Journal articles on the topic 'Platelet activation'
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1
Zhou, W., M. A. Javors, and M. S. Olson. "Platelet-activating factor as an intercellular signal in neutrophil-dependent platelet activation." Journal of Immunology 149, no. 5 (September 1, 1992): 1763–69. http://dx.doi.org/10.4049/jimmunol.149.5.1763.
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Abstract The role of platelet-activating factor (PAF) in heterotypic cell to cell interactions in a rabbit neutrophil-platelet mixture model was investigated. Platelets were exposed to each of three chemotactic agonists: PAF, leukotriene B4 (LTB4), or FMLP. Only PAF stimulated aggregation, [3H]serotonin secretion, and cytosolic Ca2+ mobilization in platelets alone. However, platelets were stimulated by LTB4 and FMLP in the presence of neutrophils. This neutrophil-dependent platelet activation was blocked by pretreatment of platelets with PAF receptor antagonists, and was prevented by desensitization of platelets to PAF. Furthermore, the time-course of platelet activation showed a positive correlation with PAF production by neutrophils stimulated with either LTB4 or FMLP. The PAF-mediated neutrophil-platelet interaction was dependent on direct cell to cell contact, as demonstrated by experiments in which the majority of newly formed PAF was neutrophil associated (rather than released). Platelet activation did not occur when the neutrophil-platelet mixture was not stirred, minimizing cell to cell contact, or when platelets were challenged with a cell-free supernatant prepared from neutrophils activated with LTB4 or FMLP. Finally, the neutrophil-platelet interaction was abolished by SC-49992, a peptidomimetic of the fibrinogen binding sequence Arg-Gly-Asp-Phe, indicating a Arg-Gly-Asp-specific recognition mechanism. Our results demonstrate that neutrophil-generated PAF plays a crucial role in neutrophil-dependent platelet activation in this model system. This type of intercellular signaling event may be important in certain inflammatory or thrombotic processes.
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Horsewood, P., CP Hayward, TE Warkentin, and JG Kelton. "Investigation of the mechanisms of monoclonal antibody-induced platelet activation." Blood 78, no. 4 (August 15, 1991): 1019–26. http://dx.doi.org/10.1182/blood.v78.4.1019.1019.
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Abstract Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).
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Horsewood, P., CP Hayward, TE Warkentin, and JG Kelton. "Investigation of the mechanisms of monoclonal antibody-induced platelet activation." Blood 78, no. 4 (August 15, 1991): 1019–26. http://dx.doi.org/10.1182/blood.v78.4.1019.bloodjournal7841019.
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Antiplatelet antibodies can activate platelets causing platelet aggregation and the release reaction. However, the pathway of activation by these antibodies is unknown and several potential mechanisms are possible. In this report, we describe studies investigating potential pathways of platelet activation by IgG antibodies. We tested 16 different IgG monoclonal antibodies (MoAbs) against a variety of platelet surface components and found that six antibodies were capable of causing platelet aggregation and release. These included MoAbs against glycoprotein (GP) IIb/IIIa, CD9, GPIV, and two other not well-characterized platelet components. There was no relationship between the number of platelet binding sites and the ability of an MoAb to activate the platelets. By adding intact and F(ab')2 preparations of the MoAb to control or Fc receptor-blocked platelets, we found that in all instances the MoAbs initiated platelet activation via interacting with the platelet Fc receptors. Clustering of the platelet protein components using a secondary antibody did not cause activation. Studies into the pathway of Fc-dependent activation demonstrated that the MoAbs were capable of activating platelets by occupying Fc receptors on adjacent platelets (interplatelet activation), as well as on the same platelet (intraplatelet activation).
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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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Speijer, Han, José W. P. Govers-Riemslag, Robert F. A. Zwaal, and Jan Rosing. "Platelet Procoagulant Properties Studied with Snake Venom Prothrombin Activators." Thrombosis and Haemostasis 57, no. 03 (1987): 349–55. http://dx.doi.org/10.1055/s-0038-1651132.
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SummaryPurified snake venom prothrombin activators were used to probe the procoagulant properties of platelet membranes. Human platelets were able to stimulate prothrombin activation by the venom activators from Oxyuranus scutellatus and Notechis scutatus, while the prothrombin activator from Echis carinatus was not affected by the presence of platelets. The prothrombinconverting activity of platelets was further studied with the venom activator from Oxyuranus scutellatus and with the factor Xa-Va complex as prothrombin activating enzymes. Stimulation of platelets with collagen, collagen plus thrombin or with the Ca-ionophore A23187 resulted in a considerable increase of platelet prothrombin converting activity probed with the factor Xa-Va complex as well as with the prothrombin activator from Oxyuranus scutellatus. The stimulatory effect of activated platelets on the rates of prothrombin activation by Oxyuranus scutellatus was similar to that determined for factor Xa-Va-catalyzed prothrombin activation. Compared to non-stimulated platelets, platelets stimulated with thrombin plus collagen exposed 20-times more procoagulant sites for as well the factor Xa-Va complex, as for the venom activator from Oxyuranus scutellatus. The actual number of procoagulant sites per platelet determined with the factor Xa-Va complex was in close agreement with the number of sites determined with the venom activator. Also the time course of appearance of procoagulant activity during platelet stimulation by collagen plus thrombin was comparable for both activator complexes. Phospholipase A2 treatment of stimulated platelets resulted in an almost complete loss of their ability to stimulate prothrombin activation by the enzyme from Oxyuranus scutellatus or by factor Xa-Va complex. The findings presented in this paper suggest: a) that the factor Xa-Va complex and the prothrombin activator from Oxyuranus scutellatus recognize the same procoagulant sites on both stimulated and unstimulated platelets and b) that negatively-charged phospholipids are essential components of these procoagulant sites.
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Quirino-Teixeira, Anna Cecíllia, Stephane Vicente Rozini, Giselle Barbosa-Lima, Diego Rodrigues Coelho, Pedro Henrique Carneiro, Ronaldo Mohana-Borges, Patrícia T. Bozza, and Eugenio D. Hottz. "Inflammatory signaling in dengue-infected platelets requires translation and secretion of nonstructural protein 1." Blood Advances 4, no. 9 (May 11, 2020): 2018–31. http://dx.doi.org/10.1182/bloodadvances.2019001169.
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Abstract Emerging evidence identifies major contributions of platelets to inflammatory amplification in dengue, but the mechanisms of infection-driven platelet activation are not completely understood. Dengue virus nonstructural protein-1 (DENV NS1) is a viral protein secreted by infected cells with recognized roles in dengue pathogenesis, but it remains unknown whether NS1 contributes to the inflammatory phenotype of infected platelets. This study shows that recombinant DENV NS1 activated platelets toward an inflammatory phenotype that partially reproduced DENV infection. NS1 stimulation induced translocation of α-granules and release of stored factors, but not of newly synthesized interleukin-1β (IL-1β). Even though both NS1 and DENV were able to induce pro-IL-1β synthesis, only DENV infection triggered caspase-1 activation and IL-1β release by platelets. A more complete thromboinflammatory phenotype was achieved by synergistic activation of NS1 with classic platelet agonists, enhancing α-granule translocation and inducing thromboxane A2 synthesis (thrombin and platelet-activating factor), or activating caspase-1 for IL-1β processing and secretion (adenosine triphosphate). Also, platelet activation by NS1 partially depended on toll-like receptor-4 (TLR-4), but not TLR-2/6. Finally, the platelets sustained viral genome translation and replication, but did not support the release of viral progeny to the extracellular milieu, characterizing an abortive viral infection. Although DENV infection was not productive, translation of the DENV genome led to NS1 expression and release by platelets, contributing to the activation of infected platelets through an autocrine loop. These data reveal distinct, new mechanisms for platelet activation in dengue, involving DENV genome translation and NS1-induced platelet activation via platelet TLR4.
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Karhausen, Jörn, Hae Woong Choi, Krishna Rao Maddipati, Joseph P. Mathew, Qing Ma, Yacine Boulaftali, Robert Hugh Lee, Wolfgang Bergmeier, and Soman N. Abraham. "Platelets trigger perivascular mast cell degranulation to cause inflammatory responses and tissue injury." Science Advances 6, no. 12 (March 2020): eaay6314. http://dx.doi.org/10.1126/sciadv.aay6314.
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Platelet responses have been associated with end-organ injury and mortality following complex insults such as cardiac surgery, but how platelets contribute to these pathologies remains unclear. Our studies originated from the observation of microvascular platelet retention in a rat cardiac surgery model. Ensuing work supported the proximity of platelet aggregates with perivascular mast cells (MCs) and demonstrated that platelet activation triggered systemic MC activation. We then identified platelet activating factor (PAF) as the platelet-derived mediator stimulating MCs and, using chimeric animals with platelets defective in PAF generation or MCs lacking PAF receptor, defined the role of this platelet-MC interaction for vascular leakage, shock, and tissue inflammation. In application of these findings, we demonstrated that inhibition of platelet activation in modeled cardiac surgery blunted MC-dependent inflammation and tissue injury. Together, our work identifies a previously undefined mechanism of inflammatory augmentation, in which platelets trigger local and systemic responses through activation of perivascular MCs.
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Coughlin, Shaun. "Protease-Activated Receptors and Platelet Function." Thrombosis and Haemostasis 82, no. 08 (1999): 353–56. http://dx.doi.org/10.1055/s-0037-1615853.
