Crossed immunoelectrophoresis using immobilized thrombin in intermediate gel. A method for demonstration of thrombin-binding platelet proteins.
A method is described in which the interaction between thrombin and platelet proteins can be studied directly. Solubilized platelet proteins were examined by crossed immunoelectrophoresis against polyspecific antiplatelet antibodies, and the interaction with thrombin observed by insertion of an intermediate gel containing thrombin coupled to Sepharose 4-B. Four immunoprecipitates were absent or showed an altered position compared to that of the control containing regular Sepharose 4-B in the intermediate gel. These were No. 1 (representing platelet factor 4), No. 13 (glycocalicin-related protein, probably GP lb), No. 19 (factor XIII), and No. 2a which has not yet been identified. Immunoprecipitates No. 16, representing a complex of GP IIb and GP IIIa, and No. 6 (albumin) as well as the other immunoprecipitates seen in the control pattern were unaffected by the presence of immobilized thrombin in the intermediate gel. The interaction of glycocalicin-related protein as well as purified glycocalicin with thrombin was confirmed by use of a monospecific antiserum. It is concluded that crossed immunoelectrophoresis using immobilized thrombin in an intermediate gel represents a new and useful approach to the investigation of the interaction between thrombin and platelet proteins. This procedure may also be extended to many other interactions between agents and cell proteins.
D001792:Blood Platelets / Q000032:analysis*; D002352:Carrier Proteins / Q000378:metabolism*; D004800:Enzymes, Immobilized; D006801:Humans; D007122:Immunoelectrophoresis / Q000379:methods*; D007123:Immunoelectrophoresis, Two-Dimensional / Q000379:methods*; D013917:Thrombin / Q000378:metabolism*
D002352:Carrier Proteins; D004800:Enzymes, Immobilized; D013917:Thrombin