Photoaffinity labeling of platelet thrombin-binding proteins.
The interaction of thrombin and platelets was studied with a heterobifunctional photoactivable crosslinking agent. Radiolabeled thrombin that was modified with ethyl-N-5-azido-2-nitrobenzoylaminoacetimidate formed two types of complex with platelet proteins: platelet-associated complexes and supernatant complexes. The platelet-associated complexes formed within 20 s. Autoradiography after electrophoresis with sodium dodecyl sulfate indicated that these complexes had apparent masses of 210, 185, 155 and 125 kDa. Formation of the complexes was blocked by hirudin; this is consistent with crosslinking that was a direct consequence of the binding of thrombin to a specific receptor, since hirudin blocks thrombin-induced platelet activation and the saturable binding of thrombin to platelets. The labeled supernatant complex had an apparent mass of about 490 kDa. It also formed in the supernatant solution of platelets after activation with a divalent cation ionophore, suggesting a complex of thrombin with a secreted protein. The supernatant complex did not involve fibrinogen or alpha 2-macroglobulin, but a similar complex was formed with partially purified secreted glycoprotein G (thrombin-sensitive protein, thrombospondin). Formation of the complex was blocked by hirudin. A similar complex was formed after prolonged (1 h) incubation without photoactivation. It is concluded that thrombin forms high-affinity, hirudin-sensitive complexes with secreted glycoprotein G, as well as with platelet surface proteins.
D001792:Blood Platelets / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007096:Imidoesters / Q000138:chemical synthesis / Q000494:pharmacology*; D008970:Molecular Weight; D010777:Photochemistry; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism*
D003432:Cross-Linking Reagents; D007096:Imidoesters; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; C018060:ethyl N-5-azido-2-nitrobenzoylaminoacetimidate; D013917:Thrombin