Thrombin-induced platelet endostatin release is blocked by a proteinase activated receptor-4 (PAR4) antagonist

Endostatin is a potent endogenous inhibitor of angiogenesis that was recent ly shown to be stored in platelets and released in response to thrombin, bu t not ADP. In the present study, we have tested the hypothesis that thrombi n-induced endostatin release from rat platelets is mediated via proteinase- activated receptor-4 (PAR4). Immunoprecipitation and Western blotting confi rmed that endostatin is contained within rat platelets. Aggregation and rel ease of endostatin could be elicited by thrombin (0.5-1.0 U ml(-1)) or by s pecific PAR4 agonist (AYPGKF-NH2; AY-NH2; 15-50 muM). Significant release o f endostatin could be induced by a dose of thrombin below that necessary fo r induction of aggregation. An adenosine diphosphate (ADP) scavenger, apyra se, inhibited the platelet aggregation induced by thrombin, but not the rel ease of endostatin. In contrast, a selective PAR4 antagonist (trans-cinnamo yl-YPGKF-NH2; tcY-NH2) prevented endostatin release and aggregation induced by thrombin or by AY-NH2. We conclude that thrombin-induced endostatin rel ease from rat platelets is PAR4-mediated via an ADP-independent mechanism t hat can occur independently of platelet aggregation.