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IntroductionPlatelet activation is critical for normal hemostasis, and platelet-dependent arterial thrombosis underlies most myocardial infarctions. Thrombin is the most potent activator of platelets.1,2 For this reason, understanding the process by which thrombin activates platelets is necessary for understanding hemostasis and thrombosis and may yield novel anti-platelet therapies. This chapter focuses on our recent studies of the receptors that mediate activation of human platelets by thrombin.3,4 Thrombin signaling is mediated, at least in part, by a family of G protein-coupled protease-activated receptors (PARs), for which PAR1 is the prototype.5,6 PAR1 is activated when thrombin binds to and cleaves its amino terminal exodomain to unmask a new receptor amino terminus.5 This new amino terminus then serves as a tethered peptide ligand, binding intramolecularly to the body of the receptor to effect transmembrane signaling.5,7,8 The synthetic peptide SFLLRN, which mimics the first six amino acids of the new amino terminus unmasked by receptor cleavage, functions as a PAR1 agonist and activates the receptor independent of thrombin and proteolysis.5,9,10 Such peptides have been used as pharmacological probes of PAR function in various cell types.Our understanding of the role of PARs in platelet activation is evolving rapidly. PAR1 mRNA and protein were detected in human platelets,5,11-13 SFLLRN-activated human platelets,5,9,10 and PAR1-blocking antibodies inhibited human platelet activation by low, but not high, concentrations of thrombin.11,12 These data suggested a role for PAR1 in activation of human platelets by thrombin but left open the possibility that other receptors contribute.Curiously, PAR1 appeared to play no role in mouse platelets.14-16 PAR1-activating peptides did not activate rodent platelets, and platelets from PAR1-deficient mice responded like wild-type platelets to thrombin.16 The latter observation prompted a search for additional thrombin receptors and led to the identification of PAR3.17 PAR3 is activated by thrombin and is expressed in mouse platelets. PAR3 blocking antibodies inhibited mouse platelet activation by low, but not high, concentrations of thrombin,18 and knockout of PAR3 abolished mouse platelet responses to low, but not high, concentrations of thrombin.3 These results established that PAR3 is necessary for normal thrombin signaling in mouse platelets but also pointed to the existence of another mouse platelet thrombin receptor. Such a receptor, PAR4, was recently identified.3,19 PAR4 appears to function in both mouse and human platelets.3 The role of PAR3 in human platelets, if any, remains to be determined, and whether still unidentified receptors contribute to thrombin activation of platelets is unknown. Nonetheless, available data suggest a testable, working model in which PAR3 and PAR4 mediate thrombin activation of mouse platelets and PAR1 and PAR4 mediate activation of human platelets.To determine the roles of PAR1, PAR3, and PAR4 in activation of human platelets by thrombin, we examined PAR mRNA and protein expression in platelets and probed PAR function using specific peptide agonists. We also examined the effect of receptor desensitization, receptor blocking antibodies, and a PAR1 antagonist, used alone and in combination, on platelet activation.4
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Bevers, Edouard, Theo Lindhout, and Johan Heemskerk. "Platelet Activation and Blood Coagulation." Thrombosis and Haemostasis 88, no. 08 (2002): 186–93. http://dx.doi.org/10.1055/s-0037-1613209.
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SummaryPlatelet activation and blood coagulation are complementary, mutually dependent processes in haemostasis and thrombosis. Platelets interact with several coagulation factors, while the coagulation product thrombin is a potent platelet-activating agonist. Activated platelets come in a procoagulant state after a prolonged elevation in cytosolic [Ca2+]i. Such platelets, e. g. when adhering to collagen via glycoprotein VI, expose phosphatidylserine (PS) at their outer surface and produce (PS-exposing) membrane blebs and microvesicles. Inhibition of aminophospholipid translocase and activation of phospholipid scramblase mediate the exposure of PS, whereas calpain-mediated protein cleavage leads to membrane blebbing and vesiculation. Surface-exposed PS strongly propagates the coagulation process by facilitating the assembly and activation of tenase and prothrombinase complexes. Factor IXa and platelet-bound factor Va support these activities. In addition, platelets can support the initiation phase of coagulation by providing binding sites for prothrombin and factor XI. They thereby take over the initiating role of tissue factor and factor VIIa in coagulation activation.
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Sadoul, Karin, Jin Wang, Boubou Diagouraga, Anne-Laure Vitte, Thierry Buchou, Thérèse Rossini, Benoît Polack, Xiaodong Xi, Patrick Matthias, and Saadi Khochbin. "HDAC6 controls the kinetics of platelet activation." Blood 120, no. 20 (November 15, 2012): 4215–18. http://dx.doi.org/10.1182/blood-2012-05-428011.
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Abstract HDAC6, a major cytoplasmic deacetylase, is shown here to fine-tune the kinetics of platelet activation, a process that must be precisely regulated to ensure hemostasis after blood vessel injury while preventing pathologic thrombus formation. The discoid shape of resting platelets in the circulation is maintained by several highly acetylated microtubules organized in a marginal band. During platelet activation, microtubules undergo major reorganizations, which contribute to the shape change of activating platelets. We show that, during these activation-induced shape changes, a dramatic HDAC6-mediated tubulin deacetylation takes place, followed by microtubule reacetylation in spread platelets. In addition, although HDAC6-controlled tubulin deacetylation is not required for platelet activation, the capacity of HDAC6 to prevent tubulin hyperacetylation influences the speed of platelet spreading. These results are particularly important in view of HDAC6 inhibitors being currently used in clinical trials and represent the first example of cell signaling by lysine acetylation in platelet biology.
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Rinder, Henry M., Jayne L. Tracey, Christine S. Rinder, David Leitenberg, and Brian R. Smith. "Neutrophil but not Monocyte Activation Inhibits P-Selectin-Mediated Platelet Adhesion." Thrombosis and Haemostasis 72, no. 05 (1994): 750–56. http://dx.doi.org/10.1055/s-0038-1648953.
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SummarySelectins are Ca2+-dependent glycoprotein receptors that mediate the adhesion of activated platelets or endothelial cells to unstimulated leukocytes. Using purified cell fractions, we examined activated neutrophil adhesion to P-selectin-expressing platelets and found that phorbol 12-myristate 13-acetate (PMA), platelet activating factor C16 (PAF), and n-formyl-met-leu-phe (fMLP) pretreatment of neutrophils inhibited activated platelet adhesion. Furthermore, PMA and PAF were capable of dissociating established resting neutrophil-activated platelet conjugates. Since L-selectin is downregulated after leukocyte activation and has been postulated as a ligand for P-selectin, we preincubated resting neutrophils with Dreg-2 and Dreg-56, blocking monoclonal antibodies (MoAb) to L-selectin; these MoAb failed to inhibit activated platelet adhesion. To more closely approximate in vivo conditions of leukocyte and platelet activation, we also employed a whole blood (WB) model of leukocyte-platelet adhesion. We found that simultaneous activation of both platelets and leukocytes by PMA caused an immediate rise in the % of P-selectin-positive platelets accompanied by a rapid increase in monocyte-platelet and neutrophil-platelet conjugates; however, the % of neutrophil-platelet conjugates subsequently declined over 30-60 min to baseline levels while monocyte-platelet adhesion remained elevated over 90 min. By contrast, selective platelet activation in WB by thrombin resulted in an increase in platelet P-selectin expression accompanied by a sustained (90 min) elevation in both monocyte- and neutrophil-platelet conjugates. This increase in leukocyte-platelet conjugates after thrombin was not inhibited by preincubation of WB with Dreg-2 or Dreg-56. We conclude that neutrophil activation decreases the expression of the ligand for platelet P-selectin within 30-60 min resulting in inhibition of neutrophil-platelet adhesion and dissociation of existing neutrophil-platelet conjugates. By contrast, monocyte activation over 90 min does not affect monocyte adhesion to activated platelets in whole blood.
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Stojanovic, Aleksandra, Matvey Gorovoy, Tatyana Voyno-Yasenetskaya, and Xiaoping Du. "The Role of LIM Kinase-1 in the Glycoprotein Ib-IX Complex-Mediated Platelet Activation and Thrombus Formation." Blood 112, no. 11 (November 16, 2008): 112. http://dx.doi.org/10.1182/blood.v112.11.112.112.
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Abstract LIM Kinase (LIMK)-1 is a member of the LIMK family of serine-threonine protein kinases that phosphorylates actin-binding protein cofilin and regulates actin cytoskeleton organization. LIMK1 is expressed in many cell types including platelets but the exact role of LIMK1 in platelet function remains unclear. To determine the role of LIMK1 in platelet activation, wild type or LIMK1 knockout mouse platelets were stimulated with platelet agonists. Platelet aggregation and granule secretion were analyzed. Integrin-dependent second wave of platelet aggregation induced by von Willebrand factor (VWF) in the presence of VWF activator botrocetin was abolished in LIMK1 knockout platelets. In contrast, platelet aggregation in response to the agonist peptide of protease-activated receptor-4 (PAR4, thrombin receptor), ADP and collagen was either not affected or enhanced in LIMK1 knockout platelets in comparison with wild type mouse platelets. Thus, LIMK appears to play an important role in platelet activation stimulated by VWF binding to its platelet receptor, glycoprotein Ib-IX complex (GPIb-IX) but had no stimulatory effect on or negatively regulate the GPIb-IX-independent platelet activation pathways mediated by PAR-4, ADP receptors and collagen receptors. To determine whether ligand binding to GPIb-IX stimulates LIMK activation and function, platelets were stimulated with VWF in the presence of either ristocetin or botrocetin, and immunoblotted with antibodies specifically recognizing phosphorylated LIMK1 (Serine 505) or cofilin (Serine 3). VWF induced phosphorylation of LIMK1 and LIMK substrate cofilin. Thus, VWF indeed stimulates LIMK1 activation and function. An important physiological role of GPIb-IX in platelets is to mediate platelet adhesion to subendothelial-bound VWF under shear stress at sites of vascular injury. To determine whether LIMK1 is important in platelet adhesion, we investigated whether LIMK1 knockout affected platelet adhesion to VWF-coated surfaces. LIMK1 knockout platelets are defective in mediating stable platelet adhesion to vWF under shear stress, suggesting that LIMK1 plays an important role in GPIb signaling and GPIb-IX-mediated integrin activation that is required for stable platelet adhesion under shear stress. Importantly, LIMK1 knockout mice showed significant delay in the formation of occlusive thrombus following FeCl3-induced carotid artery injury in comparison with wild type mice, indicating that the role of LIMK1 in GPIb-IX-mediated platelet activation is important in in vivo thrombosis. Together, our study reveals that LIMK1 plays an important role in GPIb-IX-mediated platelet activation and arterial thrombosis in vitro and in vivo.
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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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Gowert, Nina Sarah, Meike Klier, Maria Reich, Friedrich Reusswig, Lili Donner, Verena Keitel, Dieter Häussinger, and Margitta Elvers. "Defective Platelet Activation and Bleeding Complications upon Cholestasis in Mice." Cellular Physiology and Biochemistry 41, no. 6 (2017): 2133–49. http://dx.doi.org/10.1159/000475566.
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Background/Aims: Platelets are essential mediators of hemostasis to avoid excessive blood loss. Cirrhosis and chronic liver diseases are characterized by alterations in hemostasis. Alterations in the secondary hemostasis have been well studied, while defects in primary hemostasis, especially the consequences of cholestatic liver disease on platelet function are not well defined. Methods: After bile duct ligation (BDL) platelet activation and thrombus formation were analyzed in mice. Results: BDL in mice had a moderate effect on platelet counts; however, intrinsic platelet activation was strongly reduced upon activation of the collagen receptor GPVI at early time points. 7 days after bile duct ligation, platelets displayed an almost complete loss of activation with reduced agonist-triggered release of alpha and dense granules and expression of integrin αIIbβ3 on the platelet surface. This activation defects resulted in strongly reduced thrombus formation under flow, reduced platelet adhesion to fibrinogen and bleeding complications in BDL mice as measured by tail bleeding experiments. Mechanistically, elevated nitric oxide and prostacyclin levels induced phosphorylation of Vasodilator-stimulated phosphoprotein (VASP), an established inhibitor of platelet activation. Furthermore increased tissue plasminogen activator in plasma of BDL mice led to enhanced plasmin levels that might be responsible for reduced glycoprotein expression of BDL platelets. Besides, high amounts of bile acids contribute to defective signal transduction as shown in platelets from mice fed with a cholic acid diet. Conclusions: Cholestatic liver disease induces multiple platelet activation defects and impairs thrombus formation responsible for bleeding complications at least in mice.
