STUDIES OF A HIGH AFFINITY, STABLE PLATELET-THROMBIN COMPLEX FORMED DURING PLATELET ACTIVATION. A POSSIBLE MECHANISM FOR TRANSMEMBRANE SIGNAL GENERATION

C Knupp
1987 COAGULATION   unpublished
The precise mechanism of signal transmission from the platelet surface to the interior required for thrombin-induced platelet activation is not known. A stable complex between Iodo-[125l]-thrombin and platelets has been noted previously on SDS polyacrylamide gels. This 77,000 dalton complex was thought to represent the high affinity binding found in binding studies. The complex is inhibited by excess native thrombin but not by excess active site-modified thrombin. Thus, it displays the
more » ... splays the characteristics of the crucial thrombin-platelet interaction needed for platelet activation. My studies using similar methods for analysis were performed to determine if this interaction might have a role in signal generation for thrombin-induced platelet activation. Thrombin concentration studies with 3 minute incubations demonstrated that formation of the complex occurred only at thrombin doses which result in platelet activation (above 0.1 U/ml). The amount of the complex increased as the thrombin dose increased. Time course studies with thrombin 1 U/ml revealed that the reaction was rapid and was present on platelets after only 5 seconds of incubation. The complex was noted in supernatants but not until 30 seconds. This interaction was not present on platelets chilled to 4°C and was markedly diminished on platelets exposed to the metabolic inhibitors, antimycin A and 2-deoxyglu-cose. The specific thrombin inhibitors, hirudin and dansylargin-ine N-(3-ethyl-l,5-pentanediyl) amide, prevented the complex formation. Addition of either inhibitor after formation of the complex reversed its formation up to 1 minute after the addition of thrombin but not beyond this time. Treatment with trypsin but not chymotrypsin removed this complex from the platelet. It is concluded that this reaction is not simply a binding event as it requires functional platelets and is only formed at thrombin doses which activate platelets. It is an early, specific, surface reaction which requires thrombin binding and active-site domains for its formation. Formation of this complex is consistent with observation of "receptor processing" noted with specific thrombin binding. Therefore, this reaction could have an important function in the signal processing mechanism for thrombin-induced platelet activation.
doi:10.1055/s-0038-1644472 fatcat:jozwiv6imrcz7gukkjklfpbbzi