Detection of In Vivo Activated Platelets in Experimental Cerebral Thrombosis: Studies Using a New Monoclonal Antibody 2T60, Specific for Activated Human and Rabbit Platelets

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Abstract
To detect in vivo activated platelets in humans as well as in animal models of thrombosis, we developed a new murine monoclonal antibody, 2T60, specific for activated human and rabbit platelets by immunizing with human thronibin-activated platelets. 2T60 (IgG1 subclass) showed a great difference between binding to the thrombin-activated and resting human and rabbit platelets on ELISA and flow cytometer analysis. Immunoblotting analysis revealed that 2T60 reacted with a 130 or 106 kDa protein of human or rabbit platelets, respectively, only under non-reducing conditions. 125I-labeled 2T60 inserted into the intermediate gel of CIE of solubilized human platelets was incorporated into a immunoprecipitation line. 2T60 immunoprecipitated a protein of 130 or 115 kDa from human or rabbit platelets, respectively, which had been activated and 125I-surface-labeled. The N-terminal sequence of the affinity purified 2T60 antigen of human platelets was identical to that of GMP 140. There were differences in the carbohydrate chain content of GMP 140 between human and rabbit platelets. In experimental cerebral thrombosis of rabbits that had been injected with 2T60, the platelets adhering to the exposed subendothelium and contained in thrombi were found to bind 2T60 prominently. These results suggest that 2T60 may be a useful tool for clinical and experimental studies of thrombotic disease.