT1 inhibited platelet protein palmitoylation and completely blocked platelet α-granule secretion induced by SFLLRN. Similarly, the protein palmitoylation inhibitor cerulenin blocked SFLLRN-induced platelet protein palmitoylation, α-granule secretion, and platelet aggregation in intact platelets. To assess the mechanism by which protein palmitoylation affects platelet function, we evaluated the effect of inhibitors of protein palmitoylation on G protein activity. Gαq is essential to PAR-1-mediated platelet activation and is palmitoylated in an activation-dependent manner in nucleated cells. Immunoprecipitation of Gαq from [3H]-labeled platelets showed that it is palmitoylated following activation of platelets with SFLLRN. Both APT1 and cerulenin inhibited SFLLRN-induced palmitoylation of Gαq. In addition, APT1 and cerulenin inhibited SFLLRN-induced GTPase activity as detected using [γ-32P]GTP-labeled platelet lysates. These results show that palmitoylation of Gαq participates in PAR-1-mediated signal transduction. We next used intravital microscopy to determine if protein palmitoylation functions in thrombus formation in vivo. For these experiments, platelets from a donor mouse were incubated with cerulenin and labeled with calcein-AM (green) or incubated with vehicle alone and labeled with calcein-AM red-orange (red). Equal numbers of green and red labeled platelets were then infused into a recipient mouse. The accumulation of cerulenin- and vehicle-treated platelets into thrombi following laser-induced injury of the mouse cremaster muscle was quantified using high speed, digital videomicroscopy. Incubation of platelets with cerulenin resulted in an approximately 50% reduction in their ability to accumulate into thrombi. These studies show that platelet protein palmitoylation is required for thrombus formation as well as for normal platelet function.
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Schmaier, AH, HN Bradford, D. Lundberg, A. Farber, and RW Colman. "Membrane expression of platelet calpain." Blood 75, no. 6 (March 15, 1990): 1273–81. http://dx.doi.org/10.1182/blood.v75.6.1273.1273.
Full textAbstract:
Abstract Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.
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Schmaier, AH, HN Bradford, D. Lundberg, A. Farber, and RW Colman. "Membrane expression of platelet calpain." Blood 75, no. 6 (March 15, 1990): 1273–81. http://dx.doi.org/10.1182/blood.v75.6.1273.bloodjournal7561273.
Full textAbstract:
Platelet calpain has many platelet substrates, including external membrane proteins. We thus investigated whether platelet calpain II was associated with platelet membranes in unstimulated and thrombin- activated platelets. A monospecific, goat polyclonal antibody was reared to purified platelet calpain II. Sixteen whole platelet lysates were found to contain 4.5 +/- 0.7 micrograms calpain antigen II per 10(8) platelets (mean +/- SEM) as determined by a competitive enzyme- linked immunosorbent assay. Using the dipeptide fluorogenic substrate, Suc-Leu-Tyr-MCA, 17 human platelet lysates contained 3.6 +/- 0.4 micrograms calpain activity per 10(8) platelets. Platelet calpain II was associated with the Triton X-100 insoluble platelet cytoskeletons from both unstimulated and thrombin-activated platelets. When compared with the total cell content of platelet calpain II, calpain antigen (10% to 13%) and calpain activity (24% to 28%) was associated with platelet cytoskeletons in unstimulated and thrombin-activated platelets, respectively. On immunoblot, the heavy chain (80 Kd) of calpain II was detected in platelet cytoskeletons. Subcellular fractionation studies on both unstimulated and thrombin-activated platelets, revealed that half of the total platelet calpain II antigen was associated with cytosol, and the other half was associated with the membrane fraction. Platelet calpain II was not seen on the surface of unstimulated, paraformaldehyde fixed platelets by immunofluorescence. However, on thrombin-activated platelets, rim immunofluorescence was seen, indicating that activated platelets externalize their calpain. This observation was confirmed by the finding that about 2,000 molecules per platelet of an 125I-anti-calpain II Fab' specifically bound to thrombin-activated but not unstimulated platelets. Both dibucaine (1 mmol/L) and platelet activating factor (1.86 mumol/L) in the absence of external Ca++, but not collagen (5 micrograms/mL) or ionophore A23187 (2.5 mumol/L) in the absence of external Ca++, were also able to externalize platelet calpain II antigen, as indicated by a similar level of specific 125I-anti-calpain II Fab'-platelet binding. These combined studies indicate that platelet calpain II is a major protein, comprising 2% of total platelet protein, a substantial portion of which is membrane-associated. When platelets are activated by thrombin and platelet activating factor, calpain II antigen also becomes present on the external platelet surface.
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He, Ao-Di, Ming-Lu Liang, Gang Liu, Xing-Wen Da, Guang-Qiang Yao, Wen Xie, Ji-Zhou Xiang, Cunji Gao, and Zhang-Yin Ming. "The Role of FcγRIIa and TGF-β1/KLF6 Pathway in Platelet's Promoting Hepatocellular Carcinoma Cells Growth." Blood 124, no. 21 (December 6, 2014): 1429. http://dx.doi.org/10.1182/blood.v124.21.1429.1429.
