Preparation and application of a photoreactive thrombin analogue: binding to human platelets.
alpha-Thrombin has previously been shown to bind to specific, saturable glycoproteins on the platelet surface. Modification of the thrombin active site with tosyllysyl chloromethyl ketone (TosLysCH2Cl) does not alter thrombin's binding characteristics. Interaction of alpha-thrombin with high-affinity binding sites (KD = 10(-9) M) initiates the platelet response which involves proteolytic hydrolysis of this glycoprotein. Although TosLysCH2Cl--thrombin binds to and competes for the same sites as alpha-thrombin, it cannot induce platelet stimulation because it is enzymatically inactive. In this study, we describe the preparation and application of photoreactive tritium-labeled thrombin analogues. 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 saturation doses (less than or equal to 2 nM) of the thrombin derivatives in the dark and photoactivated, a single labeled complex is detected. The same experiment with greater than saturating doses (greater than or equal to 20 nM) of the thrombin derivative yields a similar complex as well as two additional ones. Molecular weight estimates of these thrombin-bound complexes were obtained by gel filtration and NaDodSO4--polyacrylamide gel electrophoresis. The low dose (high affinity) complex with TosLysCH2Cl--thrombin has an approximate molecular weight 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 molecular weights 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 two previously detected populations of thrombin binding sites on the platelet.
D001386:Azides; D001792:Blood Platelets / Q000378:metabolism*; D003538:Cystamine / Q000031:analogs & derivatives; D006801:Humans; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000302:isolation & purification / Q000378:metabolism*; D014105:Tosyl Compounds / Q000494:pharmacology
D001386:Azides; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D014105:Tosyl Compounds; C015166:DNCO; D013917:Thrombin; D003538:Cystamine