Plasmin Desensitization of the PAR1 Thrombin Receptor:  Kinetics, Sites of Truncation, and Implications for Thrombolytic Therapy

Abstract

It has been hypothesized that protease-activated receptors may be activated and attenuated by more than one protease. Here, we explore a desensitization mechanism of the PAR1 thrombin receptor by anticoagulant proteases and provide an explanation to the enigma of why plasmin/tissue plasminogen activator (t-PA) can both activate and deactivate platelets prior to thrombin treatment. By using a soluble N-terminal exodomain (TR78) as a model for the full-length receptor, we were able to unambiguously compare cleavage rates and specificities among the serum proteases. Thrombin cleaves TR78 at the R41-S42 peptide bond with a kcat of 120 s-1 and a KM of 16 microM to produce TR62 (residues 42-103). We found that, of the anticoagulant proteases, only plasmin can rapidly truncate the soluble exodomain at the R70/K76/K82 sites located on a linker region that tethers the ligand to the body of the receptor. Plasmin cleavage of the TR78 exodomain is nearly equivalent to that of thrombin cleavage at R41 with similar rates (kcat = 30 s-1) and affinity (KM = 18 microM). Specificity was demonstrated since there is no observed cleavage at the five other potential plasmin-cleavage sites. Plasmin also cleaves the TR78 exodomain at the R41 thrombin-cleavage site generating transiently activated exodomain. We directly demonstrated that plasmin cleaves these same sites in full-length membrane-embedded receptor expressed in yeast and COS7 fibroblasts. The rate of plasmin truncation is similar between the extensively glycosylated COS7-expressed receptor and the nonglycosylated yeast-produced receptor. Mutation of the R70/K76/K82 sites to A70/A76/A82 eliminates plasmin truncation and desensitization of thrombin-dependent Ca2+ signaling and converts PAR1 into a plasmin-activated receptor with full agonist activity for plasmin. Plasmin does not desensitize the Ca2+ response of platelets or COS7 cells to SFLLRN consistent with intermolecular ligand-binding sites being located to the C-terminal side of K82. Truncation of the wild-type receptor at the C-terminal plasmin-cleavage sites removes the N-terminal tethered ligand or preligand, thereby providing an effective pathway for PAR1 desensitization in vivo.

Plasmin-mediated Activation of Platelets Occurs by Cleavage of Protease-activated Receptor 4

“…Kuliopulos et al (26) showed with calcium mobilization studies that plasmin has a low affinity for the traditional thrombin cleavage site on PAR-1 and a higher affinity for a cleavage site that is further downstream; in effect, plasmin is more likely to make PAR-1 refractory to further thrombin stimulation than to stimulate calcium mobilization. In addition, platelets stimulated with plasmin after a short period failed to desensitize calcium mobilization to further simulation with SFLLRN (26), and these observations were validated in our laboratory (data not shown). Furthermore, our results explain the contradiction of previous reports concerning the ability of plasmin to inhibit thrombinand SFLLRN-induced calcium mobilization in platelets (20,21).…”

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

“…Lipofectamine 2000, dNTPs, oligo(dT) [12][13][14][15][16][17][18] primer, AccuPrime DNA polymerase, and Fluo-3 acetoxymethyl ester were all purchased from Invitrogen. Primers were designed "in house" and purchased from MWG-Biotech (Ebersberg, Germany).…”

“…trypsin and neutrophil proteinases) have been reported to activate and/or disarm PAR 1 in different cell systems (2, 4, 8, 13, 14, 16 -18). Activation and desensitization of PAR 1 must be well controlled in order to eliminate the potential pathological damage resulting from unchecked receptor signaling (14).…”

The Roles of Proteinase-Activated Receptors in the Vascular Physiology and Pathophysiology

“…20 -24 Trypsin activates PAR1 and PAR2, but also disarms PAR1. 2,[25][26][27][28] Plasmin either activates or disarms PAR1, 6,29,30 whereas it inactivates PAR2 and activates PAR4. 31,32 The activated protein C (APC) has been shown to activate PAR1 and PAR2, [33][34][35] although its functional role still remains controversial.…”