Full textAbstract:
Abstract Background: Platelet in the primary tumor microenvironment plays a crucial role in tumor cells angiogenesis, growth, and metastasis. Clinical and experimental evidences support that platelets and their extracts influence hepatocellular carcinoma (HCC) growth and biology. But the mechanism is still not fully clarified. The aim of present study was to elucidate an unperceived mechanism of the proliferative effect of platelet on HCC cells. Methods: Human blood was collected from health volunteers, washed platelets were prepared and resuspended by fresh medium. The ability of HepG2 cells to induce platelet aggregation was analyzed using a Chrono-Log Lumi-aggregometer. HepG2 cells were incubated with platelets activated by thrombin (0.08 U/ml) and collagen-related peptide (CRP, 0.8μg/ml), or releasates isolated from CRP-stimulated platelets. The effect of platelet releasate on HepG2 cell proliferation was determined with the colorimetric 3-(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide (MTT) assay. Western blot was used to measure expression of Krüppel-like factor 6 (KLF6) in HepG2 cells. Anti-FcγRIIa monoclonal antibody IV.3 (10μg/ml) and transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542 (10μM) were used. Furthermore, KLF6 gene silence was also conducted in HepG2 cells by transfected with KLF6 siRNA. Results: Our data showed HepG2 cells (1.0×105/ml) could induce human washed platelet (3.0×108/ml) aggregation in vitro, indicating that HepG2 cells could activate platelets. We further verified that releasate from CRP-activated platelets could promote the proliferation of HepG2 cells. Importantly, this effect exhibits on the down expression of KLF6 in HepG2 cells. In presence and absence of platelet stimulator thrombin (0.08 U/ml) or collagen-related peptide (CRP, 0.8μg/ml), washed platelets could reduce KLF6 expression in HepG2 cells after incubated for 12 and 24 hours. Meanwhile, supernatant from CRP-activated platelets exhibited the same effect. On the other hand, the resuspended CRP-activated platelet pellet showed no significant influence on KLF6 expression. And platelets incubated with anti- FcγRIIa monoclonal antibody IV.3 (10μg/ml) and transforming growth factor beta 1 (TGF-β1) receptor inhibitor SB431542 (10μM) abolished the effects. Furthermore, the platelet’s promoting proliferation effect was attenuated in HepG2 cells with silencing KLF6 expression. Conclusion: Tumor cells could activate platelet, and activated platelet could regulate cancer cell progression in turn. We further verified that platelet, a main source of bioavailable TGF-β1, has a promoting effect on the proliferation of HepG2 cells. Importantly, this effect exhibits on the down expression of KLF6 in HepG2 cells, in which FcγRIIa and TGF-β1 involved. These results extend our understanding of mechanisms by which platelets contribute to tumor progression, which may provide a new therapeutic target for the prevention and treatment of HCC. Disclosures No relevant conflicts of interest to declare.
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Parker, RI, and HR Gralnick. "Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets." Blood 74, no. 6 (November 1, 1989): 2016–21. http://dx.doi.org/10.1182/blood.v74.6.2016.2016.
Full textAbstract:
Abstract Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
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Parker, RI, and HR Gralnick. "Effect of aspirin on platelet-von Willebrand factor surface expression on thrombin and ADP-stimulated platelets." Blood 74, no. 6 (November 1, 1989): 2016–21. http://dx.doi.org/10.1182/blood.v74.6.2016.bloodjournal7462016.
Full textAbstract:
Platelets contain a pool of endogenous platelet-von Willebrand factor (vWF) that becomes expressed on the platelet surface when platelets are stimulated by a variety of agonists. Maximal platelet-vWF expression occurs in concert with platelet alpha-granule secretion. Aspirin (ASA) is known to impair platelet activation and alpha-granule secretion by irreversible inhibition of platelet cyclo-oxygenase. We studied native and ASA-treated platelets for their ability to mobilize and to express platelet-vWF in response to adenosine diphosphate (ADP) or thrombin. We found that each agonist was effective in promoting increased platelet- vWF surface expression on native and ASA-treated platelets. ASA-treated platelets responded identically to native platelets to low (0.01 U/mL) and high (1.0 U/mL) concentrations of thrombin, while the ADP-induced increase in ASA-treated platelets was only 50% to 60% of that for control platelets. Measurement of secreted platelet-vWF and beta- thromboglobulin indicated that the increase seen with ADP was largely independent of alpha-granule secretion. Using monoclonal antibodies (MoAbs) against the platelet glycoproteins (GP) IIb/IIIa and Ib (MoAbs 10E5 and 6D1, respectively), we demonstrated that the ADP-induced increase in platelet-vWF expression on control platelets primarily involved the binding of secreted platelet-vWF to the platelet GPIIb/IIIa. In contrast, the increase in platelet-vWF that occurred following ADP stimulation of ASA-treated platelets was largely insensitive to GPIIb/IIIa blockade. No effect of GPIb blockade in platelet-vWf expression was noted for either control or ASA-treated platelets. When platelet shape change was prevented by the addition of cytochalasin D, ADP-induced platelet-vWf surface expression on ASA- treated platelets was reduced by more than 80%. Our data indicate that platelets in which the cyclooxygenase pathway is blocked by the action of aspirin can increase surface expression of platelet-vWf as a consequence of platelet shape change. We speculate that this process exposes platelet-vWf bound to GPIIb/IIIa, or possibly GPIb, within the surface connected canalicular system.
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Goodnough, Lawrence T., David J. Kuter, Jeffrey McCullough, Sherrill J. Slichter, John DiPersio, John Romo, Randolph Peterson, et al. "Prophylactic platelet transfusions from healthy apheresis platelet donors undergoing treatment with thrombopoietin." Blood 98, no. 5 (September 1, 2001): 1346–51. http://dx.doi.org/10.1182/blood.v98.5.1346.
