Platelet Responses to Compound Interactions with Thrombin

Catalytic and noncatalytic interactions of thrombin with platelets are investigated with use of thrombin variants with altered specificities and with ligands of thrombin receptors on platelets. Both α-thrombin and weakly coagulant meizothrombin-des-fragment-1 (μ-thrombin) hydrolyze proteolytically activated receptor 1 for thrombin (rPAR1T, recombinant) with catalytic efficiencies of >107 M-1 s-1, whereas rPAR1T is not a substrate for weakly coagulant β-thrombin. In contrast, both μ-thrombin and β-thrombin are weak agonists of platelet dense body (ATP) secretion. Antibodies that block rPAR1T cleavage strongly inhibit the secretory reaction to α- and μ-thrombins but not to β-thrombin or to thrombin receptor activating peptide (TRAP). However, catalytically inactive FPR-thrombin, which binds glycoprotein Ib but does not inhibit rPAR1T cleavage, inhibits responses to TRAP as well as those to α- and μ-thrombins, which indicates that binding of the inactive enzyme to platelets influences the function of PAR1T. An antibody that inhibits binding of thrombin to platelet glycoprotein Ib inhibits secretory responses to thrombin but not to TRAP, so occupancy of glycoprotein Ib per se accounts for only part of the attenuation. All three thrombins stimulate a rise in cytosolic Ca(II), and the dose response to β-thrombin is congruent with that for ATP secretion. However, the response of cytosolic Ca(II) is 10−100 times more sensitive to μ-thrombin and α-thrombin than ATP secretion is, and is inhibited by neither anti-PAR1T Ig nor FPR-thrombin. Thus, α-thrombin appears to have an activity not shared by either μ- or β-thrombins. This activity is owed to more than coupling of independent signals from cleavage of two proteolytically activated receptors, as there is no synergism when μ-thrombin and β-thrombin costimulate secretion. It is concluded either that α-thrombin has a third interaction site on platelets with which neither μ-thrombin nor β-thrombin interacts or that dual receptors are coordinately cleaved. In either case, the strong secretory response to thrombin appears to be moderated, independently of cytosolic Ca(II), by occupancy of a noncatalytic interaction site such as glycoprotein Ib.