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The thrombin receptor, protease-activated receptor 4 (PAR4), is important for platelet activation and is the target of emerging anti-thrombotic drugs. A frequently occurring single nucleotide polymorphism (SNP; rs773902) causes a function-altering PAR4 sequence variant (NC_000019.10:p.Ala120Thr), whereby platelets from Thr120-expressing individuals are hyper-responsive to PAR4 agonists and hypo-responsive to some PAR4 antagonists than platelets from Ala120-expressing individuals. This altered pharmacology may impact PAR4 inhibitor development, yet the underlying mechanism(s) remain unknown. We tested whether PAR4 surface expression contributes to the altered receptor function. Quantitative flow cytometry was used to determine the absolute number of PAR4 on platelets from individuals subsequently genotyped at rs773902. We detected 539 ± 311 PAR4 per platelet (mean ± SD, n = 84). This number was not different across rs773902 genotypes. This first determination of cellular PAR4 numbers indicates variations in platelet surface expression do not explain the altered pharmacology of the rs773902 PAR4 sequence variant. [ABSTRACT FROM AUTHOR]

Protease-activated receptor 4 (PAR4) is a cell surface G protein-coupled receptor for serine proteases, such as thrombin. Par4-/- mice have platelets that are unresponsive to thrombin and thereby allow examination of the importance of thrombin-induced platelet activation in (patho)physiology. Par4-/- mice are protected against arterial thrombosis but show no evidence of spontaneous bleeding. This contrasts with the bleeding experienced by mice with marked thrombocytopenia, such as those with genetic deficiency of the transcription factor, nuclear factor erythroid 2 (Nfe2-/-), that have high rates of perinatal death due to hemorrhage. Given this discrepancy in spontaneous perinatal bleeding between mice without platelets and those without thrombin-induced platelet activation mechanisms, we examined in detail the immediate postnatal survival of Par4-/-pups. We observed significant postpartum loss of Par4-/- pups derived from Par4+/- intercrosses that was restricted to a dam's first litter; only 9% of surviving pups genotyped as Par4-/- in first litters and this normalized from the second litter onward (26%). A similar perinatal lethality in pups delivered by primiparous dams occurred in mice lacking platelets (Nfe2-/-; 10%) but not in those lacking fibrinogen (Fga-/-; 26%). These data,, provide the first evidence of spontaneous bleeding in Par4-/- mice, suggest that a dam's first litter provides a greater hemostatic challenge than subsequent litters, and uncovers an important role for platelets--and more specifically thrombin-induced platelet activation--in hemostasis during these more traumatic births. [ABSTRACT FROM AUTHOR]

Protease-activated receptors (PARs) are a family of four G protein-coupled receptors that exhibit increasingly appreciated differences in signaling and regulation both within and between the receptor class. By nature of their proteolytic self-activation mechanism, PARs have unique processes of receptor activation, "ligand" binding, and desensitization/resensitization. These distinctive aspects have presented both challenges and opportunities in the targeting of PARs for therapeutic benefit--the most notable example of which is inhibition of PAR1 on platelets for the prevention of arterial thrombosis. However, more recent studies have uncovered further distinguishing features of PAR-mediated signaling, revealing mechanisms by which identical proteases elicit distinct effects in the same cell, as well as how distinct proteases produce different cellular consequences via the same receptor. Here we review this differential signaling by PARs, highlight how important distinctions between PAR1 and PAR4 are impacting on the progress of a new class of anti-thrombotic drugs, and discuss how these more recent insights into PAR signaling may present further opportunities for manipulating PAR activation and signaling in the development of novel therapies. [ABSTRACT FROM AUTHOR]

Protease-activated receptor 4 (PAR4) is a cell surface G protein-coupled receptor for serine proteases, such as thrombin. Par4 -/- mice have platelets that are unresponsive to thrombin and thereby allow examination of the importance of thrombin-induced platelet activation in (patho)physiology. Par4 -/- mice are protected against arterial thrombosis but show no evidence of spontaneous bleeding. This contrasts with the bleeding experienced by mice with marked thrombocytopenia, such as those with genetic deficiency of the transcription factor, nuclear factor erythroid 2 (Nfe2 -/- ), that have high rates of perinatal death due to hemorrhage. Given this discrepancy in spontaneous perinatal bleeding between mice without platelets and those without thrombin-induced platelet activation mechanisms, we examined in detail the immediate postnatal survival of Par4 -/- pups. We observed significant postpartum loss of Par4 -/- pups derived from Par4 +/- intercrosses that was restricted to a dam's first litter; only 9% of surviving pups genotyped as Par4 -/- in first litters and this normalized from the second litter onward (26%). A similar perinatal lethality in pups delivered by primiparous dams occurred in mice lacking platelets (Nfe2 -/- ; 10%) but not in those lacking fibrinogen (Fga -/- ; 26%). These data,, provide the first evidence of spontaneous bleeding in Par4 -/- mice, suggest that a dam's first litter provides a greater hemostatic challenge than subsequent litters, and uncovers an important role for platelets-and more specifically thrombin-induced platelet activation-in hemostasis during these more traumatic births.