Full textAbstract:
Many patients receiving dose-intensive chemotherapy acquire thrombocytopenia and need platelet transfusions. A study was conducted to determine whether platelets harvested from healthy donors treated with thrombopoietin could provide larger increases in platelet counts and thereby delay time to next platelet transfusion compared to routinely available platelets given to thrombocytopenic patients. Community platelet donors received either 1 or 3 μg/kg pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) or placebo and then donated platelets 10 to 15 days later. One hundred sixty-six of these platelet concentrates were then transfused to 120 patients with platelets counts 25 × 109/L or lower. Pretransfusion platelet counts (11 × 109/L) were similar for recipients of placebo-derived and PEG-rHuMGDF–derived platelets. Early after transfusion, the median platelet count increment was higher in patients receiving PEG-rHuMGDF–derived platelets: 19 (range, −12-66) × 109/L, 41 (range, 5-133) × 109/L, and 82 (range, −4-188) × 109/L for placebo-, 1-μg/kg–, and 3-μ/kg–derived platelets, respectively. This difference was maintained 18 to 24 hours after transfusion. Transfusion-free intervals were 1.72, 2.64, and 3.80 days for the recipients of the placebo-, 1-μg/kg–, and 3-μ/kg–derived platelets, respectively. The rate of transfusion-related adverse events was not different in recipients of placebo-derived and PEG-rHuMGDF–derived platelets. Therefore, when transfused into patients with thrombocytopenia, platelets collected from healthy donors undergoing thrombopoietin therapy were safe and resulted in significantly greater platelet count increments and longer transfusion-free intervals than platelets obtained from donors treated with placebo.
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Papkoff, J., R. H. Chen, J. Blenis, and J. Forsman. "p42 mitogen-activated protein kinase and p90 ribosomal S6 kinase are selectively phosphorylated and activated during thrombin-induced platelet activation and aggregation." Molecular and Cellular Biology 14, no. 1 (January 1994): 463–72. http://dx.doi.org/10.1128/mcb.14.1.463-472.1994.
Full textAbstract:
Human platelets provide an excellent model system for the study of phosphorylation events during signal transduction and cell adhesion. Platelets are terminally differentiated cells that exhibit rapid phosphorylation of many proteins upon agonist-induced activation and aggregation. We have sought to identify the kinases as well as the phosphorylated substrates that participate in thrombin-induced signal transduction and platelet aggregation. In this study, we have identified two forms of mitogen-activated protein kinase (MAPK), p42mapk and p44mapk, in platelets. The data demonstrate that p42mapk but not p44mapk becomes phosphorylated on serine, threonine, and tyrosine during platelet activation. Immune complex kinase assays, gel renaturation assays, and a direct assay for MAPK activity in platelet extracts all support the conclusion that p42mapk but not p44mapk shows increased kinase activity during platelet activation. The activation of p42mapk, independently of p44mapk, in platelets is unique since in other systems, both kinases are coactivated by a variety of stimuli. We also show that platelets express p90rsk, a ribosomal S6 kinase that has previously been characterized as a substrate for MAPK. p90rsk is phosphorylated on serine in resting platelets, and this phosphorylation is enhanced upon thrombin-induced platelet activation. Immune complex kinase assays demonstrate that the activity of p90rsk is markedly increased during platelet activation. Another ribosomal S6 protein kinase, p70S6K, is expressed by platelets but shows no change in kinase activity upon platelet activation with thrombin. Finally, we show that the increased phosphorylation and activity of both p42mapk and p90rsk does not require integrin-mediated platelet aggregation. Since platelets are nonproliferative cells, the signal transduction pathways that include p42mapk and p90rsk cannot lead to a mitogenic signal and instead may regulate cytoskeletal or secretory changes during platelet activation.
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Sloand, EM, DM Kenney, FC Chao, J. Lawler, and JL Tullis. "Platelet antithrombin defect in malignancy: platelet protein alterations." Blood 69, no. 2 (February 1, 1987): 479–85. http://dx.doi.org/10.1182/blood.v69.2.479.479.
Full textAbstract:
Abstract Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.
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Sloand, EM, DM Kenney, FC Chao, J. Lawler, and JL Tullis. "Platelet antithrombin defect in malignancy: platelet protein alterations." Blood 69, no. 2 (February 1, 1987): 479–85. http://dx.doi.org/10.1182/blood.v69.2.479.bloodjournal692479.
Full textAbstract:
Sixty-eight patients with malignant disease were divided into two groups based on the results of the platelet antithrombin test (PAT). The normal group had a PAT clotting time ranging from 21.4 to 29.8 seconds, which was equivalent to 25% to 65% inactivation of the 2 U of thrombin added to the test system. The other group showed abnormal PAT clotting time, less than 21.4 seconds or less than 25% thrombin inactivation. The polypeptide composition of platelets from the two patient groups was analyzed by sodium dodecyl sulfate (SDS)- electrophoresis on 7.5% polyacrylamide gels. A polypeptide of 180,000 apparent mol wt was decreased or absent in both Coomassie blue- and Alcian blue-stained gels of the platelets from patients whose PAT was abnormal; this polypeptide comigrated with purified platelet thrombospondin. Tritium labeling of platelet surface glycoproteins by the periodate-borohydride method followed by two-dimensional electrophoresis was performed on platelets of seven patients with abnormal PAT. When they were compared with ten patients with normal PAT, a glycoprotein of 140,000 apparent mol wt with a pl of 4.5 to 5.2 was decreased in platelets of all seven patients with abnormal PAT. Nitrocellulose replicas of one-dimensional gels of platelets from 13 of 14 patients with abnormal PAT showed decreased reaction with an anti- human platelet glycocalicin antiserum. Platelets of these same patients also showed a decreased or absent platelet agglutination induced by ristocetin. Patients with normal PAT had a mean agglutination slope of 1.25 +/- 0.6 (n = 26) as compared with 0.37 +/- 0.34 (n = 26) for the abnormal PAT group (P less than .001). Results indicate that platelets from a subpopulation of tumor patients characterized by decreased platelet antithrombin activity have alterations in two platelet glycoproteins, identified as GPIb and thrombospondin.
