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Background:  The C-type lectin receptor CD93 is a single pass type I transmembrane glycoprotein involved in inflammation, immunity, and angiogenesis. This study investigates the role of CD93 in platelet function. CD93 knockout (KO) mice and wild-type (WT) controls were compared in this study., Methods:  Platelet activation and aggregation were investigated by flow cytometry and light transmission aggregometry, respectively. Protein expression and phosphorylation were analyzed by immunoblotting. Subcellular localization of membrane receptors was investigated by wide-field and confocal microscopy., Results:  The lack of CD93 in mice was not associated to any evident bleeding defect and no alterations of platelet activation were observed upon stimulation with thromboxane A2 analogue and convulxin. Conversely, platelet aggregation induced by stimulation of the thrombin receptor PAR4 was significantly reduced in the absence of CD93. This defect was associated with a significant reduction of α-granule secretion, integrin αIIbβ3 activation, and protein kinase C (PKC) stimulation. Resting WT and CD93-deficient platelets expressed comparable amounts of PAR4. However, upon stimulation with a PAR4 activating peptide, a more pronounced clearance of PAR4 from the platelet surface was observed in CD93-deficient platelets compared with WT controls. Confocal microscopy analysis revealed a massive movement of PAR4 in cytosolic compartments of activated platelets lacking CD93. Accordingly, platelet desensitization following PAR4 stimulation was more pronounced in CD93 KO platelets compared with WT controls., Conclusion:  These results demonstrate that CD93 supports platelet activation triggered by PAR4 stimulation and is required to stabilize the expression of the thrombin receptor on the cell surface., Competing Interests: None declared., (Thieme. All rights reserved.)

Background:  Protease-activated receptor 4 (PAR4) is a platelet thrombin receptor important for thrombosis and a target of antiplatelet drug development. A frequently occurring single-nucleotide polymorphism (rs773902) causes a PAR4 sequence variant (NC_000019.10:p.Ala120Thr) whereby platelets from Thr120-expressing individuals are hyperresponsive to PAR4 agonists versus platelets from Ala120-expressing individuals. However, whether this enhanced platelet responsiveness translates to increased thrombotic risk or decreased bleeding risk remains unknown., Objectives:  This article examines the association of rs773902 with adjudicated cardiovascular events and aspirin use in a randomized trial population of healthy older individuals., Methods:  We analyzed 13,547 participants in the ASPirin in Reducing Events in the Elderly trial. Participants had no previous cardiovascular events at enrollment and were randomized to either 100 mg daily aspirin or placebo for a median follow-up of 4.7 years. Total genotypes were 8,761 (65%) GG (Ala120 variant), 4,303 (32%) heterozygotes, and 483 (4%) AA (Thr120 variant). Cox proportional hazard regression tested the relationship between rs773902 and thrombotic events (major adverse cardiovascular events [MACE] and ischemic stroke [IS]) and bleeding (major hemorrhage [MHEM] and intracranial bleeding [ICB])., Results:  No statistically significant association was observed overall or by treatment group between rs773902 and any thrombotic or bleeding event examined. Further, there was no significant interaction between rs773902 and treatment for any of MACE, IS, MHEM, or ICB., Conclusion:  This post hoc analysis of a prospective cohort study suggests that, despite sensitizing platelet activation, the rs773902 PAR4 variant is not associated with thrombotic cardiovascular or bleeding events in a healthy older population., Competing Interests: None declared., (Thieme. All rights reserved.)

Platelet activation in response to stimulation of the Protease Activated Receptor 4 (PAR4) receptor differs by race. One factor that contributes to this difference is the expression level of Phosphatidylcholine Transfer Protein (PCTP), a regulator of platelet PAR4 function. We have conducted an expression Quantitative Trait Locus (eQTL) analysis that identifies single nucleotide polymorphisms (SNPs) linked to the expression level of platelet genes. This analysis revealed 26 SNPs associated with the expression level of PCTP at genome-wide significance (p < 5×10 -8 ). Using annotation from ENCODE and other public data we prioritised one of these SNPs, rs2912553, for functional testing. The allelic frequency of rs2912553 is racially-dimorphic, in concordance with the racially differential expression of PCTP. Reporter gene assays confirmed that the single nucleotide change caused by rs2912553 altered the transcriptional potency of the surrounding genomic locus. Electromobility shift assays, luciferase assays, and overexpression studies indicated a role for the megakaryocytic transcription factor GATA1. In summary, we have integrated multi-omic data to identify and functionalise an eQTL. This, along with the previously described relationship between PCTP and PAR4 function, allows us to characterise a genotype-phenotype relationship through the mechanism of gene expression.