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Ed Nignpense, Borkwei, Kenneth A. Chinkwo, Christopher L. Blanchard, and Abishek B. Santhakumar. "Black Sorghum Phenolic Extract Modulates Platelet Activation and Platelet Microparticle Release." Nutrients 12, no. 6 (June 12, 2020): 1760. http://dx.doi.org/10.3390/nu12061760.
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Platelet hyper-activation and platelet microparticles (PMPs) play a key role in the pathogenesis of cardiovascular diseases. Dietary polyphenols are believed to mimic antiplatelet agents by blunting platelet activation receptors via its antioxidant phenomenon. However, there is limited information on the anti-platelet activity of grain-derived polyphenols. The aim of the study is to evaluate the effects of sorghum extract (Shawaya short black 1 variety), an extract previously characterised for its high antioxidant activity and reduction of oxidative stress-related endothelial dysfunction, on platelet aggregation, platelet activation and PMP release. Whole blood samples collected from 18 healthy volunteers were treated with varying non-cytotoxic concentrations of polyphenol-rich black sorghum extract (BSE). Platelet aggregation study utilised 5 µg/mL collagen to target the GPVI pathway of thrombus formation whereas adenine phosphate (ADP) was used to stimulate the P2Y1/P2Y12 pathway of platelet activation assessed by flow cytometry. Procaspase-activating compound 1 (PAC-1) and P-selectin/CD62P were used to evaluate platelet activation- related conformational changes and degranulation respectively. PMPs were isolated from unstimulated platelets and quantified by size distribution and binding to CD42b. BSE treatment significantly reduced both collagen-induced platelet aggregation and circulatory PMP release at 40 µg/mL (p < 0.001) when compared to control. However, there was no significant impact of BSE on ADP-induced activation-dependent conformational change and degranulation of platelets. Results of this study suggest that phenolic rich BSE may confer cardio-protection by modulating specific signalling pathways involved in platelet activation and PMP release.
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OFOSU, Frederick A., John FREEDMAN, Lori DEWAR, Yinqi SONG, and John W. FENTON. "A trypsin-like platelet protease propagates protease-activated receptor-1 cleavage and platelet activation." Biochemical Journal 336, no. 2 (December 1, 1998): 283–85. http://dx.doi.org/10.1042/bj3360283.
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Protease-activated receptor-1 (PAR-1) is a G-protein-linked receptor on platelets and perivascular cells activated by α-thrombin and the PAR-1-activating peptide, SFLLRN. α-Thrombin activates PAR-1 by cleaving it at R41–S42 to release the 41-residue peptide TR(1–41). Unexpectedly, platelet activation with SFLLRN was also associated with PAR-1 cleavage and the release of TR(1–41). Both PAR-1 cleavage and platelet activation resulting from SFLLRN addition to platelets were markedly inhibited by the serine protease inhibitor 4,2-(aminoethyl)-benzene sulphonylfluoride·HCl (pefabloc SC) and soybean trypsin inhibitor, but not by inhibitors of calpain, cysteine proteases or metalloproteases. Thus, a trypsin-like platelet protease propagates SFLLRN-dependent PAR-1 cleavage and platelet activation.
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Winters, KJ, PR Eisenberg, AS Jaffe, and SA Santoro. "Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+." Blood 76, no. 8 (October 15, 1990): 1546–57. http://dx.doi.org/10.1182/blood.v76.8.1546.1546.
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Abstract The effects of activation of plasminogen by streptokinase and tissue- type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca2+, conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca2+. The binding of activation- specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation.
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Winters, KJ, PR Eisenberg, AS Jaffe, and SA Santoro. "Dependence of plasmin-mediated degradation of platelet adhesive receptors on temperature and Ca2+." Blood 76, no. 8 (October 15, 1990): 1546–57. http://dx.doi.org/10.1182/blood.v76.8.1546.bloodjournal7681546.
Full textAbstract:
The effects of activation of plasminogen by streptokinase and tissue- type-plasminogen activator on platelet activation and the membrane glycoproteins (GPs) that mediate platelet adhesion and aggregation are not yet fully defined. To clarify effects on platelets during activation of plasminogen in vitro, we used monoclonal antibodies (MoAbs), flow cytometry, and platelets surface-labeled with 125I to characterize changes in receptors for fibrinogen (GPIIb-IIIa), von Willebrand factor (GPIb), and collagen (GPIa-IIa). Activation of plasminogen in plasma with pharmacologic concentrations of plasminogen activators did not degrade GPIIb-IIIa or GPIb, and caused only a modest decrease in GPIa. In washed platelets GPIIb-IIIa was extensively degraded by plasmin at 37 degrees C in the absence of exogenous Ca2+, conditions that destabilize the IIb-IIIa complex. Degradation of GPIb in washed platelets displayed a similar although less-marked dependence on temperature and the absence of Ca2+. The binding of activation- specific MoAbs did not increase during activation of plasminogen in plasma. We conclude that during pharmacologic fibrinolysis, reported inhibition of platelet function in plasma is not due to degradation of platelet-adhesive receptors. In addition, platelet activation observed during thrombolytic therapy does not appear to be a direct consequence of plasminogen activation.
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Estevez, Brian, Michael Keegan Delaney, Aleksandra Stojanovic-Terpo, and Xiaoping Du. "A Signaling Mechanism By Which Platelet Glycoprotein Ib-IX Promotes Thrombin-Induced Platelet Activation." Blood 124, no. 21 (December 6, 2014): 2759. http://dx.doi.org/10.1182/blood.v124.21.2759.2759.
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Abstract Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. We reconstituted GPIb-IX (GPIb) /Protease-activated receptor (PAR) cooperativity in response to thrombin in Chinese Hamster Ovary (CHO) cells expressing PAR1. Thrombin-induced PAR1-dependent calcium signaling was significantly enhanced by GPIb expression, and this effect of GPIb appears to require GPIb signaling, as deletion of the cytoplasmic binding site for an intracellular signaling molecule, 14-3-3, in GPIbα abolished the stimulatory effect of GPIb. The importance of GPIb-14-3-3 interaction in promoting thrombin-induced platelet activation was also shown in human platelets, in which pretreatment with MPαC, an inhibitory peptide based on a critical 14-3-3 binding site in the C-terminus of the GPIbα, inhibited thrombin-induced platelet activation. Furthermore, 14-3-3 binding site deletion in GPIba or MPαC-pretreatment inhibited thrombin-induced activation of Rac1 and phosphorylation of LIMK1, both of which have been shown to mediate von Willebrand factor-induced GPIb signaling, and the role of GPIb in promoting thrombin signaling was abolished with a Rac-inhibitor, NSC23766 or in Rac1-/- platelets. Importantly, LIMK1-/- platelets display defective thrombin-induced platelet activation but enhanced PAR4-activating peptide induced platelet activation. Disclosures No relevant conflicts of interest to declare.
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Schafer, AI, GB Zavoico, J. Loscalzo, and AK Maas. "Synergistic inhibition of platelet activation by plasmin and prostaglandin I2." Blood 69, no. 5 (May 1, 1987): 1504–7. http://dx.doi.org/10.1182/blood.v69.5.1504.1504.
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Abstract Endothelial cell prostacyclin (PGI2) inhibits platelet activation by raising platelet cyclic AMP. Previously, platelet activation was also shown to be blocked by plasmin formed by endothelium-derived tissue plasminogen activator (TPA). We have now studied interactions between PGI2 and plasmin in the control of platelet function. PGI2 and plasmin cause synergistic inhibition of thrombin- and ADP-induced aggregation of washed platelets. Inhibition by PGI2 is similarly potentiated by TPA added to platelet-rich plasma to generate plasmin. Thrombin-stimulated rise in platelet cytosolic Ca2+, measured by fura2 fluorescence, and thromboxane A2 formation, measured by radioimmunoassay (RIA), are likewise synergistically inhibited by PGI2 and plasmin. Plasmin neither increases nor potentiates PGI2-stimulated increases in platelet cyclic AMP. Thus, PGI2 and plasmin cause synergistic inhibition of platelet activation by both cyclic AMP-dependent and independent mechanisms. This interaction between two different endothelium-derived products may play an important role in localizing the hemostatic plug to a site of vascular injury by preventing further thrombin-mediated accrual of platelets.
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Schafer, AI, GB Zavoico, J. Loscalzo, and AK Maas. "Synergistic inhibition of platelet activation by plasmin and prostaglandin I2." Blood 69, no. 5 (May 1, 1987): 1504–7. http://dx.doi.org/10.1182/blood.v69.5.1504.bloodjournal6951504.
Full textAbstract:
Endothelial cell prostacyclin (PGI2) inhibits platelet activation by raising platelet cyclic AMP. Previously, platelet activation was also shown to be blocked by plasmin formed by endothelium-derived tissue plasminogen activator (TPA). We have now studied interactions between PGI2 and plasmin in the control of platelet function. PGI2 and plasmin cause synergistic inhibition of thrombin- and ADP-induced aggregation of washed platelets. Inhibition by PGI2 is similarly potentiated by TPA added to platelet-rich plasma to generate plasmin. Thrombin-stimulated rise in platelet cytosolic Ca2+, measured by fura2 fluorescence, and thromboxane A2 formation, measured by radioimmunoassay (RIA), are likewise synergistically inhibited by PGI2 and plasmin. Plasmin neither increases nor potentiates PGI2-stimulated increases in platelet cyclic AMP. Thus, PGI2 and plasmin cause synergistic inhibition of platelet activation by both cyclic AMP-dependent and independent mechanisms. This interaction between two different endothelium-derived products may play an important role in localizing the hemostatic plug to a site of vascular injury by preventing further thrombin-mediated accrual of platelets.
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Nolfi-Donegan, Deirdre, Gowtham K. Annarapu, Cheryl A. Hillery, and Sruti Shiva. "HMGB1-Mediated Platelet Activation Is Independent of Platelet Mitochondrial Reactive Oxygen Species Generation." Blood 136, Supplement 1 (November 5, 2020): 6. http://dx.doi.org/10.1182/blood-2020-141160.
