Preparation and application of a photoreactive thrombin analog: binding to human platelets

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Abstract
.alpha.-Thrombin binds to specific, saturable glycoproteins on the platelet surface. The preparation and application of photoreactive tritium-labeled thrombin analogs are described. The .alpha.-thrombin derivative retains its platelet-stimulating and enzymatic activities and, upon photoactivation, covalently binds to specific platelet membrane components. When freshly washed human platelets are exposed to less than saturating doses (.ltoreq. 2 nM) of the thrombin derivatives in the dark and photoactivated, a single labeled complex is detected. The same experiment with greater than saturation doses (.gtoreq. 20 nM) of the thrombin derivative yields a similar complex as well as 2 additional ones. MW estimates of these thrombin-bound complexes were obtained by gel filtration and NaDodSO4[sodium dodecyl sulfate]-polyacrylamide gel electrophoresis. The low dose (high affinity) complex with TosLysCH2Cl[tosyllysyl chloromethyl ketone]-thrombin has an approximate MW of 200,000, while that with active .alpha.-thrombin is smaller, approximately 120,000, due to enzymatic cleavage. The additional complexes detected with the high thrombin dose had estimated MW of 400,000 and 46,000, respectively, and appeared to be the same for TosLysCH2Cl-thrombin and for the .alpha.-thrombin coupled platelets. These isolated complexes appear to correspond to the 2 previously detected populations of thrombin binding sites on the platelet.