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Battinelli, Elisabeth M. "Platelet and Megakaryocytic Regulation of Tumor Progression." Blood 130, Suppl_1 (December 7, 2017): SCI—26—SCI—26. http://dx.doi.org/10.1182/blood.v130.suppl_1.sci-26.sci-26.
Full textAbstract:
Traditionally viewed as the bandaids of the blood, the contribution of platelets to the progression of malignancy is emerging as a compelling focus for therapeutic intervention. Complex interactions between tumor cells, and circulating platelets play an important role in tumor growth and dissemination, and a growing body of data supports a role for platelet activation and release of chemokines in metastases and neovascularization. Supporting this concept is the evidence that elevated platelet counts (thrombocytosis) at time of diagnosis with malignancy is a harbinger of an aggressive cancer with a poor prognosis. One very interesting and provocative connection between cancer and platelets is the increasing evidence that tumor cells hijack platelets to promote a more pro-malignant phenotype to drive disease progression. Our laboratories have been instrumental in establishing the pro-malignant role of platelets in metastasis and neovascularization. We have demonstrated that tumor cells can instruct platelets to release key cytokines that promote angiogenesis and metastasis of tumor cells. Perhaps the most compelling clinical evidence of the link between platelets and malignancy is the finding that anti-platelet agents can have a profound impact on malignancy. We have demonstrated previously, anti-platelet agents such as aspirin and anticoagulants suppress release of key neovascularization factors from platelets and suppress the neovascularization potential. Aspirin also suppresses the invasive properties of platelets in mouse metastasis models as well as in vitro metastasis assays. Similarly, we have also demonstrated that tamoxifen, a selective estrogen receptor modulator often used to treat breast cancer, can also diminish the ability of platelets to support malignancy by diminishing the platelet's role in promoting neovascularization as well as metastasis. Although much is understood regarding how tumors communicate with platelets less is understood about how platelets manipulate tumor cells. Our laboratory has elucidated the role of key chemokines released from platelets in response to tumor cells and how these factors promote tumor growth and metastasis. We have recently discovered that tumor cells can instruct platelets to release CCL5, a known driver of tumor cell invasion and metastasis, and have expanded the role of CCL5 not only as a regulator of metastasis but also as a central controller of platelet production. Despite this progress many questions still remain regarding the interaction between tumor cells and platelets. We are particularly interested in how tumor cells instruct megakaryocytes to increase platelet production. In addition malignancy may also reprogram megakaryocytes thereby manipulating the platelet phenotype to support tumor growth and metastasis. Because most cancer therapies focus on the tumor itself, the idea of targeting platelets in the tumor microenvironment to arrest tumor growth and metastatic spread represents a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.
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Liu, Fang, Graciela Gamez, David R. Myers, Wilbur Lam, and Shawn M. Jobe. "Mitochondrially-Mediated Platelet Integrin AlphaIIbbeta3 Inactivation Limits Platelet Recruitment and Thrombus Growth,." Blood 118, no. 21 (November 18, 2011): 3251. http://dx.doi.org/10.1182/blood.v118.21.3251.3251.
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Abstract Abstract 3251 Platelet stimulation with strong agonist(s) results in the formation of a phenotypically unique platelet subpopulation known as procoagulant platelets. All platelets stimulated in strongly-activating conditions undergo granule release, activation of integrin αIIbβ3, and spreading. However, minutes later a subpopulation of these activated platelets undergo additional phenotypic changes including phosphatidylserine (PS) exposure, adoption of a vesiculated and balloon-like morphology, and an altered integrin αIIbβ3 conformation that has a decreased binding affinity for activation-dependent antibodies such as PAC-1 or JON/A. Deletion of the mitochondrial protein cyclophilin D (CypD), a critical regulator of mitochondrial permeability transition pore (mPTP) formation, results in marked abrogation of procoagulant platelet formation. We have previously demonstrated remarkable impairment of dual-agonist-initiated PS exposure and procoagulant activity in CypD−/− platelets (Jobe et al, Blood 2008). In this study the functional importance of the morphological and adhesive changes that occur in procoagulant platelets were examined using CypD−/− platelets and mice with platelet-specific deficiency of CypD. (CypDplt−/−). The morphology and phenotype of fibrinogen-adherent CypD+/+ (WT) and CypD−/− platelets were examined following dual stimulation with thrombin and the GPVI agonist convulxin. In dual-stimulated WT platelets, platelets initially spread; then after three minutes, nearly half of the platelets demonstrated lamellopodial and filipodial retraction with platelet rounding and vesiculation, and these changes were associated with increased PS exposure and decreased binding of JON/A. These changes were delayed in CypD−/− platelets, but eventually started to be seen 30 minutes subsequent to stimulation. Adhesion and platelet recruitment to strongly-stimulated platelets were studied in flow conditions. Increased platelet accumulation was noted when unstimulated platelets were flowed over strongly-stimulated adherent CypD−/− platelets relative to WT platelets. Similarly, increased platelet