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Background: Sickle cell disease (SCD) is a hemolytic disorder that exhibits pathologic platelet activation. Notably, hemolysis is tightly associated with platelet activation and thrombotic complications of SCD such as stroke, leg ulceration, and pulmonary hypertension. To this end, we and others have shown that free hemoglobin (Hb) released into the plasma via hemolysis directly activates healthy platelets ex vivo in a concentration-dependent manner. Treatment with Hb stimulates the production of mitochondrial reactive oxygen species (mtROS) within the platelet, resulting in thrombotic activation. Accordingly, scavenging mtROS prevents Hb-induced platelet activation. While it is established that hemolysis mediates platelet activation, the factors that modulate this response remain unknown. High mobility group box 1 (HMGB1) is an inflammatory mediator, which signals through toll-like receptor-4 (TLR4), to instigate thrombosis. Notably, we previously showed that HMGB1 is elevated in the plasma of SCD patients and augments platelet activation on its own. Importantly, complementary interactions between Hb and other pro-thrombotic plasma components such as HMGB1 have not been investigated. Preliminary data show that HMGB1, like Hb, stimulates platelet mtROS generation. Thus, we hypothesized that elevated levels of HMGB1 and Hb synergize in SCD to stimulate platelet mtROS production and downstream platelet activation. Methods: Whole blood samples were collected from healthy human control subjects or SCD subjects in steady state (n=5 per group). Plasma HMGB1 concentrations were quantified by ELISA. Platelets were isolated and treated with reagents including HMGB1, ferric Hb, or the mtROS scavenger MitoTEMPO. Platelet activation was measured by flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and two markers of platelet activation: PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa), and APC to detect exposure of surface CD62P (P-selectin). Platelet mtROS were estimated using MitoSOX Red and fluorescence spectroscopy. Data was analyzed using FlowJo software and nonparametric statistical tests. Results: The baseline level of platelet activation in SCD was 7-fold higher than in controls (P=0.009). However, there was no difference in platelet activation levels between isolated SCD and control platelets in response to ex vivo treatment with agonists ADP (5 uM), collagen (50 ug/ml), or thrombin (0.1 U/ml) (P=0.38), suggesting that SCD platelets have the same agonist-induced response as control platelets when they are removed from their native plasma. We found that the concentration of HMGB1 in SCD plasma is 1.5 fold higher than controls (P=0.04), and that plasma HMGB1 concentrations positively correlate with increased platelet activation (P=0.04). Co-incubation of isolated healthy control platelets with HMGB1 (10 ug/ml) and major hemolytic byproduct Hb (10 uM, 30 uM, 50 uM) enhanced platelet activation at all doses of Hb by 3-fold compared to that of Hb alone. Mechanistically, platelets exposed to Hb alone generated mtROS which are necessary for Hb-induced platelet activation. We found that HMGB1 similarly induced platelet mtROS generation, but scavenging with MitoTEMPO did not prevent HMGB1-mediated platelet activation. However the addition of MitoTEMPO to platelets co-treated with HMGB1 + Hb attenuated platelet activation 2-fold, indicating that scavenging mtROS neutralizes the enhanced effect of Hb + HMGB1. Conclusions: We show that HMGB1 synergizes with Hb to enhance platelet activation in SCD, but unlike Hb, HMGB1-dependent platelet activation is independent of mtROS generation. Accordingly, we observed that MitoTEMPO partially attenuates the enhanced activation from co-stimulation by HMGB1 + Hb. These data characterize the synergy between HMGB1 and Hb for the first time and demonstrate differential platelet signaling between the two agonists. Disclosures No relevant conflicts of interest to declare.
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Kunapuli, Satva P. "P2 Receptors and Platelet Activation." Scientific World JOURNAL 2 (2002): 424–33. http://dx.doi.org/10.1100/tsw.2002.106.
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Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. In platelets, the classical P2T receptor is now resolved into three P2 receptor subtypes: the P2Y1, the P2Y12, and the P2X1 receptors. Both pharmacological and molecular biological approaches have confirmed the role of the P2Y1 and P2Y12 receptors in the ADP-induced platelet fibrinogen receptor activation. The P2Y1 and the P2X1 receptors independently contribute to platelet shape change. Whereas the P2Y12 receptor mediates the potentiation of dense granule release reaction, both the P2Y1 and P2Y12 receptors play an important role in the ADP-induced phospholipase A2 activation. The signaling events downstream of these receptors leading to the physiological effects remain elusive, and they are yet to be delineated.
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Rubinstein, E., C. Boucheix, I. Urso, and R. C. Carroll. "Fc gamma receptor-mediated interplatelet activation by a monoclonal antibody against beta 2 microglobulin." Journal of Immunology 147, no. 9 (November 1, 1991): 3040–46. http://dx.doi.org/10.4049/jimmunol.147.9.3040.
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Abstract Three different mAb directed against beta 2 microglobulin (two IgG1 and one IgG2a) were tested for their ability to activate human platelets. Although all three antibodies bound to platelets, only one of them, B2.62.2, of the IgG1 subclass, induced platelet activation. This activation is similar to the activation by SYB-1, a CD9 antibody of the same subclass previously described as activating platelets through platelet Fc gamma R. These similarities include serotonin secretion, a lag time preceding aggregation and the induction of a strong intracellular calcium mobilization from storage pools. As with CD9 antibodies, the F(ab')2 fragments of B2.62.2 did not induce activation but blocked the activation by the native antibody, by preventing the binding to beta 2 microglobulin. Also, this activation was inhibited by pretreating the platelet with IV-3, a mAb that blocks the Fc binding site of the FcR. Inasmuch as the same antibody does not prevent the binding of B2.62.2 on platelets, we conclude that the activation by B2.62.2 is mediated by the FcR. Nevertheless, there were differences with the activation by SYB-1. B2.62.2 activation was more dependent on thromboxane A2 formation and no cytoplasmic alkalinization was detected. Finally, contrary to SYB-1, B2.62.2 activation proved to be sensitive to platelet count, suggesting that it involves the formation of immune complexes consisting of antibodies and platelets, that activate nearby platelets.
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Holinstat, Michael, W. James Hudson, Anita M. Preininger, and Heidi E. Hamm. "Irreversible Platelet Activation Requires PAR1 Regulation of Phosphatidylinositol Phosphates (PIPns) Activation of Rap1." Blood 110, no. 11 (November 16, 2007): 3889. http://dx.doi.org/10.1182/blood.v110.11.3889.3889.
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Abstract Thrombin is the most potent activator of platelets, through reversible and then irreversible aggregation and mediates a diverse range of vascular effects which may result in acute coronary syndrome and stroke. We demonstrate that PAR1 signals human platelet activation through generation of a phosphatidylinositol phosphate signaling pathway; inhibition of this pathway blocked the formation of a stable platelet aggregate. Early Rap1 activation and platelet aggregation are insensitive to perturbations along the PI-kinase pathway, while stable aggregate formation requires this pathway. Computational lipidomics identified a single phosphatidic acid species required for PAR1-, but not PAR4-mediated Rap1 activation and platelet aggregation. Both PI5K and PI3Kg were required for stable PAR1-mediated platelet aggregation, and we show that these enzymes are present in a complex with activated Rap1. This novel lipid signaling pathway is a potential target for anti-platelet therapy.
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Verheul, Henk M. W., Anita S. Jorna, Klaas Hoekman, Henk J. Broxterman, Martijn F. B. G. Gebbink, and Herbert M. Pinedo. "Vascular endothelial growth factor–stimulated endothelial cells promote adhesion and activation of platelets." Blood 96, no. 13 (December 15, 2000): 4216–21. http://dx.doi.org/10.1182/blood.v96.13.4216.
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Abstract Coagulation abnormalities, including an increased platelet turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested the hypothesis that platelets contribute to angiogenesis. Stimulation with vascular endothelial growth factor (VEGF, 25 ng/mL) of human umbilical vein endothelial cells (HUVECs) promoted adhesion of nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs with basic fibroblast growth factor (bFGF) did not promote platelet adhesion. By blocking tissue factor (TF) activity, platelet adhesion was prevented and antibodies against fibrin(ogen) and the platelet-specific integrin, αIIbβ3, inhibited platelet adhesion for 70% to 90%. These results indicate that VEGF-induced platelet adhesion to endothelial cells is dependent on activation of TF. The involvement of fibrin(ogen) and the αIIbβ3 integrin, which exposes a high-affinity binding site for fibrin(ogen) on platelet activation, indicates that these adhering platelets are activated. This was supported by the finding that the activity of thrombin, a product of TF-activated coagulation and a potent platelet activator, was required for platelet adhesion. Finally, platelets at physiologic concentrations stimulated proliferation of HUVECs, indicative of proangiogenic activity in vivo. These results support the hypothesis that platelets contribute to tumor-induced angiogenesis. In addition, they may explain the clinical observation of an increased platelet turnover in cancer patients. Platelets may also play an important role in other angiogenesis-dependent diseases in which VEGF is involved, such as diabetes and autoimmune diseases.
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Verheul, Henk M. W., Anita S. Jorna, Klaas Hoekman, Henk J. Broxterman, Martijn F. B. G. Gebbink, and Herbert M. Pinedo. "Vascular endothelial growth factor–stimulated endothelial cells promote adhesion and activation of platelets." Blood 96, no. 13 (December 15, 2000): 4216–21. http://dx.doi.org/10.1182/blood.v96.13.4216.h8004216_4216_4221.
Full textAbstract:
Coagulation abnormalities, including an increased platelet turnover, are frequently found in patients with cancer. Because platelets secrete angiogenic factors on activation, this study tested the hypothesis that platelets contribute to angiogenesis. Stimulation with vascular endothelial growth factor (VEGF, 25 ng/mL) of human umbilical vein endothelial cells (HUVECs) promoted adhesion of nonactivated platelets 2.5-fold. In contrast, stimulation of HUVECs with basic fibroblast growth factor (bFGF) did not promote platelet adhesion. By blocking tissue factor (TF) activity, platelet adhesion was prevented and antibodies against fibrin(ogen) and the platelet-specific integrin, αIIbβ3, inhibited platelet adhesion for 70% to 90%. These results indicate that VEGF-induced platelet adhesion to endothelial cells is dependent on activation of TF. The involvement of fibrin(ogen) and the αIIbβ3 integrin, which exposes a high-affinity binding site for fibrin(ogen) on platelet activation, indicates that these adhering platelets are activated. This was supported by the finding that the activity of thrombin, a product of TF-activated coagulation and a potent platelet activator, was required for platelet adhesion. Finally, platelets at physiologic concentrations stimulated proliferation of HUVECs, indicative of proangiogenic activity in vivo. These results support the hypothesis that platelets contribute to tumor-induced angiogenesis. In addition, they may explain the clinical observation of an increased platelet turnover in cancer patients. Platelets may also play an important role in other angiogenesis-dependent diseases in which VEGF is involved, such as diabetes and autoimmune diseases.
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Zhou, Junsong, Yi Wu, Lubica Rauova, Gavin Koma, Lu Wang, Mortimer Poncz, Hong Li, et al. "A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice." Blood 139, no. 13 (March 31, 2022): 2050–65. http://dx.doi.org/10.1182/blood.2021012055.
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Abstract Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has 3 CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, adenosine triphosphate (ATP) release, and αIIbβ3 activation. ERp46 protein potentiated αIIbβ3 activation, platelet aggregation, and ATP release, whereas inactive ERp46 inhibited these processes. ERp46 knockout mice had prolonged tail-bleeding times and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbβ3 activation, platelet aggregation, ATP release, and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the 3 active sites. Platelet aggregation stimulated by an αIIbβ3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbβ3 by surface plasmon resonance but poorly to platelets lacking αIIbβ3 and physically associated with αIIbβ3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the β3 subunit than other PDIs and in contrast to PDI, generated thiols in β3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in β3, implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in β3, implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbβ3 activation by targeting disulfide bonds.