Thrombin-induced platelet activation via PAR1 and PAR4 is an important event in haemostasis. Although the underlying mechanisms responsible for ensuring efficient PAR1 activation by thrombin have been extensively studied, the potential involvement of recognitions sites outside the active site of the protease in thrombin-induced PAR4 activation is largely unknown. In this study, we developed a new assay to assess the importance of exosite I and II for PAR4 activation with α - and γ-thrombin. Surprisingly, we found that exosite II is critical for activation of PAR4. We also show that this dependency on exosite II likely represents a new mechanism, as it is unaffected by blockage of the previously known interaction between thrombin and glycoprotein Ibα.

To identify a physiological agonist of PAR3, we used PAR4 null murine platelets, which were known to express only PAR3. In this study, we tested several proteases and found that trypsin, but not heat-inactivated trypsin, activated PAR4 null murine platelets. Even at high concentrations, trypsin caused shape change without increasing intracellular calcium levels in PAR4 null murine platelets. Consistent with this result, the Gq inhibitor YM-254890 had no effect on trypsin-induced shape change. However, trypsin-induced platelet shape change was abolished by either p160ROCK inhibitor, Y27632 or H1152. Furthermore, trypsin caused phosphorylation of myosin light chain (Thr18), but not Akt or Erk. Surprisingly, trypsin caused a similar shape change in PAR4-desensitised PAR3 null murine platelets as in PAR4null murine platelets, indicating that trypsin did not activate PAR3 to cause shape change. More interestingly, the Src family kinase (SFK) inhibitor PP2 abolished trypsin-induced, but not AYPGKF-induced, shape change. Hence, trypsin activated a novel signalling pathway through RhoA/p160ROCK and was regulated by SFKs. In conclusion, our study demonstrates a novel protease signalling pathway in platelets that is independent of PARs. This protease-induced novel signalling pathway regulates platelet shape change through SFKs and p160ROCK.

Extravascular activation of the coagulation cascade in the lung is commonly observed in pulmonary fibrosis. Coagulation proteases may exert profibrotic cellular effects via protease-activated receptors (PARs)-1 and -2. Here, we investigated the potential role of two other members of the PAR family, namely PAR-3 and PAR-4, in the pathobiology of lung fibrosis. Elevated expression of PAR-3, but not PAR-4, was detected in the lungs of idiopathic pulmonary fibrosis (IPF) patients and in bleomycin-induced lung fibrosis in mice. Increased PAR-3 expression in fibrotic lungs was mainly attributable to alveolar type II (ATII) cells. Stimulation of primary mouse ATII, MLE15 and A549 cells with thrombin (FIIa) - that may activate PAR-1, PAR-3 and PAR-4 - induced epithelial-mesenchymal transition (EMT), a process that has been suggested to be a possible mechanism underlying the expanded (myo)fibroblast pool in lung fibrosis. EMT was evidenced by morphological alterations, expression changes of epithelial and mesenchymal phenotype markers, and functional changes. Single knockdown of FIIa receptors, PAR-1, PAR-3, or PAR-4, had no major impact on FIIa-induced EMT. Simultaneous depletion of PAR-1 and PAR-3, however, almost completely inhibited this process, whereas only a partial effect on FIIa-mediated EMT was observed when PAR-1 and PAR-4, or PAR-3 and PAR-4 were knocked down. PAR-1 and PAR-3 co-localise within ATII cells with both being predominantely plasma membrane associated. In conclusion, our study indicates that PARs synergise to mediate FIIa-induced EMT and provides first evidence that PAR-3 via its ability to potentiate FIIa-triggered EMT could potentially contribute to the pathogenesis of pulmonary fibrosis.

In our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

Developing novel anti-platelet therapies is an important clinical strategy for the prevention of arterial thromboses which cause heart attacks and most strokes. Thrombin activates platelets via protease-activated receptors (PARs), and PAR antagonists are currently under investigation as antithrombotics. Yet despite these clinical advances, the importance of PARs to platelet activation during thromboses formed under pathological conditions has not been investigated. To this end, we examined the role of PAR-dependent platelet activation in thrombus formation in the presence of elevated blood shear rates. We used two in vivo thrombosis models and an ex vivo whole blood flow approach in PAR4(-/-) mice, whose platelets are unresponsive to thrombin, to show that the contribution of PAR-mediated platelet activation to thrombosis is diminished at pathological blood shear rates as a direct result of decreased incorporation of thrombin-activated platelets into growing thrombi. Our ex vivo observations were replicated in human whole blood treated with a PAR1 antagonist. These results define a novel, shear-regulated role for thrombin/PAR-dependent platelet activation during thrombosis and provide important insights into the conditions under which PAR antagonists may best be used for the prevention of acute coronary syndromes.