accumulation of CypD−/− platelets was noted on a collagen surface compared to WT platelets. In platelet aggregation assays, dual stimulated WT platelets demonstrated a chaotic pattern of aggregation two to three minutes after activation consistent with aggregate disruption, a pattern that was not observed in CypD−/− platelets. Together these studies indicate that procoagulant platelet formation results in integrin inactivation. Since this process is impaired in the absence of CypD, CypD−/− platelets demonstrate increased platelet accumulation in flow assays and stable aggregate formation. Adhesive and cytoskeletal proteins were investigated in strongly-stimulated platelets. Western blot analysis demonstrated significant proteolytic cleavage of both talin and the cytoplasmic domain of integrin β3 in WT platelets stimulated with thrombin plus convulxin, and these events were only minimally observed in single-agonist stimulated platelets. Proteolytic cleavage of both talin and integrin β3 were markedly decreased in CypD−/− platelets and in WT platelets treated with calpain inhibitors. CypD is a ubiquitously expressed protein; therefore, in vivo thrombosis and hemostasis were tested in CypDplt−/− mice. Following photochemically mediated mesenteric endothelial injury, time to occlusion was shortened in CypDplt−/− mice (723 ± 111 sec in CypDplt+/− vs. 371 ± 106 sec in CypDplt−/− mice), and complete arterial occlusion was increased in CypDplt−/− mice (55% in CypDplt+/− vs. 86% in CypDplt−/−) (p=0.02). Our in vitro and in in vivo results are consistent with the hypothesis that in strongly-stimulated platelets mitochondrially-mediated events initiate proteolytic cleavage of talin and integrin β3 accompanied by platelet integrin αIIbβ3 inactivation, and that this process limits platelet recruitment and thrombus growth. Since these events are limited to platelets stimulated by strong agonist(s), these results suggest a novel negative feedback mechanism initiated by accumulation of multiple or strong agonist(s) that limits thrombotic occlusion in vivo. Disclosures: No relevant conflicts of interest to declare.
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Polak, Dawid, Marcin Talar, Cezary Watala, and Tomasz Przygodzki. "Intravital Assessment of Blood Platelet Function. A Review of the Methodological Approaches with Examples of Studies of Selected Aspects of Blood Platelet Function." International Journal of Molecular Sciences 21, no. 21 (November 6, 2020): 8334. http://dx.doi.org/10.3390/ijms21218334.
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Platelet biology owes to intravital studies not only a better understanding of platelets’ role in primary hemostasis but also findings that platelets are important factors in inflammation and atherosclerosis. Researchers who enter the field of intravital platelet studies may be confused by the heterogeneity of experimental protocols utilized. On the one hand, there are a variety of stimuli used to activate platelet response, and on the other hand there are several approaches to measure the outcome of the activation. A number of possible combinations of activation factors with measurement approaches result in the aforementioned heterogeneity. The aim of this review is to present the most often used protocols in a systematic way depending on the stimulus used to activate platelets. By providing examples of studies performed with each of the protocols, we attempt to explain why a particular combination of stimuli and measurement method was applied to study a given aspect of platelet biology.
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O'Sullivan, Brian P., Matthew D. Linden, Andrew L. Frelinger, Marc R. Barnard, Michele Spencer-Manzon, James E. Morris, Raneem O. Salem, Michael Laposata, and Alan D. Michelson. "Platelet activation in cystic fibrosis." Blood 105, no. 12 (June 15, 2005): 4635–41. http://dx.doi.org/10.1182/blood-2004-06-2098.
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Abstract Cystic fibrosis (CF) is caused by a mutation of the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). We examined platelet function in CF patients because lung inflammation is part of this disease and platelets contribute to inflammation. CF patients had increased circulating leukocyte-platelet aggregates and increased platelet responsiveness to agonists compared with healthy controls. CF plasma caused activation of normal and CF platelets; however, activation was greater in CF platelets. Furthermore, washed CF platelets also showed increased reactivity to agonists. CF platelet hyperreactivity was incompletely inhibited by prostaglandin E1 (PGE1). As demonstrated by Western blotting and reverse-transcriptase-polymerase chain reaction (RT-PCR), there was neither CFTR nor CFTR-specific mRNA in normal platelets. There were abnormalities in the fatty acid composition of membrane fractions of CF platelets. In summary, CF patients have an increase in circulating activated platelets and platelet reactivity, as determined by monocyte-platelet aggregation, neutrophil-platelet aggregation, and platelet surface P-selectin. This increased platelet activation in CF is the result of both a plasma factor(s) and an intrinsic platelet mechanism via cyclic adenosine monophosphate (cAMP)/adenylate cyclase, but not via platelet CFTR. Our findings may account, at least in part, for the beneficial effects of ibuprofen in CF. (Blood. 2005;105:4635-4641)
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Fressinaud, E., D. Baruch, C. Rothschild, HR Baumgartner, and D. Meyer. "Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen." Blood 70, no. 4 (October 1, 1987): 1214–17. http://dx.doi.org/10.1182/blood.v70.4.1214.1214.
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Abstract Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.