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Vara, Dina S., Michelangelo Campanella, Ilaria Canobbio, Warwick B. Dunn, Giuseppe Pizzorno, Michio Hirano, and Giordano Pula. "Autocrine amplification of integrin αIIbβ3 activation and platelet adhesive responses by deoxyribose-1-phosphate." Thrombosis and Haemostasis 109, no. 06 (2013): 1108–19. http://dx.doi.org/10.1160/th12-10-0751.
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SummaryUsing direct injection mass spectrometry (DIMS) we discovered that deoxyribose-1-phosphate (dRP) is released by platelets upon activation. Interestingly, the addition of exogenous dRP to human platelets significantly increased platelet aggregation and integrin αIIbβ3 activation in response to thrombin. In parallel, genetically modified platelets with double genetic deletion of thymidine phosphorylase and uridine phosphorylase were characterised by reduced release of dRP, impaired aggregation and decreased integrin αIIbβ3 activation in response to thrombin. In vitro platelet adhesion onto fibrinogen and collagen under physiological flow conditions was potentiated by treatment of human platelets with exogenous dRP and impaired in transgenic platelets with reduced dRP release. Human and mouse platelets responded to dRP treatment with a sizeable increase in reactive oxygen species (ROS) generation and the pre-treament with the antioxidant apocynin abolished the effect of dRP on aggregation and integrin activation. Experiments directly assessing the activation of the small G protein Rap1b and protein kinase C suggested that dRP increases the basal levels of activity of these two pivotal platelet-activating pathways in a redox-dependent manner. Taken together, we present evidence that dRP is a novel autocrine amplifier of platelet activity, which acts on platelet redox levels and modulates integrin αIIbβ3.
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Lomazova, K. D., A. M. Polyakova, O. S. Astrina, and D. B. Tsukerman. "Platelet activating factor and endotoxin-induced platelet activation." Bulletin of Experimental Biology and Medicine 107, no. 5 (May 1989): 611–14. http://dx.doi.org/10.1007/bf00841762.
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Brandt, John T., Carmen J. Julius, Jeanne M. Osborne, and Clark L. Anderson. "The Mechanism of Platelet Aggregation Induced by HLA-Related Antibodies." Thrombosis and Haemostasis 76, no. 05 (1996): 774–79. http://dx.doi.org/10.1055/s-0038-1650659.
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SummaryImmune-mediated platelet activation is emerging as an important pathogenic mechanism of thrombosis. In vitro studies have suggested two distinct pathways for immune-mediated platelet activation; one involving clustering of platelet FcyRIIa, the other involving platelet-associated complement activation. HLA-related antibodies have been shown to cause platelet aggregation, but the mechanism has not been clarified. We evaluated the mechanism of platelet aggregation induced by HLA-related antibodies from nine patients. Antibody to platelet FcyRIIa failed to block platelet aggregation with 8/9 samples, indicating that engagement of platelet FcyRIIa is not necessary for the platelet aggregation induced by HLA-related antibodies. In contrast, platelet aggregation was blocked by antibodies to human C8 (5/7) or C9 (7/7). F(ab’)2 fragments of patient IgG failed to induce platelet activation although they bound to HLA antigen on platelets. Intact patient IgG failed to aggregate washed platelets unless aged serum was added. The activating IgG could be adsorbed by incubation with lymphocytes and eluted from the lymphocytes. These results indicate that complement activation is involved in the aggregation response to HLA-related antibodies. This is the first demonstration of complement-mediated platelet aggregation by clinical samples. Five of the patients developed thrombocytopenia in relationship to blood transfusion and two patients developed acute thromboembolic disease, suggesting that these antibodies and the complement-dependent pathway of platelet aggregation may be of clinical significance.
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Żmigrodzka, M., M. Guzera, and A. Winnicka. "Flow cytometric assessment of activation of peripheral blood platelets in dogs with normal platelet count and asymptomatic thrombocytopenia." Polish Journal of Veterinary Sciences 19, no. 2 (June 1, 2016): 407–14. http://dx.doi.org/10.1515/pjvs-2016-0051.
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AbstractPlatelets play a crucial role in hemostasis. Their activation has not yet been evaluated in healthy dogs with a normal and low platelet count. The aim of this study was to determine the influence of activators on platelet activation in dogs with a normal platelet count and asymptomatic thrombocytopenia. 72 clinically healthy dogs were enrolled. Patients were allocated into three groups. Group 1 consisted of 30 dogs with a normal platelet count, group 2 included 22 dogs with a platelet count between 100 and 200×109/land group 3 consisted of 20 dogs with a platelet count lower than 100×109/l. Platelet rich-plasma (PRP) was obtained from peripheral blood samples using tripotassium ethylenediaminetetraacetic acid (K3-EDTA) as anticoagulant. Next, platelets were stimulated using phorbol-12-myristate-13-acetate or thrombin, stabilized using procaine or left unstimulated. The expression of CD51 and CD41/CD61 was evaluated. Co-expression of CD41/CD61 and Annexin V served as a marker of platelet activation. The expression of CD41/CD61 and CD51 did not differ between the 3 groups. Thrombin-stimulated platelets had a significantly higher activity in dogs with a normal platelet count than in dogs with asymptomatic thrombocytopenia. Procaine inhibited platelet activity in all groups. In conclusion, activation of platelets of healthy dogsin vitrovaried depending on the platelet count and platelet activator.
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Huynh, Angela, Donald M. Arnold, James W. Smith, Taylor D. Elliott, Hina Bhakta, John G. Kelton, and Ishac Nazy. "Fluid-Phase Immune Complexes Can Assemble and Activate Platelets in Heparin-Induced Thrombocytopenia." Blood 132, Supplement 1 (November 29, 2018): 3746. http://dx.doi.org/10.1182/blood-2018-99-115037.
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Abstract Introduction: Heparin-induced thrombocytopenia (HIT) is a complication of heparin therapy that is caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Several studies have reported that in order for these immune complexes to be pathogenic, they must assemble on the platelet surface. When bound to the platelet surface, the conformation of PF4 allows for optimal presentation of the epitope for antibody binding and subsequent activation of Fc-receptors on platelets and monocytes. To what degree pathogenic HIT immune complexes can form and activate platelets in fluid-phase as with other immune complex diseases (systemic lupus erythematosus, glomerulonephritis, and rheumatoid arthritis) is not known. We used mutated PF4 proteins that can no longer bind the platelet surface to evaluate anti-PF4/heparin antibody induced platelet activation. We hypothesized that the epitopes required for PF4 binding of HIT antibodies and subsequent platelet activation can be formed in fluid-phase. Methods: Each of the 70 amino acids of PF4 were mutated previously by alanine scanning mutagenesis where non-alanine residues were mutated to alanine or alanine residues to valine. We selected 14 PF4 mutants that affected KKO (a platelet-activating murine monoclonal HIT-like antibody) binding in a heparin-capture assay for this study. Mutant and wild-type PF4 were overexpressed in Escherichia coli and affinity purified. To confirm binding to platelets, biotin-conjugated PF4 mutants were incubated with donor platelets and PF4 platelet binding was measured using streptavidin-FITC by flow cytometry. Platelet activation was measured using a modified 14C-serotonin-release assay, where excess wild-type or mutant PF4 (0, 50 and 100 μg/mL) was added to 14C-serotonin-labelled donor platelets and activation was measured after incubation with KKO. ≥20% 14C-serotonin release was considered positive for platelet activation. Platelet activation was correlated with platelet surface binding to identify mutants that could form surface-bound or fluid-phase antigenic complexes. Results: Of the 14 PF4 mutants tested, 7 bound to platelet surfaces and 11 supported platelet activation by KKO. These PF4 mutants were further characterized into 3 categories: PF4 mutants that bound to the platelet surface and induced platelet activation (n=6); PF4 mutants that did not bind to the platelet surface but induced platelet activation (n=5); and PF4 mutants that bound to the platelet surface but did not induce platelet activation (n=1). These results indicate that certain PF4 mutants were able to bind KKO and induce platelet activation in fluid-phase. These data suggest that specific epitopes in fluid-phase PF4/heparin immune complexes can mediate platelet activation in HIT, without the need for surface assembly on the platelet. Conclusions: Using point mutations of PF4, we have identified that the HIT antigenic complexes can be formed in fluid-phase and induce platelet activation. Further studies are required to investigate the role of a fluid-phase HIT antigen complex in the development of thrombocytopenia, inflammation and thrombosis of HIT. This study was funded by the Canadian Institutes for Health Research. Disclosures Arnold: Bristol Myers Squibb: Research Funding; Amgen: Consultancy, Research Funding; UCB: Consultancy; UCB: Consultancy; Novartis: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Bristol Myers Squibb: Research Funding.
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Jahn, Kristin, Thomas P. Kohler, Lena-Sophie Swiatek, Sergej Wiebe, and Sven Hammerschmidt. "Platelets, Bacterial Adhesins and the Pneumococcus." Cells 11, no. 7 (March 25, 2022): 1121. http://dx.doi.org/10.3390/cells11071121.
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Systemic infections with pathogenic or facultative pathogenic bacteria are associated with activation and aggregation of platelets leading to thrombocytopenia and activation of the clotting system. Bacterial proteins leading to platelet activation and aggregation have been identified, and while platelet receptors are recognized, induced signal transduction cascades are still often unknown. In addition to proteinaceous adhesins, pathogenic bacteria such as Staphylococcus aureus and Streptococcus pneumoniae also produce toxins such as pneumolysin and alpha-hemolysin. They bind to cellular receptors or form pores, which can result in disturbance of physiological functions of platelets. Here, we discuss the bacteria-platelet interplay in the context of adhesin–receptor interactions and platelet-activating bacterial proteins, with a main emphasis on S. aureus and S. pneumoniae. More importantly, we summarize recent findings of how S. aureus toxins and the pore-forming toxin pneumolysin of S. pneumoniae interfere with platelet function. Finally, the relevance of platelet dysfunction due to killing by toxins and potential treatment interventions protecting platelets against cell death are summarized.
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Kanaji, Taisuke, Sachiko Kanaji, Shailendra B. Patel, and Peter J. Newman. "Platelet Activation In Sitosterolemia Mice." Blood 116, no. 21 (November 19, 2010): 2022. http://dx.doi.org/10.1182/blood.v116.21.2022.2022.
