Differential activation and inhibition of human platelet thrombin receptors by structurally distinct α-, β- and γ-thrombin

Abstract

The development of drugs to neutralize the action of thrombin has to date focused on the a form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active α-thrombin which may be further hydrolyzed to β- and γ-thrombin. While all three forms of the enzyme retain catalytic activities, only α-thrombin is presumed to be physiologically important. The β- and γ-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that β- and γ-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173-21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. β-Thrombin, like γ-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the β-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For β-thrombin and γ-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that α-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that α-thrombin may be rapidly converted to β-thrombin and/or γ-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by α-, β;- and γ-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits α-thrombin, is a slightly weaker inhibitor of β- thrombin and a very weak inhibitor of γ-thrombin- induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, α1-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only β- and γ-thrombin, primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like β- and γ-thrombin, activates PAR-4 and is also inactive with TRAP-4, desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to γ-thrombin than to α-thrombin. The analysis of the crystalline structures of α-, γ-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.