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Fressinaud, E., D. Baruch, C. Rothschild, HR Baumgartner, and D. Meyer. "Platelet von Willebrand factor: evidence for its involvement in platelet adhesion to collagen." Blood 70, no. 4 (October 1, 1987): 1214–17. http://dx.doi.org/10.1182/blood.v70.4.1214.bloodjournal7041214.
Full textAbstract:
Although it is well established that plasma von Willebrand Factor (vWF) is essential to platelet adhesion to subendothelium at high shear rates, the role of platelet vWF is less clear. We studied the respective role of both plasma and platelet vWF in mediating platelet adhesion to fibrillar collagen in a parallel-plate perfusion chamber. Reconstituted blood containing RBCs, various mixtures of labeled washed platelets and plasma from controls or five patients with severe von Willebrand disease (vWD), was perfused through the chamber for five minutes at a shear rate of 1,600 s-1. Platelet-collagen interactions were estimated by counting the radioactivity in deposited platelets and by quantitative morphometry. When the perfusate consisted of normal platelets suspended in normal plasma, platelet deposition on the collagen was 24.7 +/- 3.6 X 10(6)/cm2 (mean +/- SEM, n = 6). Significantly less deposition (16 +/- 2.3) was observed when vWD platelets were substituted for normal platelets. In mixtures containing vWD plasma, significantly greater deposition (9 +/- 2.2) was obtained with normal than with vWD platelets (1 +/- 0.4) demonstrating a role for platelet vWF in mediating the deposition of platelets on collagen. Morphometric analysis confirmed these data. Our findings indicate that platelet, as well as plasma, vWF mediates platelet-collagen interactions at a high shear rate.
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Hardisty, R. "Platelet Heterogeneity. Biology and Pathology." Journal of Clinical Pathology 44, no. 5 (May 1, 1991): 440. http://dx.doi.org/10.1136/jcp.44.5.440-c.
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Koseoglu, Secil, and Robert Flaumenhaft. "Advances in platelet granule biology." Current Opinion in Hematology 20, no. 5 (September 2013): 464–71. http://dx.doi.org/10.1097/moh.0b013e3283632e6b.
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Luo, Xu‐ling, Jin‐yong Jiang, Zhen Huang, and Lin‐xi Chen. "Autophagic regulation of platelet biology." Journal of Cellular Physiology 234, no. 9 (February 4, 2019): 14483–88. http://dx.doi.org/10.1002/jcp.28243.
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Cimmino, Giovanni, and Paolo Golino. "Platelet Biology and Receptor Pathways." Journal of Cardiovascular Translational Research 6, no. 3 (January 10, 2013): 299–309. http://dx.doi.org/10.1007/s12265-012-9445-9.
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Moers, Alexandra, Nina Wettschureck, Sabine Grüner, Bernhard Nieswandt, and Stefan Offermanns. "Unresponsiveness of Platelets Lacking Both Gαqand Gα13." Journal of Biological Chemistry 279, no. 44 (August 23, 2004): 45354–59. http://dx.doi.org/10.1074/jbc.m408962200.
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The diffusible platelet stimuli ADP and thromboxane A2activate multiple G protein-mediated signaling pathways and function as important secondary mediators of platelet activation as they are released from activated platelets. Because they can also increase their own formation and release, their effects are amplified; eventually, all major G protein-mediated signaling pathways are activated. The multiple positive feedback mechanisms operating during platelet activation have obscured the exact analysis of the roles individual G protein-mediated signaling pathways play during the platelet activation process. In this report, we show that platelets lacking Gqand G13are completely unresponsive to diffusible stimuli such as ADP, thromboxane A2, or thrombin, even when applied at very high concentrations in combination, whereas all stimuli are able to induce platelet aggregation, shape change, and RhoA activation in platelets lacking only one Gα subunit. This shows that Gqor G13is required to induce some platelet activation, whereas the activation of Gi-mediated signaling alone is not sufficient to induceactivation of mouse platelets. In addition, platelets lacking Gαqand Gα13adhered normally to collagen under high shearbut did not aggregate any more in response to collagen, indicating that collagen-induced platelet activation but not platelet adhesion requires intact G protein-mediated signaling pathways.
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Cheepala, Satish B., Kazumasa Takenaka, Tamara I. Pestina, Carl W. Jackson, and John D. Schuetz. "The Role of ABC Transporter Abcc4 in Platelets Physiologic Function and Its Impact On Collagen Meditated Platelet Aggregation." Blood 120, no. 21 (November 16, 2012): 1063. http://dx.doi.org/10.1182/blood.v120.21.1063.1063.