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Abstract Abstract 2022 Introduction: Sitosterolemia is a rare, autosomal recessive disorder characterized by the accumulation of plant sterols in blood and tissues, and is caused by mutations in one of the adenosine triphosphate-binding cassette (ABC) transporter ABCG5 or ABCG8 genes. Patients with mutations in either of these sterol transport proteins, which normally form a heterodimer sterol egress channel, frequently develop tendon and cutaneous xanthomas and, most importantly, are at risk of developing premature coronary atherosclerosis. Other clinical manifestations include hematological abnormalities such as hemolytic anemia, macrothrombocytopenia, and loss of ristocetin-induced platelet agglutination – a measure of the ability of platelet glycoprotein (GP) Ib to function as a adhesion receptor for von Willebrand factor (VWF). Mice genetically deficient in ABCG5 or ABCG8 fully recapitulate the macrothrombocytopenia and loss of platelet function seen in human sitosterolemia, a condition that can be corrected by treatment with the sterol-absorption inhibitor, ezetimibe. Because the mechanism by which accumulated plant sterols affects platelet size, production, and function is incompletely understood, we further analyzed these traits in Abcg5- and Abcg8-deficient mice in an animal model of sitosterolemia. Methods: Blood was collected every 1–2 weeks and CBC monitored in Abcg5- and Abcg8-deficient mice that had been fed either a high or low plant sterol diet. Expression of platelet receptors was analyzed by both flow cytometry and Western blotting. GPIbα null mice were used as controls since they have similarly enlarged platelets. Platelet microparticles were analyzed by labeling with a mAb specific for GPIIb and Annexin V. Plasma samples were analyzed for von Willebrand factor (VWF) antigen and multimer patterns. Results: Following onset of a high sterol diet, the platelet count decreased over a two week period to less than 30% of that of normal controls in both Abcg5-/- and Abcg8-/- mice. At the same time, the mean platelet volume gradually increased over a 4 week period from a normal value of approximately 7 fl to approximately 10 fl. In addition, an increased number of platelet-derived microparticles were detected in Abcg5-/- and Abcg8-/- mice kept on high sterol diet over a 9 week period, suggesting that low-grade platelet activation was occurring in these mice. Unexpectedly, the surface expression of the GPIIb integrin subunit was decreased in both Abcg5-/- and Abcg8-/- mouse platelets compared to that of similarly large platelets derived from GPIbα-null (Bernard-Soulier) platelets. Intracellular staining revealed that the GPIIb in Abcg8-/- platelets was being internalized and retained within the cell. Both surface-bound and total platelet fibrinogen was increased in Abcg5-/- and Abcg8-/- mice fed a high sterol diet. Finally, plasma VWF antigen levels decreased by approximately 50% within two weeks following switching Abcg5-/- mice from a low-fat to a high-fat diet. Multimer analysis showed loss of high molecular weight multimers in some of the plasma samples. Conclusions: This study demonstrates a heightened state of platelet activation in a murine model of sitosterolemia, leading to VWF and fibrinogen binding, with concomitant internalization of GPIIb. This might explain, at least in part, the cause of macrothorombocytopenia and absence of ristocetin-induced platelet aggregation in sitosterolemia patients. Platelet activation may also lead to calpain activation, resulting filamin A and talin degradation and microparticle production. Future studies aims at identifying the mechanism by which plant sterols or their metabolites elicit these effects on platelets may provide important clues to the ability of dietary lipids to affect platelet activation and the development of atherosclerosis. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.
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Stalker, Timothy J. "Platelet Activation Gradients During Thrombus Formation." Blood 126, no. 23 (December 3, 2015): SCI—13—SCI—13. http://dx.doi.org/10.1182/blood.v126.23.sci-13.sci-13.
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The hemostatic response requires the tightly regulated interaction of the coagulation system, platelets, other blood cells and components of the vessel wall at a site of vascular injury. The dysregulation of this response may result in excessive bleeding if the response is impaired, and pathologic thrombosis with vessel occlusion and tissue ischemia if the response is overly robust. Extensive studies over the past decades have sought to unravel the regulatory mechanisms that coordinate the multiple biochemical and cellular responses in time and space to ensure that an optimal response to vascular damage is achieved. We and others have observed that platelet activation at a site of injury in vivo is heterogeneous, with a gradient of platelet activation extending from the site of injury. Platelets immediately adjacent to the injured vessel wall are densely packed and fully activated forming a stably adherent core region. This stable core is overlaid by a shell of less activated platelets that are more loosely packed. Genetic and pharmacologic studies have shown that the formation of these regions is dependent on partially overlapping gradients of distinct platelet agonists, with ADP serving as a mediator of platelet recruitment and retention in the shell region, and thrombin necessary for full platelet activation in the core region. The distribution of platelet agonists and other plasma solutes in time and space is in turn determined in part by their transport in the plasma microenvironments that evolve as platelets accumulate. Platelet mass consolidation and the subsequent narrowing of the gaps between platelets are important mechanisms by which plasma solutes are retained within the platelet mass to promote platelet activation. Consolidation also regulates the escape of plasma and platelet-derived bioactive molecules into the extravascular space. These studies and others examining how cellular, biochemical and physical factors are integrated to shape the optimal response to vascular injury in vivo will be discussed. Disclosures No relevant conflicts of interest to declare.
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Zhang, Guoying, Binggang Xiang, Anping Dong, Radek C. Skoda, Alan Daugherty, Susan S. Smyth, Xiaoping Du, and Zhenyu Li. "Biphasic roles for soluble guanylyl cyclase (sGC) in platelet activation." Blood 118, no. 13 (September 29, 2011): 3670–79. http://dx.doi.org/10.1182/blood-2011-03-341107.
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AbstractNitric oxide (NO) stimulates cGMP synthesis by activating its intracellular receptor, soluble guanylyl cyclase (sGC). It is a currently prevailing concept that No and cGMP inhibits platelet function. However, the data supporting the inhibitory role of NO/sGC/cGMP in platelets have been obtained either in vitro or using whole body gene deletion that affects vessel wall function. Here we have generated mice with sGC gene deleted only in megakaryocytes and platelets. Using the megakaryocyte- and platelet-specific sGC-deficient mice, we identify a stimulatory role of sGC in platelet activation and in thrombosis in vivo. Deletion of sGC in platelets abolished cGMP production induced by either NO donors or platelet agonists, caused a marked defect in aggregation and attenuated secretion in response to low doses of collagen or thrombin. Importantly, megakaryocyte- and platelet-specific sGC deficient mice showed prolonged tail-bleeding times and impaired FeCl3-induced carotid artery thrombosis in vivo. Interestingly, the inhibitory effect of the NO donor SNP on platelet activation was sGC-dependent only at micromolar concentrations, but sGC-independent at millimolar concentrations. Together, our data demonstrate important roles of sGC in stimulating platelet activation and in vivo thrombosis and hemostasis, and sGC-dependent and -independent inhibition of platelets by NO donors.
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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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O’Brien, Kelly A., Aleksandra Stojanovic, Nissim Hay, and Xiaoping Du. "The Role of Akt3 in Platelet Activation." Blood 112, no. 11 (November 16, 2008): 2853. http://dx.doi.org/10.1182/blood.v112.11.2853.2853.
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Abstract Akt is a family of serine/threonine kinases that are activated downstream of Phosphoinositide 3 Kinases (PI3K). There are three known Akt isoforms: Akt1, Akt2, and Akt3. Both Akt1 and Akt2 have been shown to be expressed in platelets and play important roles in PI3K-dependent platelet activation signaling. Previous studies have concluded that Akt3 is not expressed in platelets; however, we show that Akt3 is in fact a major Akt isoform present in platelets, and demonstrate that Akt3 plays an important role in platelet activation. In order to determine if Akt3 mRNA is present in platelets, RNA was isolated from platelets and RT-PCR experiments were performed using Akt3 specific primers. Akt3 mRNA was detected in both human and mouse platelets, and the possibility that Akt3 mRNA came from leukocyte contamination was excluded because Akt3 was not detected with RT-PCR using RNA purified from the same number of leukocytes as that in platelet preparation. To assess if Akt3 protein is present in platelets, western blot analysis was performed using an Akt3 specific antibody. Results revealed that Akt3 is highly expressed in human and mouse platelets. In order to assess the relative amount of Akt3 present in platelets, total Akt levels and phosphorylated Akt levels were measured in Akt3 knockout mouse platelets in comparison with wild type mouse platelets. Akt3 knockout mouse platelets showed a significant decrease in total Akt, and also a significant reduction in phosphorylated Akt, as indicated by Western blot analysis of phospho-Thr308-Akt and phospho-Ser473-Akt antibodies. Thus, our data indicates Akt3 is a major Akt isoform expressed in platelets. To determine the role of Akt3 in platelet activation, platelets from wild type mice and Akt3 knockout mice were stimulated with low concentrations of platelet agonists. Akt3 knockout mouse platelets exhibited impaired aggregation and ATP secretion in response to low dose thrombin and collagen compared with wild type controls, indicating that Akt3 plays an important role in promoting platelet activation. In order to assess the signaling molecules downstream from Akt3, Akt3 knockout and wild type mouse platelets were stimulated with low dose thrombin and phosphorylation of GSK-3β, an enzyme recently reported to negatively regulate platelet function downstream from Akt1 or Akt2 in platelets, was assessed. Western blot for phospho-Ser9 of GSK-3β in Akt3 knockout mouse platelets stimulated with thrombin showed less phosphorylation compared to Akt1 or Akt2 knockout mouse platelets, indicating that Akt3 is the major Akt isoform responsible for GSK-3β phosphorylation. Thus, our data shows that Akt3 is a major Akt isoform in platelets and plays an important role in platelet activation. Since phosphorylation of GSK-3β at Ser9 has been reported to reverse the inhibitory effect of GSK-3β on platelet function, our data also suggest that the stimulatory role of Akt3 in platelet activation may be mediated by negative regulation of GSK-3β function.
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Jen, C. J., and S. J. Hu. "Direct platelet-fibrin interaction that does not require platelet activation." American Journal of Physiology-Heart and Circulatory Physiology 253, no. 4 (October 1, 1987): H745—H750. http://dx.doi.org/10.1152/ajpheart.1987.253.4.h745.
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Interactions between platelets and fibrin are important in hemostasis but often confused with platelet-fibrinogen interactions. Fibrin solubilized in NaBr-acetic acid formed polymers in neutral pH buffers. A stirred mixture of solubilized fibrin and washed human platelets at neutral pH range showed drastic reduction in turbidity and concomitant platelet adhesion onto newly formed fibrin strands. Optimally, more than 99% of platelets could be associated with fibrin, whereas only 3% of erythrocytes were trapped in the fibrin network under similar conditions. This platelet-fibrin interaction was fibrin concentration dependent and polymerization stage dependent. It preceded any detectable platelet release of serotonin. Gly-Pro-Arg-Pro (250 times in excess) retarded the kinetics of fibrin polymerization as well as that of platelet-fibrin interaction. However, factors affecting platelet activation showed little effect on platelet-fibrin interaction. Our results support the existence of a platelet-fibrin interaction that does not require platelet activation.
