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

Abstract

The activation of plasmin from its circulating precursor plasminogen is the mechanism of several clot-busting drugs used to clinically treat patients who have suffered a stroke; however, plasmin thus generated has been shown to activate platelets directly. There has been speculation as to whether plasmin interacts with the protease-activated receptors (PARs) because of its similarity in amino acid specificity with the classic platelet activator thrombin. We have investigated whether plasmin activates platelets via PAR activation through multiple complementary approaches. At concentrations sufficient to induce human platelet aggregation, plasmin released very little calcium compared with that induced by thrombin, the PAR-1 agonist peptide SFLLRN, or the PAR-4 agonist peptide AYPGKF. Stimulation of platelets with plasmin initially failed to desensitize additional stimulation with SFLLRN or AYPGKF, but a prolonged incubation with plasmin desensitized platelets to further stimulation by thrombin. The desensitization of PAR-1 had no effect on plasmininduced platelet aggregation and yielded an aggregation profile that was similar to plasmin in response to a low dose of thrombin. However, PAR-4 desensitization completely eliminated aggregation in response to plasmin. Inclusion of the PAR-1-specific antagonist BMS-200261 inhibited platelet aggregation induced by a low dose of thrombin but not by plasmin. Additionally, mouse platelets naturally devoid of PAR-1 showed a full aggregation response to plasmin in comparison to thrombin. Furthermore, human and mouse platelets treated with a PAR-4 antagonist, as well as platelets isolated from PAR-4 homozygous null mice, failed to aggregate in response to plasmin. Finally, a proteaseresistant recombinant PAR-4 was refractory to activation by plasmin. We conclude that plasmin induces platelet aggregation primarily through slow cleavage of PAR-4.

Proteinase-activated Receptors, Targets for Kallikrein Signaling

“…5 and 8), kallikreins would promote the release of platelet endostatin and suppress the release of platelet vascular endothelial growth factor (51). The preferential activation of PAR 4 versus PAR 1 mirrors the action of plasmin on human platelets, wherein plasmin can both activate and disarm PAR 1 (60,61). Whether PAR 1 would be activated or disarmed by hK14 (Fig.…”

The Cardiovascular Actions of Protease-Activated Receptors

“…As noted, multiple PARs also could enable responses to a wide repertoire of proteases (in addition to thrombin). Indeed, PAR-4 has been characterized variably as a cellular receptor for trypsin and plasmin (Sambrano et al, 2000;Quinton et al, 2004). In the case of plasmin, the kinetics of PAR-4 cleavage are relatively slow.…”

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

“…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. 36 In the following sections, I would like to discuss the role of PARs in regulating the vascular functions and in the pathophysiology of vascular diseases, with some focus on thrombin and its major receptor PAR1.…”