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Background: Protease-activated receptor 4 (PAR4) mediates thrombin signaling on platelets and other cells. Our recent structural studies demonstrated that a single nucleotide polymorphism in extracellular loop 3 and PAR4-P310L (rs2227376) leads to a hyporeactive receptor., Objectives: The goal of this study was to determine how the hyporeactive PAR4 variant in extracellular loop 3 impacts platelet function in vivo using a novel knock-in mouse model (PAR4-322L)., Methods: A point mutation was introduced into the PAR4 gene F2rl3 via CRISPR/Cas9 to create PAR4-P322L, the mouse homolog to human PAR4-P310L. Platelet response to PAR4 activation peptide (AYPGKF), thrombin, ADP, and convulxin was monitored by αIIbβ3 integrin activation and P-selectin translocation using flow cytometry or platelet aggregation. In vivo responses were determined by the tail bleeding assay and the ferric chloride-induced carotid artery injury model., Results: PAR4-P/L and PAR4-L/L platelets had a reduced response to AYPGKF and thrombin measured by P-selectin translocation or αIIbβ3 activation. The response to ADP and convulxin was unchanged among genotypes. In addition, both PAR4-P/L and PAR4-L/L platelets showed a reduced response to thrombin in aggregation studies. There was an increase in the tail bleeding time for PAR4-L/L mice. The PAR4-P/L and PAR4-L/L mice both showed an extended time to arterial thrombosis., Conclusion: PAR4-322L significantly reduced platelet responsiveness to AYPGKF and thrombin, which is in agreement with our previous structural and cell signaling studies. In addition, PAR4-322L had prolonged arterial thrombosis time. Our mouse model provides a foundation to further evaluate the role of PAR4 in other pathophysiological contexts., Competing Interests: Declaration of competing interests There are no competing interests to disclose, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Thromboelastography (TEG) is used for real-time determination of hemostatic status in patients with acute risk of bleeding. Thrombin is thought to drive clotting in TEG through generation of polymerized fibrin and activation of platelets through protease-activated receptors (PARs). However, the specific role of platelet agonist receptors and signaling in TEG has not been reported., Objectives: Here, we investigated the specific receptors and signaling pathways required for platelet function in TEG using genetic and pharmacologic inhibition of platelet proteins in mouse and human blood samples., Methods: Clotting parameters (R time, α-angle [α], and maximum amplitude [MA]), were determined in recalcified, kaolin-triggered citrated blood samples using a TEG 5000 analyzer., Results: We confirmed the requirement of platelets, platelet contraction, and αIIbβ3 integrin function for normal α and MA. Loss of the integrin adaptor Talin1 in megakaryocytes/platelets (Talin1 mKO ) also reduced α and MA, but only minimal defects were observed in samples from mice lacking Rap1 GTPase signaling. PAR4 mKO samples showed impaired α but normal MA. However, impaired TEG traces similar to those in platelet-depleted samples were observed with samples from PAR4 mKO mice depleted of glycoprotein VI on platelets or with addition of a Syk inhibitor. We reproduced these results in human blood with combined inhibition of PAR1, PAR4, and Syk., Conclusion: Our results demonstrate that standard TEG is not sensitive to platelet signaling pathways critical for integrin inside-out activation and platelet hemostatic function. Furthermore, we provide the first evidence that PARs and glycoprotein VI play redundant roles in platelet-mediated clot contraction in TEG., Competing Interests: Declaration of competing interests There are no competing interests to disclose., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Human and mouse platelets both express protease-activated receptor (PAR) 4 but sequence alignment reveals differences in several functional domains. These differences may result in functional disparities between the receptors which make it difficult to translate PAR4 studies using mice to human platelet physiology., Objectives: To generate transgenic mice that express human, but not mouse, PAR4 and directly compare human and mouse PAR4 function in the same platelet environment., Methods: Transgenic mice were made using a genomic clone of the F2RL3 gene (encoding PAR4) and backcrossed with Par4 KO mice. For certain experiments, mice were bred with GRK6 KO mice. Tail bleeding time and platelet function in response to PAR4-activating peptide were assessed., Results: Human F2RL3 was successfully integrated into the mouse genome, transgenic mice were crossed to the mPar4 KO background (PAR4 tg/KO), and PAR4 was functionally expressed on platelets. Compared to WT, PAR4 tg/KO mice exhibited shortened tail bleeding time and their platelets were more responsive to PAR4-AP as assessed by α-granule release and integrin activation. The opposite was observed with thrombin. Knocking out GRK6 had no effect on human PAR4-expressing platelets, unlike mouse Par4-expressing platelets. PAR4 tg/KO platelets exhibited greater Ca 2+ area under the curve and more robust extracellular vesicle release than WT stimulated with PAR4-AP., Conclusion: These data suggest that (1) human PAR4- and mouse Par4-mediated signaling are different and (2) the feedback regulation mechanisms of human and mouse PAR4 are different. These functional differences are important to consider when interpreting PAR4 studies done with mice., (© 2022 International Society on Thrombosis and Haemostasis.)

