CHARACTERIZATION OF IN-VITRO AND IN-VIVO PLATELET RESPONSES TO THROMBIN AND THROMBIN RECEPTOR-ACTIVATING PEPTIDES IN GUINEA-PIGS

Guinea pig platelets are similar to human platelets in their responsiv eness to thrombin receptor-activating peptides and other agonists. The refore. guinea pigs anesthetized with Inactin (90 mg/kg i.p.) were use d to assess in vivo activities of thrombin and thrombin receptor-activ ating peptides (TRAPs) using In-111-labeled platelets and a microcompu ter-based system. The aggregatory responses are expressed as percent c hange for a 20 min period over basal radioactivity (AUC). Reversible a ccumulation of platelets occurred in the pulmonary microcirculation in response to stimuli. Human thrombin (50 and 100 U/kg i.v.) caused a d ose-related platelet accumulation. Responses of similar magnitude were induced by SFLLRN (TRAP-(1-6)) and Ala-Phe(p-F)-Arg-Cha-HArg-Tyr-NH2 (high-affinity thrombin receptor-activating peptide, 0.03, 0.1 and 0.3 mg/kg i.v.). High-affinity thrombin receptor-activating peptide, a ne w synthetic oligopeptide agonist, is about 3-fold more potent than TRA P-(1-6), a wild-type sequence. Similarly, high-affinity thrombin recep tor activating peptide is about 4 times more potent than TRAP-(1-6) in the radioligand binding study using platelet membrane. By comparison, high-affinity thrombin receptor-activating peptide manifested an aggr egatory activity (EC(60) = 1.2 mu M) about 15 times more potent than t hat of TRAP-(1-6) (EC(60) = 18.6 mu M) in washed guinea pig platelets. The intrapulmonary platelet aggregation in response to thrombin, TRAP -(1-6) and high-affinity thrombin receptor-activating peptide was char acterized by long duration (similar to 30 min); a reduction in respons e (18-54%) tended to occur with repeated challenges, presumably due to desensitization and consumption. The response to thrombin (100 U/kg) was greatly inhibited by (D)-Phe-Pro-Arg-chloromethyl ketone (PPACK), a potent thrombin inhibitor (250 mu g/kg + 6 mu g/kg per min i.v. x 30 ): AUC, 150 +/- 552 vs. 7171 +/- 1052 in the control period (n = 8, P < 0.05). The response to high-affinity thrombin receptor-activating pe ptide (0.03 mg/kg), which acts on thrombin receptor directly, was not affected by PPACK. It is concluded that guinea pigs are an appropriate preparation for evaluation of in vivo activity of thrombin inhibitors as well as thrombin receptor agonists and antagonists.