Protease signaling in cells elicits multiple physiologically important responses via protease-activated receptors (PARs). There are 4 members of this family of G-protein–coupled receptors (PAR1-4). PARs are activated by proteolysis of the N terminus to reveal a tethered ligand. The rate-limiting step of PAR signaling is determined by the efficiency of proteolysis of the N terminus, which is regulated by allosteric binding sites, cofactors, membrane localization, and receptor dimerization. This ultimately controls the initiation of PAR signaling. In addition, these factors also control the cellular response by directing signaling toward G-protein or β-arrestin pathways. PAR1 signaling on endothelial cells is controlled by the activating protease and heterodimerization with PAR2 or PAR3. As a consequence, the genetic and epigenetic control of PARs and their cofactors in physiologic and pathophysiologic conditions have the potential to influence cellular behavior. Recent studies have uncovered polymorphisms that result in PAR4 sequence variants with altered reactivity that interact to influence platelet response. This further demonstrates how interactions within the plasma membrane can control the physiological output. Understanding the structural rearrangement following PAR activation and how PARs are allosterically controlled within the plasma membrane will determine how best to target this family of receptors therapeutically. The purpose of this article is to review how signaling from PARs is influenced by alternative cleavage sites and the physical interactions within the membrane. Going forward, it will be important to relate the altered signaling to the molecular arrangement of PARs in the cell membrane and to determine how these may be influenced genetically.

Treatment of bleeding disorders using transfusion of donor-derived platelets faces logistical challenges due to their limited availability, high risk of contamination, and short (5 to 7 days) shelf life. These challenges could be potentially addressed by designing platelet mimetics that emulate the adhesion, aggregation, and procoagulant functions of platelets. To this end, we created liposome-based platelet-mimicking procoagulant nanoparticles (PPNs) that can expose the phospholipid phosphatidylserine on their surface in response to plasmin. First, we tested PPNs in vitro using human plasma and demonstrated plasmin-triggered exposure of phosphatidylserine and the resultant assembly of coagulation factors on the PPN surface. We also showed that this phosphatidylserine exposed on the PPN surface could restore and enhance thrombin generation and fibrin formation in human plasma depleted of platelets. In human plasma and whole blood in vitro, PPNs improved fibrin stability and clot robustness in a fibrinolytic environment. We then tested PPNs in vivo in a mouse model of thrombocytopenia where treatment with PPNs reduced blood loss in a manner comparable to treatment with syngeneic platelets. Furthermore, in rat and mouse models of traumatic hemorrhage, treatment with PPNs substantially reduced bleeding and improved survival. No sign of systemic or off-target thrombotic risks was observed in the animal studies. These findings demonstrate the potential of PPNs as a platelet surrogate that should be further investigated for the management of bleeding.

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.

Protease activated receptor 4 (PAR4) mediates sustained thrombin signaling in platelets and is required for a stable thrombus. PAR4 is activated by proteolysis of the N-terminus to expose a tethered ligand. The structural basis for PAR4 activation and the location of its ligand binding site (LBS) are unknown. Using hydrogen/deuterium exchange (H/D exchange), computational modeling and signaling studies, we determined the molecular mechanism for tethered ligand-mediated PAR4 activation. H/D exchange identified that the LBS is composed by transmembrane domain 3 (TM3) and TM7. Unbiased computational modeling further predicted an interaction between Gly48 from the tethered ligand and Thr153 from the LBS. Mutating Thr153 significantly decreased PAR4 signaling. H/D exchange and modeling also showed that extracellular loop 3 (ECL3) serves as a gatekeeper for the interaction between the tethered ligand and LBS. A naturally occurring sequence variant (P310L, rs2227376) and two experimental mutations (S311A and P312L) determined that the rigidity conferred by prolines in ECL3 are essential for PAR4 activation. Finally, we examined the role of the polymorphism at position 310 in venous thromboembolism (VTE) using the INVENT consortium multi-ancestry GWAS meta-analysis. Individuals with the PAR4 Leu310 allele had a 15% relative risk reduction for VTE (odds ratio [OR]: 0.85; 95% CI: 0.77-0.94) compared to the Pro310 allele. These data are consistent with our H/D exchange, molecular modeling, and signaling studies. In conclusion, we have uncovered the structural basis for PAR4 activation and identified a previously unrecognized role for PAR4 in VTE.