Background: Protease-activated receptor 4 (PAR4) is expressed by a wide variety of cells, including megakaryocytes/platelets, immune cells, cardiomyocytes, and lung epithelial cells. It is the only functional thrombin receptor on murine platelets. A global deficiency of PAR4 is associated with impaired hemostasis and reduced thrombosis., Objective: We aimed to generate a mouse line with a megakaryocyte/platelet-specific deletion of PAR4 (PAR4 fl/fl ;PF4 Cre+ ) and use the mouse line to investigate the role of platelet PAR4 in hemostasis and thrombosis in mice., Methods: Platelets from PAR4 fl/fl ;PF4 Cre+ were characterized in vitro. Arterial and venous thrombosis was analyzed. Hemostatic plug formation was analyzed using a saphenous vein laser injury model in mice with global or megakaryocyte/platelet-specific deletion of PAR4 or wild-type mice treated with thrombin or glycoprotein VI (GPVI) inhibitors., Results: PAR4 fl/fl ;PF4 Cre+ platelets were unresponsive to thrombin or specific PAR4 stimulation but not to other agonists. PAR4 -/- and PAR4 fl/fl ;PF4 Cre+ mice both exhibited a similar reduction in arterial thrombosis compared to their respective controls. More importantly, we show for the first time that platelet PAR4 is critical for venous thrombosis in mice. In addition, PAR4 -/- mice and PAR4 fl/fl ;PF4 Cre+ mice exhibited a similar impairment in hemostatic plug stability in a saphenous vein laser injury model. Inhibition of thrombin in wild-type mice gave a similar phenotype. Combined PAR4 deficiency on platelets with GPVI inhibition did not impair hemostatic plug formation but further reduced plug stability., Conclusion: We generated a novel PAR4 fl/fl ;PF4 Cre+ mouse line. We used this mouse line to show that PAR4 signaling in platelets is critical for arterial and venous thrombosis and hemostatic plug stability., (© 2021 International Society on Thrombosis and Haemostasis.)

Essentials The rs773902 SNP results in differences in platelet protease-activated receptor (PAR4) function. The functional consequences of rs773902 were analyzed in human platelets and stroke patients. rs773902 affects thrombin-induced platelet function, PAR4 desensitization, stroke association. Enhanced PAR4 Thr120 effects on platelet function are blocked by ticagrelor. SUMMARY: Background F2RL3 encodes protease-activated receptor (PAR) 4 and harbors an A/G single-nucleotide polymorphism (SNP) (rs773902) with racially dimorphic allelic frequencies. This SNP mediates an alanine to threonine substitution at residue 120 that alters platelet PAR4 activation by the artificial PAR4-activation peptide (PAR4-AP) AYPGKF. Objectives To determine the functional effects of rs773902 on stimulation by a physiological agonist, thrombin, and on antiplatelet antagonist activity. Methods Healthy human donors were screened and genotyped for rs773902. Platelet function in response to thrombin was assessed without and with antiplatelet antagonists. The association of rs773902 alleles with stroke was assessed in the Stroke Genetics Network study. Results As compared with rs773902 GG donors, platelets from rs773902 AA donors had increased aggregation in response to subnanomolar concentrations of thrombin, increased granule secretion, and decreased sensitivity to PAR4 desensitization. In the presence of PAR1 blockade, this genotype effect was abolished by higher concentrations of or longer exposure to thrombin. We were unable to detect a genotype effect on thrombin-induced PAR4 cleavage, dimerization, and lipid raft localization; however, rs773902 AA platelets required a three-fold higher level of PAR4-AP for receptor desensitization. Ticagrelor, but not vorapaxar, abolished the PAR4 variant effect on thrombin-induced platelet aggregation. A significant association of modest effect was detected between the rs773902 A allele and stroke. Conclusion The F2RL3 rs773902 SNP alters platelet reactivity to thrombin; the allelic effect requires P2Y 12 , and is not affected by gender. Ticagrelor blocks the enhanced reactivity of rs773902 A platelets. PAR4 encoded by the rs773902 A allele is relatively resistant to desensitization and may contribute to stroke risk., (© 2018 International Society on Thrombosis and Haemostasis.)

