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 alpha form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active alpha-thrombin which may be further hydrolyzed to beta- and gamma-thrombin. While all three forms of the enzyme retain catalytic activities, only alpha-thrombin is presumed to be physiologically important. The beta- and gamma-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that beta- and gamma-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. Beta-thrombin, like gamma-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 beta-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For beta-thrombin and gamma-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 alpha-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that alpha-thrombin may be rapidly converted to beta-thrombin and/or gamma-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by alpha-, beta- and gamma-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 alpha-thrombin , is a slightly weaker inhibitor of beta-thrombin and a very weak inhibitor of gamma-thrombin -induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, alpha(1)-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only beta- and gamma-thrombin , primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like beta- and gamma-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 gamma-thrombin than to alpha-thrombin. The analysis of the crystalline structures of alpha-, gamma-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.

Activation of factor XI by products of prothrombin activation

“…␤-IIa and ␥-IIa at nanomolar concentrations are also activators of PAR-4 on platelets. 39 The importance of ABE I is obvious in thrombin-triggered clotting assays run in the absence of calcium. Here, the added thrombin must covert fibrinogen to fibrin.…”

“…41 Based on an analysis of crystal structures, Soslau et al proposed that conversion of ␣-IIa to ␤-IIa and then ␥-IIa is accompanied by a progressive opening of the active site to take on a more trypsin-like conformation. 39 Physiologic roles for ␤-IIa and ␥-IIa have not been established, but several possibilities have been discussed. Although possessing low activity toward fibrinogen and PAR-1, these proteases activate FXIII 42 and are inhibited by antithrombin.…”

“…on May 11, 2018. by guest www.bloodjournal.org From magnitude lower affinity for the inhibitor hirudin. 39,40 Conversion of ␤-IIa to ␥-IIa involves cleavage in the ␥-loop, further impairing fibrinogen cleavage and hirudin binding. 9,10,[38][39][40] Dang et al prepared an ␣-IIa variant lacking the ␥-loop ("loopless") and noted a 240-fold reduction in fibrinogen cleavage compared with ␣-IIa.…”

“…39,40 Conversion of ␤-IIa to ␥-IIa involves cleavage in the ␥-loop, further impairing fibrinogen cleavage and hirudin binding. 9,10,[38][39][40] Dang et al prepared an ␣-IIa variant lacking the ␥-loop ("loopless") and noted a 240-fold reduction in fibrinogen cleavage compared with ␣-IIa. 41 Based on an analysis of crystal structures, Soslau et al proposed that conversion of ␣-IIa to ␤-IIa and then ␥-IIa is accompanied by a progressive opening of the active site to take on a more trypsin-like conformation.…”

The dimeric structure of factor XI and zymogen activation

“…Clotting was induced with b-thrombin, 14,17 which converts fibrinogen to fibrin poorly due to disruption of anion-binding exosite I. 31 As this exosite is not involved in fXI activation, b-thrombin and a-thrombin activate fXI similarly. 14 In the reactions shown in Figure 7D, b-thrombin activates fXI bound to poly-P, leading to a-thrombin generation from plasma prothrombin and clot formation.…”