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Lenich, Catherine, Jian-Ning Liu, and Victor Gurewich. "Thrombin Stimulation of Platelets Induces Plasminogen Activation Mediated by Endogenous Urokinase-Type Plasminogen Activator." Blood 90, no. 9 (November 1, 1997): 3579–86. http://dx.doi.org/10.1182/blood.v90.9.3579.
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Abstract Gene knockout mice studies indicate that urokinase-type plasminogen activator (u-PA) is importantly involved in fibrinolysis, but its physiologic mechanism of action remains poorly understood. We postulated that platelets may be involved in this mechanism, as they carry a novel receptor for u-PA and a portion of the single-chain u-PA (scu-PA) intrinsic to blood is tightly associated with platelets. Therefore, plasminogen activation by platelet-associated u-PA was studied. When washed platelets were incubated with plasminogen, no plasmin was generated as detected by plasmin synthetic substrate (S2403) hydrolysis; however, after the addition of thrombin, but not other agonists, platelet-dependent plasminogen activation occurred. Plasminogen activation was surface-related, being inhibited by blocking platelet fibrinogen receptors or by preventing plasminogen binding to the thrombin-activated platelet surface. U-PA was identified as the only plasminogen activator responsible and enrichment of platelets with exogenous scu-PA significantly augmented plasminogen activation. These findings appeared paradoxical because thrombin inactivates scu-PA. Indeed, zymograms showed inactivation of scu-PA during the first hour of incubation with even the lowest dose of thrombin used (1 u/mL). However, this was followed by a thrombin dose-dependent (1 to 10 u/mL) partial return of u-PA activity. Reactivation of u-PA was not due to the direct action of thrombin, but required platelets and was found to be related to a platelet lysosomal thiol protease, consistent with cathepsin C. In conclusion, a new pathway of plasminogen activation by platelet-associated endogenous or exogenous scu-PA was demonstrated, which is specifically triggered by thrombin activation of platelets. These findings may help explain u-PA–mediated physiological fibrinolysis and have implications for therapeutic thrombolysis with scu-PA.
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Perry, Seth W., Jason A. Hamilton, Larry W. Tjoelker, Ghassan Dbaibo, Kirk A. Dzenko, Leon G. Epstein, Yusuf Hannun, J. Steven Whittaker, Stephen Dewhurst, and Harris A. Gelbard. "Platelet-activating Factor Receptor Activation." Journal of Biological Chemistry 273, no. 28 (July 10, 1998): 17660–64. http://dx.doi.org/10.1074/jbc.273.28.17660.
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Gawaz, Meinrad. "Leukocyte Adhesion and Activation in Atherothrombosis." Blood 122, no. 21 (November 15, 2013): SCI—52—SCI—52. http://dx.doi.org/10.1182/blood.v122.21.sci-52.sci-52.
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Abstract Platelets interact with a variety of cells, including leukocytes and progenitor cells. Upon activation, platelets release chemokines such as CXCL12/SDF-1 that modulate important functional aspects of vascular inflammation and atherogenesis. Platelet-derived SDF-1 and CyPA regulate chemotaxis, migration, and cell activation in the microenvironment of accumulating platelets. Previously we have shown that platelets are the major source of CXCL12/SDF-1. Platelet-derived SDF-1 provides a strong signal for recruitment of circulating progenitor cells and leukocytes towards vascular and myocardial injury. Patients with severe coronary artery disease have enhanced levels of plasma and platelet-associated SDF-1 that correlates with an enhanced number of circulating progenitor cells and the development of cardiovascular diseases. In patients with both acute myocardial infarction platelet-SDF-1 and an increase in circulating platelet/progenitor cell aggregates, the combination is associated with myocardial recovery and improved prognosis, indicating that platelet-SDF-1 is critical for repair and regeneration of vascular or myocardial injury. Administration of a stable recombinant SDF-1 fusion protein reduces infarct size and preserves myocardial infarction in mice. We showed that platelet-derived SDF-1 is an important survival factor both for platelets (in an autocrine manner) and for surrounding cells. SDF-1 released from activated platelets binds to its receptor CXCR4 and induces internalization of the SDF-1/CXCR4 complex. SDF-1-ligation of CXCR4 augments surface expression of CXCR7; a process coupled via ERK1/2 and cyclophilin A. SDF-1α caused downstream phosphorylation of Erk1/2 and cyclophilin A (CyPA). NIM-811 a CyPA-PPIase-activity inhibitor significantly abolished SDF-1α-driven CXCR7 surface exposure. Moreover, Cypa-/-murine platelets failed to show SDF-1α/CXCR4-mediated CXCR7 translocation. SDF-1α-induced ubiquitination of CXCR7 was essential for its surface translocation and was dependent on Erk1/2 and CyPA-PPIase activity, inhibited by U0126 and NIM-811, respectively. In contrast to wild-type, Cypa-/- murine platelets failed to exhibit a dynamic change in CXCR7 ubiquitination status upon SDF-1α exposure and thus failed in subsequent CXCR7 externalization. SDF-1/CXR4-driven CXCR7 translocation inhibits activation-induced apoptosis of platelets and prolongs platelet survival in vitro and in vivo. Similarly, platelet SDF-1 interacts with monocytes, reduces apoptosis and promotes differentiation of monocytes into macrophages. Inhibition of platelet release and adhesion to endothelial cells reduces monocyte migration and differentiation into macrophages in vitro and attenuates vascular inflammation and atheroprogression in apoE-deficient mice. In conclusion, platelets and platelet interaction with leukocytes are critical in regulation of vascular inflammation and atheroprogression and may offer promising targets for modulating inflammation in disease states. Disclosures: No relevant conflicts of interest to declare.
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Schulz, Christian, Nina V. Leuschen, Thomas Fröhlich, Michael Lorenz, Susanne Pfeiler, Christian A. Gleissner, Elisabeth Kremmer, et al. "Identification of novel downstream targets of platelet glycoprotein VI activation by differential proteome analysis: implications for thrombus formation." Blood 115, no. 20 (May 20, 2010): 4102–10. http://dx.doi.org/10.1182/blood-2009-07-230268.
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Abstract Platelets play a key role in hemostasis and various diseases including arterial thrombosis. Glycoprotein VI (GPVI) mediates adhesion to collagen structures exposed at sites of vascular injury and subsequent platelet activation. We determined the effects of specific activation of GPVI on the human platelet proteome. Isolated human platelets were stimulated with an activating monoclonal antibody specific for GPVI. Platelet proteins were analyzed by 2-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry. We identified 8 differentially abundant proteins associated with cell signaling, metabolism, organization and rearrangement of the cytoskeleton, and membrane trafficking. Differentially abundant proteins included aldose reductase (AR), beta-centractin, charged multivesicular body protein 3, Src substrate cortactin, ERp57, and pleckstrin. Importantly, GPVI-modulated protein abundance was functionally relevant. Correspondingly, AR enzyme activity significantly increased upon GPVI activation and inhibition of AR resulted in reduced platelet aggregation. Furthermore, ERp57 was released upon ligation of platelet GPVI and increased the activity of tissue factor, a major initiator of blood coagulation. In summary, GPVI activation results in differential changes in abundance of platelet proteins, including AR and ERp57, which support platelet aggregation and platelet-dependent coagulation. These results provide further insight into the mechanisms that underlie platelet activation through the GPVI receptor and may help to identify novel pharmacologic targets.
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Ma, Liping, Da-Nian Nie, Xiu-Ju Wang, Shuang-Fen Xie, Yi-Qing Li, Yu-Dan Wu, Zhi-Xin Pei, Cheng Zhang, and Song-Mei Yin. "The Function of Toll-Like Receptor 4 Expressions on Human Platelet in Platelet Activation." Blood 112, no. 11 (November 16, 2008): 5361. http://dx.doi.org/10.1182/blood.v112.11.5361.5361.
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Abstract Lipopolysaccsharide (LPS) is a principal outer membrane component of gram-negative bacteria. It initiates an inflammatory response to infection by activating Toll-like receptor-4 (TLR4) in host. Infection increases risk for hemostasis, thrombosis, DIC, and tissue repair. Platelet contributes to the inflammation process through respond to invading pathogens, membrane adhesion molecule (P-selectin) is one of the indexes to determine platelet activation. Experiment was designed to study whether TLR4 is expressed on human platelet, and what is the function of TLR4 in platelet activation induced with LPS. Platelet suspensions from 10 heath people were treated with LPS of different concentrations for 1 hour, which were 0mg/L(control),0.1mg/L,0.5mg/L,1mg/L and 5mg/L, respectively. The expressions of TLR4, P-select on platelets and PAI-1 in platelets were detected through flow cytometry (FCM) and western blot(WB) methods. ADP-induced platelet aggregation was measured by LG-PABER aggregometer. Compared with control, the expressions of TLR4,P-selectin on platelets and PAI-1 in platelets after treated with LPS of 0.5mg/L,1mg/L and 5mg/L were increased (P<0.05). positive correlation was observed between TLR4 and P-selectin on platelets, but between TLR4 and PAI-1 in platelets. LPS of all concentrations did not affected ADP-induced platelet aggregation. Therefore, it is evident that functional TLR4 is expressed on human platelet. TLR4 on platelet might be one of the important intermedia between platelet activation and LPS or bacteria, and contribute to the inflammatory process in host. It is also worthy to study whether LPS affect platelet aggregation induced by other inductors.
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Loza, Jean-Pierre, Victor Gurewich, Michael Johnstone, and Ralph Pannell. "Platelet-Bound Prekallikrein Promotes Pro-Urokinase-Induced Clot Lysis: A Mechanism for Targeting the Factor XII Dependent Intrinsic Pathway of Fibrinolysis." Thrombosis and Haemostasis 71, no. 03 (1994): 347–52. http://dx.doi.org/10.1055/s-0038-1642441.
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SummaryClots formed from platelet rich plasma were found to be lysed more readily by low concentrations of pro-urokinase (pro-UK) than clots formed from platelet poor plasma. This was not a non-specific effect since the reverse occurred with tissue plasminogen activator. A mechanical explanation due to platelet-mediated clot retraction was excluded by experiments in which retraction was inhibited with cyto-chalasin B. Therefore, a platelet-mediated enzymatic mechanism was postulated to explain the promotion of fibrinolysis. Casein autography of isolated platelets revealed a ≈ 90 kDa band of activity which comigrated with plasma prekallikrein (PK)/kallikrein, a known activator of pro-UK. Furthermore, treatment of platelets with plasma PK activator (PPA), consisting essentially of factor XIIa, induced activation of pro-UK and of chromomgenic substrate for kallikrein (S-2302). This activity corresponded to approximately 40-200 pM kallikrein per 10 8 washed and gel filtered platelets per ml. The activation of pro-UK by PPA-pretreated platelets was dose-dependent and inhibited by soybean trypsin inhibitor but not by bdellin, a specific inhibitor of plasmin, nor by the corn inhibitor of factor XIIa. Kinetic analysis of pro-UK activation by kallikrein showed promotion of the reaction by platelets. The KM of the reaction was reduced by platelets by ≈ 7-fold, while the kcat was essentially unchanged. In conclusion, PK was shown to be tightly associated with platelets where it can be activated by factor XIIa during clotting. The activation of pro-UK by platelet-bound kallikrein provides an explanation for the observed platelet mediated promotion of pro-UK-induced clot lysis. Since pro-UK and plasminogen have also been shown to be associated with platelets, the present findings suggest a mechanism by which the factor Xlla-dependent intrinsic pathway of fibrinolysis can be localized and targeted to a thrombus.