Essentials Roles of the two thrombin receptors in platelet signaling are poorly understood. Computational systems biology modeling was used together with continuous flow cytometry. Dual-receptor system has wide-range sensitivity to thrombin and optimal response dynamics. Procoagulant platelet formation is determined by donor-specific activities of the two receptors., Summary: Background Activation of human platelets with thrombin proceeds via two protease-activated receptors (PARs), PAR1 and PAR4, that have identical main intracellular signaling responses. Although there is evidence that they have different cleavage/inactivation kinetics (and some secondary variations in signaling), the reason for such redundancy is not clear. Methods We developed a multicompartmental stochastic computational systems biology model of dual-receptor thrombin signaling in platelets to gain insight into the mechanisms and roles of PAR1 and PAR4 functioning. Experiments employing continuous flow cytometry of washed human platelets were used to validate the model and test its predictions. Activity of PAR receptors in donors was evaluated by mRNA measurement and by polymorphism sequencing. Results Although PAR1 activation produced rapid and short-lived response, signaling via PAR4 developed slowly and propagated in time. Response of the dual-receptor system was both rapid and prolonged in time. Inclusion of PAR1/PAR4 heterodimer formation promoted PAR4 signaling in the medium range of thrombin concentration (about 10 nm), with little contribution at high and low thrombin. Different dynamics and dose-dependence of procoagulant platelet formation in healthy donors was associated with individual variations in PAR1 and PAR4 activities and particularly by the Ala120Thr polymorphism in the F2RL3 gene encoding PAR4. Conclusions The dual-receptor combination is critical to produce a response combining three critical advantages: sensitivity to thrombin concentration, rapid onset and steady propagation; specific features of the protease-activated receptors do not allow combination of all three in a single receptor., (© 2016 International Society on Thrombosis and Haemostasis.)

Background: The high allelic frequency of the prothrombotic Leiden polymorphism in human blood coagulation factor V (FV) has been speculated to reflect positive selection during evolution. Heterozygous Leiden carriers enrolled in the placebo arm of the PROWESS sepsis trial and heterozygous Leiden mice challenged with endotoxin both showed reduced mortality, whereas homozygous Leiden mice were not protected from lethal endotoxemia. Follow-up analyses of clinical outcomes and of mouse models of infection with various pathogens remained inconclusive., Objective: To establish whether activated protein C resistance of FV Leiden modifies the outcome of bacterial infection in murine sepsis models., Methods: Homozygous and heterozygous FV Leiden mice were subjected to gram-positive (S. aureus) or gram-negative (Y. pestis; E. coli) septic peritonitis or polymicrobial, focal septic peritonitis induced by cecal ligation and puncture. The effect of FV Leiden on 7-day survival and bacterial dissemination was assessed. Outcomes were compared with the sepsis survival of mice with genetically impaired hemostasis (hemophilia A, thrombocytopenia, thrombin receptor PAR4 [protease activated receptor 4] deficiency, endothelial protein C receptor [ProcR/EPCR] deficiency)., Results: Heterozygous, but not homozygous, Leiden mice were protected from lethal infection with highly virulent S. aureus and Y. pestis strains. FV Leiden did not affect the outcome of sepsis induced by cecal ligation and puncture, staphylokinase-deficient S. aureus, Pla-deficient Y. pestis, or E. coli. Thrombocytopenia, deficiency of PAR1 or PAR4 did not affect S. aureus sepsis survival, whereas hemophilia A increased mortality. ProcR deficiency selectively abolished the survival advantage of heterozygous Leiden mice., Conclusions: In mice, heterozygous FV Leiden carriers are protected from sepsis mortality after infection with clinically relevant human bacterial pathogens., (© 2015 International Society on Thrombosis and Haemostasis.)