The vast majority of thrombin (>95%) is generated after clotting is completed, suggesting that thrombin formation serves purposes beyond coagulation, such as tissue repair after vessel injury. Two types of vascular thrombin binding sites exist: protease-activated receptors (PARs) and thrombomodulin (TM). Their expression is low in contractile vascular smooth muscle cells (SMC), the dominating subendothelial cell population, but becomes markedly up-regulated upon injury. In human SMC, PAR-1, PAR-3, and PAR-4 mediate thrombin-induced proliferation, migration and matrix biosynthesis as well as generation of inflammatory and growth-promoting mediators. Thrombin-responsive PARs are transcriptionally down-regulated in human vascular SMC by vasodilatory prostaglandins (PGI2/PGE2). For PAR-1 and PAR-3 this mechanism involves cAMP-dependent inactivation of the transcription factor NFAT. The human PAR-4 promoter does not possess NFAT recognition motifs suggesting involvement of other cAMP-regulated effectors. Unlike PARs, TM is induced in SMC exposed to vasodilatory prostaglandins. Enhanced thrombin binding to TM might ameliorate PAR-mediated SMC stimulation. Also expressed in human SMC is the endothelial protein C receptor (EPCR), which serves as an anchor to facilitate generation of activated protein C (aPC) by TM-bound thrombin. Whether prostaglandins affect aPC-generation is not known. In SMC, thrombin and aPC act synergistically via PAR-1 to modify tissue remodelling, in contrast to their antagonistic interaction in the coagulation pathways. Overall, this will contribute to plaque stability and wound healing. The processes outlined here are likely to become clinically relevant after up-regulation of vascular cyclooxygenase2, the rate limiting step in vascular PGE2/PGI2 biosynthesis, such as in advanced atherosclerosis and acute coronary syndromes.

The present study investigates whether vascular smooth muscle cells of the human saphenous vein (SMC) express a functionally active protease-activated receptor-3 (PAR-3). PAR-3 mRNA was detected by RT-PCR. In the presence of thrombin, a rapid and transient increase in PAR-3 mRNA was observed. Stimulation of SMC with thrombin or the synthetic PAR-3-activating peptide, TFRGAP, resulted in transient mobilization of intracellular calcium. After a preceding challenge with thrombin, the calcium signal to TFRGAP was abolished, suggesting cleavage and subsequent desensitization of PAR-3 by thrombin. Activation of PAR-3 by TFRGAP elicited a time-dependent activation of the extracellular-signal-regulated kinase (ERK)-1/2 with a maximum response 10-20 min after stimulation. At 200 microM, TFRGAP increased [3H]-thymidine incorporation into cellular DNA about two-fold. These data indicate that PAR-3 is expressed in human SMC and triggers intracellular signaling. Thus, in the SMC PAR-3 might contribute to thrombin-induced responses.

Putative binding sites in a platelet thrombin receptor (PAR-1) for the activating peptide SFLLRNPNDKYEPF (AP) and for the bradykinin analogue MKRPPGFSPFRSSRIG were revealed using a computer program for identifying complementary peptide segments. The program is based on the assumption that interactions of agonist's peptides and protein's receptors can be elucidated by complementary average hydropathies as much as possible equal by size and opposite by sign. Some of the computer-found putative binding sites were close to the supposed AP-PAR-1 contacts in the amino-terminal exodomain and in the second extracellulary loop of PAR-1. Peptides complementary to these binding sites were also computer-designed and were synthesized. They mostly inhibited the aggregation of gel filtered platelets by thrombin (0.025 U/mL) with IC50 in a high micromolar range of concentrations. The peptide complementary to site L258-Y269 of PAR-1 induced aggregation of gel filtered platelets with EC50 = 98 [micromol/L] related to thrombin (0.025 U/mL) aggregation response.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

The identification of the human thrombin receptor has prompted considerable interest in the development of receptor antagonists that can inhibit the effects of thrombin on cells without affecting fibrin formation. Increasing information about the biology of the receptor highlights some of the potential pitfalls in the development and application of such agents. However, it also provides additional insights into the potential mechanisms by which an antagonist might work beyond the original approach of developing competitive inhibitors of the tethered ligand domain. Thrombin receptors have proved to be present on a variety of cells other than platelets, including other vascular cells and cells within the central nervous system. Although most of the recent emphasis in thinking about the need for anti-thrombin receptor agents has focused on their use as anti-platelet agents, it is worth considering that the best clinical uses of such drugs may ultimately prove to have little to do with platelets.