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Berrettini, Mauro, Pasquale Parise, Vincenzo Costantini, Serena Grasselli, and Giuseppe G. Nenci. "Platelet Activation in Psoriasis." Thrombosis and Haemostasis 53, no. 02 (1985): 195–97. http://dx.doi.org/10.1055/s-0038-1661271.
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SummaryRecent epidemiological studies have suggested that psoriasis represents a risk factor for thrombotic vascular diseases. In order to evaluate the possible role of hemostatic changes in the development of thrombotic episodes in psoriasis, some parameters of the hemostatic “balance” were investigated in 22 male psoriatic patients and compared to those of 22 male control subjects. Incidence of known risk factors for vascular diseases (diabetes, hypertension, smoking, dyslipidemia) was comparable in the two study groups. There were no statistically significant differences in platelet count, circulating platelet aggregates, platelet production of malondialdehyde (MDA), total plasma anti thrombin and fibrinolytic activities. In patients with psoriasis the incidence of spontaneous platelet hyperaggregability and plasma levels of β-thromboglobulin were significantly higher than in control subjects. Platelet regeneration time, measured as MDA recovery after aspirin ingestion, was significantly shorter in psoriatic patients. These data suggest that an in vivo platelet activation occurs in patients with psoriasis and could contribute to the development of thrombotic complications. The release of mitogenic and inflammatory substances by activated platelets may play a role in the histogenesis of psoriatic lesions.
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Wang, Xiang, Yun-Feng Fu, Xiao Liu, Guo Feng, Dan Xiong, Guang-Fu Mu, and Fang-Ping Chen. "ROS Promote Ox-LDL-Induced Platelet Activation by Up-Regulating Autophagy Through the Inhibition of the PI3K/AKT/mTOR Pathway." Cellular Physiology and Biochemistry 50, no. 5 (2018): 1779–93. http://dx.doi.org/10.1159/000494795.
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Background/Aims: Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in patients with dyslipidemic disorders. Although oxLDL stimulates activating signaling, researchers have not clearly determined how these events drive accelerated thrombosis. Here, we describe the mechanism by which ROS regulate autophagy during ox-LDL-induced platelet activation by modulating the PI3K/AKT/mTOR signaling pathway. Methods: For in vitro experiments, ox-LDL, the ROS scavenger N-acetylcysteine (NAC), the mTOR inhibitor rapamycin and the autophagy inhibitor 3-MA were used alone or in combination with other compounds to treat platelets. Then, platelet aggregation was evaluated on an aggregometer and platelet adhesion was measured under shear stress. The levels of a platelet activation marker (CD62p) were measured by flow cytometry, reactive oxygen species (ROS) levels were then quantified by measuring DCFH-DA fluorescence intensity via flow cytometry. Nitric oxide (NO) and superoxide (O2·-) levels were determined by the nitric acid deoxidize enzyme method and lucigenin-enhanced chemiluminescence (CL), respectively. Transmission electron microscopy was used to observe the autophagosome formation, immunofluorescence staining was employed to detect LC3 expression and western blotting was used to measure the levels of PI3K/AKT/mTOR pathway- and autophagy-related proteins. Results: Ox-LDL-induced platelets showed a significant increase in platelet aggregation and adhesion, CD62p expression, ROS level and O2·- content, with an elevated LC3II/LC3I ratio and Beclin1 expression, but a dramatic reduction in the levels of p62 and pathway-related proteins (all P < 0.05). However, platelet activation and autophagy were aggravated by the Rapamycin treatment, and decreased following treatment with NAC, 3-MA, or NAC and 3-MA, together with increased activity of the PI3K/AKT/mTOR pathway. Additionally, decreased platelet activation and autophagy were observed in platelets treated with NAC and Rapamycin or Rapamycin and 3-MA compared with platelets treated with Rapamycin alone, suggesting that both NAC and 3-MA reversed the effects of Rapamycin. Conclusion: Inhibition of ROS production may reduce autophagy to suppress ox-LDL-induced platelet activation by activating PI3K/AKT/mTOR pathway.
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Nolfi-Donegan, Deirdre, Sruti Shiva, and Cheryl A. Hillery. "HMGB1 As a Novel Platelet Agonist That Acts Synergistically with ADP to Activate Platelets in Sickle Cell Disease." Blood 132, Supplement 1 (November 29, 2018): 1073. http://dx.doi.org/10.1182/blood-2018-99-110269.
Full textAbstract:
Abstract Background: Sickle cell disease (SCD) is a proinflammatory and prothrombotic disorder that exhibits increased platelet activation. High mobility group box 1 (HMGB1) is a nuclear protein that can mediate inflammation when released from inflammatory or ischemic cells. HMGB1 is increased in many inflammatory disease states including SCD. Recent data suggests HMGB1 activates platelets and may work synergistically with potent platelet agonists such as collagen and thrombin, but little is known regarding HMGB1-platelet interactions in combination with weaker agonists like ADP, or in isolated platelets. Moreover, the effect of HMGB1 on platelet activation has not been evaluated in SCD. We hypothesized that the in vitro addition of low-dose recombinant HMGBI (rHMBG1) to isolated platelets will lower the threshold dose of physiologic agonists required to achieve platelet activation, and that this effect is exaggerated in SCD. Methods: Platelets were isolated from healthy controls (n=4) and patients with hemoglobin SS disease (SCD; n=5). The level of platelet activation was assessed after treatment with ADP at concentrations of 0 μM, 0.5 μM, 2 μM, and 5 μM with the addition of either low-dose rHMGB1 (10 μg/mL) or the same volume of vehicle. Percent platelet activation was measured via flow cytometry using PE antibody to GPIIb (CD41) to select for platelets, and PAC1 to detect the activation-dependent conformational change in integrin αIIbβ3 (GP IIb-IIIa). Platelet activation was interpreted as percent of platelets that bound PAC1. Data was analyzed using FlowJo software and nonparametric statistical tests. Results: Mean baseline platelet activation was 1.5% (range 0.4-3.3%) for control platelets and 7.3% (1.4-17.7%) for SCD platelets (p=0.19). In the SCD group, the addition of low-dose rHMGB1 (10 μg/mL) increased the mean percent of activated platelets from 7.3% to 26.5% (10.9-43%) (p=0.01). In comparison, mean activation of control platelets increased from only 1.5% to 19.5% (8.3-42.7%) after addition of rHMGB1 (p=0.12). Having illustrated that rHMGB1 can activate washed SCD platelets, we then compared the synergistic effect of rHMGB1 with ADP. There was increased platelet activation observed when ADP was added to rHMGB1 in SCD platelets: ADP 0.5 μM increased mean platelet activation from 13.8% (range 0.3-25.3%) to 54.4% (6.7-84.9%) with the addition of rHMGB1 (p=0.02); ADP 2 μM increased platelet activation from 14.1% (2.8-23.8%) to 56.2% (22.2-88.6%) with rHMGB1 (p=0.006); and ADP 5 μM increased platelet activation from 21.4% (2.5-30.1%) to 65.3% (31.7-85.9%) after adding rHMGB1 (p=0.004) (Fig 1; * and **, diamonds vs stars). We did not find a similar statistically significant synergistic effect in the control samples treated with ADP compared to combined HMGB1 + ADP, except at ADP dose 2 μM where platelet activation increased from 14.94% (4.6-28.6%) to 39.1% (19.5-56.0%) after the addition of rHMGB1 (p=0.04; Fig 1; #, square vs circle). Activation of platelets with just ADP was not different comparing control with SCD platelets (Fig 1; circles vs stars). Similarly, activation of platelets with both ADP and rHMGB1 was not significantly different comparing control with SCD platelets except for a trend at 0.5 μM ADP + rHMGB1 10 μg/mL with 19.29% (6.6-38.7) in controls vs 54.44% (6.7-84.9) in the SCD group (p=0.07) (Fig 1; diamonds vs squares). Summary: We found that rHMGB1 acts both independently and synergistically with ADP to increase platelet activation in SCD platelets. In our small cohort, SCD platelets had increased responsiveness to low dose-rHMGB1 compared to control platelets. Moreover, combining rHMGB1 with ADP greatly enhanced platelet activation in SCD but not control platelets. Our data suggest that SCD platelets are sensitized to HMGB1 in the presence of weaker agonists such as ADP. This heightened responsiveness of SCD platelets to HMGB1 may explain the enhanced platelet activation and inflammation associated with SCD in vivo. With further study, HMGB1 could be a target of clinical drug-directed therapy in SCD patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Lagarrigue, Frederic, Alexandre R. Gingras, David S. Paul, Andrew J. Valadez, Monica N. Cuevas, Hao Sun, Miguel A. Lopez-Ramirez, et al. "Rap1 binding to the talin 1 F0 domain makes a minimal contribution to murine platelet GPIIb-IIIa activation." Blood Advances 2, no. 18 (September 21, 2018): 2358–68. http://dx.doi.org/10.1182/bloodadvances.2018020487.
Full textAbstract:
Abstract Activation of platelet glycoprotein IIb-IIIa (GPIIb-IIIa; integrin αIIbβ3) leads to high-affinity fibrinogen binding and platelet aggregation during hemostasis. Whereas GTP-bound Rap1 GTPase promotes talin 1 binding to the β3 cytoplasmic domain to activate platelet GPIIb-IIIa, the Rap1 effector that regulates talin association with β3 in platelets is unknown. Rap1 binding to the talin 1 F0 subdomain was proposed to forge the talin 1–Rap1 link in platelets. Here, we report a talin 1 point mutant (R35E) that significantly reduces Rap1 affinity without a significant effect on its structure or expression. Talin 1 head domain (THD) (R35E) was of similar potency to wild-type THD in activating αIIbβ3 in Chinese hamster ovary cells. Coexpression with activated Rap1b increased activation, and coexpression with Rap1GAP1 reduced activation caused by transfection of wild-type THD or THD(R35E). Furthermore, platelets from Tln1R35E/R35E mice showed similar GPIIb-IIIa activation to those from wild-type littermates in response to multiple agonists. Tln1R35E/R35E platelets exhibited slightly reduced platelet aggregation in response to low doses of agonists; however, there was not a significant hemostatic defect, as judged by tail bleeding times. Thus, the Rap1–talin 1 F0 interaction has little effect on platelet GPIIb-IIIa activation and hemostasis and cannot account for the dramatic effects of loss of Rap1 activity on these platelet functions.