Background: The coagulation cascade has been shown to participate in chronic liver injury and fibrosis, but the contribution of various thrombin targets, such as protease activated receptors (PARs) and fibrin(ogen), has not been fully described. Emerging evidence suggests that in some experimental settings of chronic liver injury, platelets can promote liver repair and inhibit liver fibrosis. However, the precise mechanisms linking coagulation and platelet function to hepatic tissue changes following injury remain poorly defined., Objectives: To determine the role of PAR-4, a key thrombin receptor on mouse platelets, and fibrin(ogen) engagement of the platelet αII b β3 integrin (αIIb β3 ) in a model of cholestatic liver injury and fibrosis., Methods: Biliary and hepatic injury was characterized following 4 week administration of the bile duct toxicant α-naphthylisothiocyanate (ANIT) (0.025%) in PAR-4-deficient mice, mice expressing a mutant form of fibrin(ogen) incapable of binding integrin αII b β3 (Fibγ(Δ5) ), and wild-type mice., Results: Elevated plasma thrombin-antithrombin and serotonin levels, hepatic fibrin deposition, and platelet accumulation in liver accompanied hepatocellular injury and fibrosis in ANIT-treated wild-type mice. PAR-4 deficiency reduced plasma serotonin levels, increased serum bile acid concentration, and exacerbated ANIT-induced hepatocellular injury and peribiliary fibrosis. Compared with PAR-4-deficient mice, ANIT-treated Fibγ(Δ5) mice displayed more widespread hepatocellular necrosis accompanied by marked inflammation, robust fibroblast activation, and extensive liver fibrosis., Conclusions: Collectively, the results indicate that PAR-4 and fibrin-αII b β3 integrin engagement, pathways coupling coagulation to platelet activation, each exert hepatoprotective effects during chronic cholestasis., (© 2014 International Society on Thrombosis and Haemostasis.)

Platelets from protease-activated receptor 4 (PAR4)-deficient mice are unresponsive to thrombin, and Par4-/- mice have prolonged bleeding times and are protected against thrombosis. However, in addition to its role in platelets, PAR4 contributes to thrombin signaling in cells in the blood vessel wall that might participate in hemostasis and thrombosis, such as endothelial cells. To determine whether the hemostatic and thrombotic phenotypes of Par4-/- mice were due to loss of PAR4 function in hematopoietic vs. other cell types, tail bleed times and thromboplastin-induced pulmonary embolism were examined in lethally irradiated mice reconstituted with Par4+/+ or Par4-/- bone marrow. In Par4+/+ and Par4-/- mice reconstituted with Par4+/+ marrow, the median tail bleed times were 2.0 and 1.7 min, respectively, vs. > 10 min for both Par4+/+ and Par4-/- mice reconstituted with Par4-/- marrow. In the pulmonary embolism model, Par4+/+ and Par4-/- mice reconstituted with Par4+/+ marrow survived a median of 3.7 and 2.8 min, respectively, after administration of thromboplastin, vs. > 20 min for both Par4+/+ and Par4-/- mice reconstituted with Par4-/- marrow. Further, the phenotype of mice reconstituted with Par4-/- marrow was almost as dramatic as that seen in Nf-e2-/- mice, which lack platelets. These data strongly suggest that increased tail bleed times and protection against thrombosis in Par4-/- mice are accounted for by lack of PAR4 function in platelets, emphasize the importance of thrombin signaling in platelets among the multiple pathways and cell types that govern hemostasis and thrombosis.

Unlabelled: Coagulation factors (F)VIIa, FXa and thrombin are implicated in cellular responses in vascular, mesenchymal and inflammatory cells. Fibroblasts are the most abundant cells in connective tissue, and damage to blood vessels places coagulation factors in contact with these and other cell types., Objectives: To investigate cellular responses of primary dermal fibroblasts to FVIIa, FXa and thrombin by following changes in expression of candidate proteins: monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF), and to determine the expression of receptors implicated in signaling by these coagulation factors., Methods: Steady-state mRNA levels were quantified by RNase protection assay, and protein secretion by ELISA. PAR gene expression was assessed by ribonuclease protection assay and conventional and quantitative reverse-transcription-polymerase chain reaction., Results: FVIIa did not induce the candidate genes. In contrast, FXa and thrombin induced MCP-1 mRNA and protein secretion strongly, IL-8 moderately, and IL-6 weakly. Neither FXa nor thrombin induced VEGF mRNA or protein secretion, although FXa induced VEGF protein secretion in lung fibroblasts. Comparison of the presence of candidate receptors in the two fibroblast subtypes demonstrated higher levels of PAR-1 and PAR-3 in lung fibroblasts relative to their dermal counterparts and the additional expression of PAR-2., Conclusions: FXa and thrombin induce expression of MCP-1, IL-8 and IL-6, and distribution and expression of PARs on dermal fibroblasts is reduced relative to their lung counterparts. Tissue origin may influence the cellular response of fibroblasts to coagulation proteases.