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Abstract Abstract 1063 Platelet activation is a highly regulated process, and cyclic nucleotide mediated signaling pathways are crucial to effective platelet activation. Vascular injury produces, exposed collagen which binds circulating platelets through the platelet's “collagen” receptor, GPVI, resulting in the activation of guanyly/adenlyl cyclases. These interactions result in the rapid alterations in the cyclic nucleotide concentration inside the platelets leading to activation of protein kinase A and G signaling pathways to modulate platelet function. While, ABCC4 functions as a plasma membrane transporter for cyclic nucleotides its contribution to platelet activation has been obscured because it was reportedly as primarily intracellular in the platelets dense granules. This original report (Jedlitschky, Tirschmann et al. 2004) evaluated ABCC4 localization by immune-fluorescence of platelets attached to collagen coated coverslips. However, attachment via collagen produces platelet activation leading to mobilization and fusion of alpha and dense granules to the plasma membrane, thus under these conditions distinguishing between plasma membrane and dense granules is not possible. We resolved this problem by labeling quiescent platelets with a cell impermeable biotinylating agent (EZ-Link Sulfo-NHS-LC-LC Biotin). Isolation of membrane and internal fraction demonstrated that of over ninety percent of Abcc4 localizes to the plasma membrane. Furthermore, confocal microscopy of platelets stained with specific antibodies against Abcc4 confirmed Abcc4 localization to the plasma membrane. We extended these studies to the Abcc4- knockout (KO) mouse model. The Abcc4- KO mouse does not have any change in the number of platelet or dense granules compared to the wild type mouse. Platelet activation in vivo can be initiated by interaction with collagen through the GPVI receptor that is expressed at the plasma membrane of the platelets. At the molecular level, the initiation of platelet activation by collagen results in an increase in the cyclic nucleotide concentration leading to activation of signaling cascade through protein kinase A or G. Expose of Abcc4-KO platelets to collagen and revealed impaired activation in response to collagen. However, Abcc4-KO platelets activated by either thrombin or ADP (which activate either G-coupled PAR receptors or P2Y12 receptor respectively) shows an aggregation profile almost identical to wildtype platelets, thus indicating the defect in Abcc4 -KO platelet aggregation is specific to the collagen pathway. To understand the basis for the impaired collagen aggregation of Abcc4-KO platelets, we investigated the collagen receptor (GPVI) signaling pathway in Abcc4-KO platelets. Interestingly, in the Abcc4-KO platelets after the platelet activation with collagen, cyclic nucleotide dependent phosphorylation of VASP through protein kinase A or G at Ser-157 or Ser-239 respectively is reduced compared to the wildtype. Notably, Abcc4-KO platelets had reduced GPVI surface expression that correlated with the reduced phosphorylation of VASP after collagen stimulation. The similar, protein levels of Syk and Plcg2, (downstream signaling molecules of GPVI signaling pathway), in the Abcc4 wildtype and KO platelets implies that GPVI expression is the primary defect in Abcc4 deficiency. These results suggest that Abcc4 plays a crucial role in regulating cyclic nucleotides in response to GPVI activation by collagen. These findings suggest ABCC4/Mrp4 loss of function or inhibition (by drugs) may disrupt platelet aggregation under conditions of vascular injury. As, many antiplatelet drugs are potent inhibitors of Abcc4 (e.g., Dipyridamole and Sildenafil) these conclusions have strong implications for not just the development of antiplatelet drugs, but also for further exploring the role of Abcc4 in regulating intracellular nucleotide levels and platelet biology. Disclosures: No relevant conflicts of interest to declare.
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Quach, M. Edward, Wenchun Chen, and Renhao Li. "Mechanisms of platelet clearance and translation to improve platelet storage." Blood 131, no. 14 (April 5, 2018): 1512–21. http://dx.doi.org/10.1182/blood-2017-08-743229.
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Abstract Hundreds of billions of platelets are cleared daily from circulation via efficient and highly regulated mechanisms. These mechanisms may be stimulated by exogenous reagents or environmental changes to accelerate platelet clearance, leading to thrombocytopenia. The interplay between antiapoptotic Bcl-xL and proapoptotic molecules Bax and Bak sets an internal clock for the platelet lifespan, and BH3-only proteins, mitochondrial permeabilization, and phosphatidylserine (PS) exposure may also contribute to apoptosis-induced platelet clearance. Binding of plasma von Willebrand factor or antibodies to the ligand-binding domain of glycoprotein Ibα (GPIbα) on platelets can activate GPIb-IX in a shear-dependent manner by inducing unfolding of the mechanosensory domain therein, and trigger downstream signaling in the platelet including desialylation and PS exposure. Deglycosylated platelets are recognized by the Ashwell-Morell receptor and potentially other scavenger receptors, and are rapidly cleared by hepatocytes and/or macrophages. Inhibitors of platelet clearance pathways, including inhibitors of GPIbα shedding, neuraminidases, and platelet signaling, are efficacious at preserving the viability of platelets during storage and improving their recovery and survival in vivo. Overall, common mechanisms of platelet clearance have begun to emerge, suggesting potential strategies to extend the shelf-life of platelets stored at room temperature or to enable refrigerated storage.
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Sen Gupta, Anirban. "Synthetic Platelets for Treatment of Traumatic Hemorrhage and Thrombocytopenia." Blood 134, Supplement_1 (November 13, 2019): SCI—37—SCI—37. http://dx.doi.org/10.1182/blood-2019-121079.
