Glycoprotein Ib-V-IX, a Receptor for von Willebrand Factor, Couples Physically and Functionally to the Fc Receptor y-Chain, Fyn, and Lyn to Activate Human Platelets

The adhesion molecule von Willebrand factor (vWF) activates platelets upon binding 2 surface receptors, glycoprotein (GP) Ib-V-IX and integrin aIIbb3. We have used 2 approaches to selectively activate GP Ib using either the snake venom lectin alboaggregin-A or mutant recombinant forms of vWF (DA1-vWF and RGGS-vWF) with selective binding properties to its 2 receptors. We show that activation of GP Ib induces platelet aggregation, secretion of 5-hydroxy tryptamine (5-HT), and an increase in cytosolic calcium. Syk becomes tyrosine phosphorylated and activated downstream of GP Ib, and associates with several tyrosinephosphorylated proteins including the Fc receptor g-chain through interaction with Syk SH2 domains. GP Ib physically associates with the g-chain in GST-Syk-SH2 precipitates from platelets stimulated through GP Ib, and 2 Src family kinases, Lyn and Fyn, also associate with this signaling complex. In addition, GP Ib stimulation couples to tyrosine phosphorylation of phospholipase Cg2. The Src familyspecific inhibitor PP1 dose-dependently inhibits phosphorylation of Syk, its association with tyrosine-phosphorylated g-chain, phosphorylation of PLCg2, platelet aggregation, and 5-HT release. The results indicate that, upon activation, GP Ib is physically associated with FcR g-chain and members of the Src family kinases, leading to phosphorylation of the g-chain, recruitment, and activation of Syk. Phosphorylation of PLCg2 also lies downstream of Src kinase activation and may critically couple early signaling events to functional platelet responses

Do platelets promote cardiac recovery after myocardial infarction:Roles beyond occlusive ischemic damage

Our understanding of platelet function has traditionally focused on their roles in physiological hemostasis and pathological thrombosis, with the latter being causative of vessel occlusion and subsequent ischemic damage to various tissues. In particular, numerous in vivo studies have implicated causative roles for platelets in the pathogenesis of ischemia-reperfusion (I/R) injury to the myocardium. However, platelets clearly have more complex pathophysiological roles, particularly as a result of the heterogeneous nature of biologically active cargo secreted from their granules or contained within released microparticles or exosomes. While some of these released mediators amplify platelet activation and thrombosis through autocrine or paracrine amplification pathways, they can also regulate diverse cellular functions within the localized microenvironment and recruit progenitor cells to the damage site to facilitate repair processes. Notably, there is evidence to support cardioprotective roles for platelet mediators during I/R injury. As such, it is becoming more widely appreciated that platelets fulfill a host of physiological and pathological roles beyond our basic understanding. Therefore, the purpose of this perspective is to consider whether platelets, through their released mediators, can assume a paradoxically beneficial role to promote cardiac recovery after I/R injury. </jats:p

Absence of platelet phenotype in mice lacking the motor protein myosin Va.

BACKGROUND: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/-) platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIb)β(3) activation, Ca(2+) signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/-) platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. CONCLUSION: Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets

Genetic analysis of the role of protein kinase Ctheta in platelet function and thrombus formation.

BACKGROUND: PKCtheta is a novel protein kinase C isozyme, predominately expressed in T cells and platelets. PKCtheta(-/-) T cells exhibit reduced activation and PKCtheta(-/-) mice are resistant to autoimmune disease, making PKCtheta an attractive therapeutic target for immune modulation. Collagen is a major agonist for platelets, operating through an immunoreceptor-like signalling pathway from its receptor GPVI. Although it has recently been shown that PKCtheta positively regulates outside-in signalling through integrin alpha(IIb)beta(3) in platelets, the role of PKCtheta in GPVI-dependent signalling and functional activation of platelets has not been assessed. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we assessed static adhesion, cell spreading, granule secretion, integrin alpha(IIb)beta(3) activation and platelet aggregation in washed mouse platelets lacking PKCtheta. Thrombus formation on a collagen-coated surface was assessed in vitro under flow. PKCtheta(-/-) platelets exhibited reduced static adhesion and filopodia generation on fibrinogen, suggesting that PKCtheta positively regulates outside-in signalling, in agreement with a previous report. In contrast, PKCtheta(-/-) platelets also exhibited markedly enhanced GPVI-dependent alpha-granule secretion, although dense granule secretion was unaffected, suggesting that PKCtheta differentially regulates these two granules. Inside-out regulation of alpha(IIb)beta(3) activation was also enhanced downstream of GPVI stimulation. Although this did not result in increased aggregation, importantly thrombus formation on collagen under high shear (1000 s(-1)) was enhanced. CONCLUSIONS/SIGNIFICANCE: These data suggest that PKCtheta is an important negative regulator of thrombus formation on collagen, potentially mediated by alpha-granule secretion and alpha(IIb)beta(3) activation. PKCtheta therefore may act to restrict thrombus growth, a finding that has important implications for the development and safe clinical use of PKCtheta inhibitors

