Protease-activated receptors in hemostasis

Abstract

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.

Figure 1

PAR signaling is initiated by proteolysis of the N terminus to irreversibly activate the receptor by exposing the tethered ligand. (A-B) Proteases have distinct cleavage sites that generate unique tethered ligands that can direct the downstream signaling toward specific pathways. The PAR1 cleavage sites for thrombin, APC, MMP1, and MMP13 are shown as a representative example.

Figure 2

PARs use cofactors to regulate the specificity and rates of cleavage. (A) The current model of PAR4 activation on platelets. PAR1 serves as a cofactor to enhance the rate of PAR4 cleavage by thrombin. The PAR1-PAR4 cooperation is based on the model originally proposed for PAR3-PAR4 on mouse platelets by Nakanishi-Matsui et al. (B) PAR1 cleavage by APC requires the EPCR as a cofactor.

Figure 3

PAR family members interact with one another and other GPCRs as homodimers and heterodimers. (A) In addition to homodimers, PAR1 and PAR4 form heterodimers that influence the rate of PAR4 activation by thrombin. (B) Thrombin-cleaved PAR1 can transactivate an adjacent PAR2. The PAR1-PAR2 heterodimer traffic within the cell as a unit and activate Rac1 pathways. (C) PAR1-PAR3 heterodimers may regulate cytoprotective signaling on endothelial cells. (D) In platelets, PAR4-P2Y12 form heterodimers that recruit β-arrestin-2 to PAR4 where it serves as a scaffold for AKT signaling.

Figure 4

Polymorphisms in the PAR4 gene (f2rl3) give rise to sequence variants that have altered reactivities. (A) PAR4-120T is hyperreactive compared with PAR4-120A. The rare PAR4 variant, PAR4-296V, has low reactivity regardless of the amino acid at 120. (B) Individuals heterozygous for PAR4-296V have low PAR4 reactivity and subsequent low platelet response to PAR4 agonists. A potential mechanism is that the hyporeactive allele sequesters the hyperreactive allele as a dominant-negative receptor.

Figure 5

Therapeutic strategies for targeting PARs. PARs have been targeted with traditional orthosteric antagonists, such as vorapaxar for PAR1. In addition, PAR signaling may be inhibited at the initiation step by preventing the protease from cleaving the N terminus with blocking antibodies. Alternatively, pepducins target the intracellular face of the receptor to interfere with G-protein signaling. Parmodulins target the C-terminal eighth helix and have been selected to direct signaling to the cytoprotective pathways (β-arrestin) while blocking G_αq signaling.