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Platelets are primarily responsible for staunching bleeding by forming a 'platelet plug' and further amplifying thrombin generation on its surface to facilitate fibrin formation, leading to hemostatic clot formation at the site of vascular breach. Therefore, platelet transfusions are clinically used to mitigate bleeding risks in thrombocytopenia (prophylactic transfusion) and to mitigate hemorrhage in traumatic injuries (emergency transfusion). Currently these transfusions utilize donor-derived platelets, stored at 20-24oC with gentle agitation. In this condition, platelets have high risk of bacterial contamination and very short shelf-life (~ 5 days), which severely limit their logistical availability and use. Several parallel strategies are currently undergoing research to address these issues, including platelet storage at reduced temperatures (chilled or freeze-dried), pathogen reduction technologies and bioreactor-based in vitro platelet production from precursor cells. An alternative (and complimentary) approach that is the focus of our research is the engineering of I.V.-administrable synthetic hemostat nanoparticles that functionally mimic platelet's clotting mechanisms. These 'synthetic platelet' nanoparticle systems can be manufactured at large scale, sterilized without compromising functions and stored for long periods of time (6-9 months), thereby allowing significant logistical advantages in transfusion applications. Here we present in vitro and in vivo evaluation of such technology. For these studies, the 'synthetic platelet' nanoparticles were manufactured by decorating liposomes with a combination of VWF-binding, collagen-binding and fibrinogen-mimetic peptides, for integrative mimicry of platelet's hemostasis-relevant adhesive and aggregatory mechanisms. The nanoparticles were stored at room temperature in aqueous suspension as well as lyophilized powder, and particle stability was assessed over 6-9 months by dynamic light scattering (DLS). The nanoparticles were also exposed to E-beam sterilization, and particle stability as well platelet-mimetic bioactivity was assessed by DLS, aggregometry, microfluidics and rotational thromboelastometry (ROTEM). The systemic safety and targeted hemostatic efficacy of I.V.-administered nanoparticles were evaluated in mouse model of thrombocytopenia, and in mouse, rat and pig models of traumatic hemorrhage. DLS and electron microscopy confirmed that the synthetic platelet nanoparticles have a size of 150-200 nm diameter, and they remain stable over 6-9 months in storage. Microfluidic studies showed that these nanoparticles could rapidly adhere to 'vWF + collagen'-coated surfaces and enhance the recruitment and aggregation of active platelets on these surfaces. Aggregometry studies showed that the nanoparticles did not affect resting platelets but enhanced aggregation of ADP- or collagen-activated platelets (i.e. no thrombotic risk towards resting platelets). Flow cytometry studies confirmed this specificity of nanoparticle binding to active platelets. ROTEM studies showed that the 'synthetic platelet' nanoparticles significantly improved clot kinetics and firmness. In vivo, in all animal models, the nanoparticles showed no systemic pro-thrombotic effects, as assessed by hemodynamics as well as organ histology. In thrombocytopenic mice, prophylactically administered 'synthetic platelet' nanoparticles dose-dependently reduced tail bleeding time. In mouse, rat and pig trauma models, post-injury administration of 'synthetic platelet' nanoparticles reduced blood loss, stabilized blood pressure, delayed hypotension and thereby significantly improved survival. The nanoparticles could be further utilized as a platform for targeted presentation of phosphatidylserine (PS) to augment thrombin generation, or targeted delivery of tranexamic acid (TXA) for anti-fibrinolytic effect or delivery of inorganic polyphosphate (PolyP) to augment clot stability. These studies not only establish the potential of these nanoparticles as a platelet surrogate for transfusion applications, but also demonstrate their utilization as a platform for modular augmentation of various hemostatic outputs in prophylactic and emergency applications. Figure Disclosures Sen Gupta: Haima Therapeutics LLC: Equity Ownership.
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Tuszynski, GP, VL Rothman, A. Murphy, K. Siegler, and KA Knudsen. "Thrombospondin promotes platelet aggregation." Blood 72, no. 1 (July 1, 1988): 109–15. http://dx.doi.org/10.1182/blood.v72.1.109.109.
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Abstract Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.
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Tuszynski, GP, VL Rothman, A. Murphy, K. Siegler, and KA Knudsen. "Thrombospondin promotes platelet aggregation." Blood 72, no. 1 (July 1, 1988): 109–15. http://dx.doi.org/10.1182/blood.v72.1.109.bloodjournal721109.
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Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.
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Krötz, Florian, Hae Young Sohn, Torsten Gloe, Stefan Zahler, Tobias Riexinger, Thomas M. Schiele, Bernhard F. Becker, Karl Theisen, Volker Klauss, and Ulrich Pohl. "NAD(P)H oxidase–dependent platelet superoxide anion release increases platelet recruitment." Blood 100, no. 3 (August 1, 2002): 917–24. http://dx.doi.org/10.1182/blood.v100.3.917.
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Abstract Platelets, although not phagocytotic, have been suggested to release O2−. Since O2−-producing reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases can be specifically activated by certain agonists and are found in several nonphagocytotic tissues, we investigated whether such an enzyme is the source of platelet-derived O2−. We further studied which agonists cause platelet O2−release and whether platelet-derived O2− influences thrombus formation in vitro. Collagen, but not adenosine 5′-diphosphate (ADP) or thrombin, increased O2− formation in washed human platelets. This was a reduced nicotinamide adenine dinucleotide (NADH)–dependent process, as shown in platelet lysates. Consistent with a role of a platelet, NAD(P)H oxidase expression of its subunits p47phox and p67phoxand inhibition of platelet O2− formation by diphenylene-iodoniumchloride (DPI) and by the specific peptide-antagonist gp91ds-tat were observed. Whereas platelet-derived O2− did not influence initial aggregation, platelet recruitment to a preformed thrombus following collagen stimulation was significantly attenuated by superoxide dismutase (SOD) or DPI. It was also inhibited when ADP released during aggregation was cleaved by the ectonucleotidase apyrase. ADP in supernatants of collagen-activated platelets was decreased in the presence of SOD, resulting in lower ADP concentrations available for recruitment of further platelets. Exogenous O2−increased ADP- concentrations in supernatants of collagen-stimulated platelets and induced irreversible aggregation when platelets were stimulated with otherwise subthreshold concentrations of ADP. These results strongly suggest that collagen activation induces NAD(P)H oxidase–dependent O2− release in platelets, which in turn enhances availability of released ADP, resulting in increased platelet recruitment.