Carbonic Anhydrase Inhibitors Suppress Platelet Procoagulant Responses and In Vivo Thrombosis

Carbonic anhydrase (CA) inhibitors have a long history of safe clinical use as mild diuretics, in the treatment of glaucoma and for altitude sickness prevention. In this study, we aimed to determine if CA inhibition may be an alternative approach to control thrombosis. We utilized a high-resolution dynamic imaging approach to provide mechanistic evidence that CA inhibitors may be potent anti-procoagulant agents in vitro and effective anti-thrombotics in vivo. Acetazolamide and methazolamide, while sparing platelet secretion, attenuated intracellular chloride ion entry and suppressed the procoagulant response of activated platelets in vitro and thrombosis in vivo. The chemically similar N-methyl acetazolamide, which lacks CA inhibitory activity, did not affect platelet procoagulant response in vitro. Outputs from rotational thromboelastometry did not reflect changes in procoagulant activity and reveal the need for a suitable clinical test for procoagulant activity. Drugs specifically targeting procoagulant remodeling of activated platelets, by blockade of carbonic anhydrases, may provide a new way to control platelet-driven thrombosis without blocking essential platelet secretion responses

Blood platelets stimulate cancer extravasation through TGFβ-mediated downregulation of PRH/HHEX

Cancer cells go through a process known as epithelial–mesenchymal transition (EMT) during which they acquire the ability to migrate and invade extracellular matrix. Some cells also acquire the ability to move across a layer of endothelial cells to enter and exit the bloodstream; intra- and extravasation, respectively. The transcription factor PRH/HHEX (proline-rich homeodomain/haematopoietically expressed homeobox) controls cell proliferation and cell migration/invasion in a range of cell types. Our previous work showed that PRH activity is downregulated in prostate cancer cells owing to increased inhibitory PRH phosphorylation and that this increases cell proliferation and invasion. PRH inhibits migration and invasion by prostate and breast epithelial cells in part by activating the transcription of Endoglin, a transforming growth factor β (TGFβ) co-receptor. Here we show that depletion of PRH in immortalised prostate epithelial cells results in increased extravasation in vitro. We show that blood platelets stimulate extravasation of cells with depleted PRH and that inhibition of TGFβ signalling blocks the effects of platelets on these cells. Moreover, TGFβ induces changes characteristic of EMT including decreased E-Cadherin expression and increased Snail expression. We show that in prostate cells PRH regulates multiple genes involved in EMT and TGFβ signalling. However, both platelets and TGFβ increase PRH phosphorylation. In addition, TGFβ increases binding of its effector pSMAD3 to the PRH/HHEX promoter and downregulates PRH protein and mRNA levels. Thus, TGFβ signalling downregulates PRH activity by multiple mechanisms and induces an EMT that facilitates extravasation and sensitises cells to TGFβ

Syntaxin 8 regulates platelet dense granule secretion, aggregation, and thrombus stability.

Platelet secretion not only drives thrombosis and hemostasis, but also mediates a variety of other physiological and pathological processes. The ubiquitous SNARE machinery and a number of accessory proteins have been implicated in regulating secretion in platelet. Although several platelet SNAREs have been identified, further members of the SNARE family may be needed to fine-tune platelet secretion. In this study we identified expression of the t-SNARE syntaxin 8 (STX8) (Qc SNARE) in mouse and human platelets. In mouse studies, whereas STX8 was not essential for α-granule or lysosome secretion, Stx8(-/-) platelets showed a significant defect in dense granule secretion in response to thrombin and CRP. This was most pronounced at intermediate concentrations of agonists. They also showed an aggregation defect that could be rescued with exogenous ADP and increased embolization in Stx8(-/-) mice in vivo consistent with an important autocrine and paracrine role for ADP in aggregation and thrombus stabilization. STX8 therefore specifically contributes to dense granule secretion and represents another member of a growing family of genes that play distinct roles in regulating granule release from platelets and thus platelet function in thrombosis and hemostasis