Detection of thrombin-activated rat platelets by FACS analysis with the anti-RV4 polyclonal antibody and MoAb RPM.9. Washed rat platelets (2 × 105) were incubated with 2 μg of anti-RV4 followed by FITC-labeled goat antirabbit antibody (A) or with 2 μg of MoAb RPM.9 followed by FITC-labeled goat antimouse antibody (B) after treatment with 5 U/mL thrombin (dashed line) or without treatment (solid line). (A) Anti-RV4 did not bind to resting platelets and gave a fluorescence signal equivalent to that of a nonimmune serum (data not shown). Treatment with thrombin resulted in a positive shift in fluorescence suggesting that anti-RV4 recognizes the new NH2-terminus generated by cleavage of rat GPV. (B) On the contrary, MoAb RPM.9 positively labeled resting rat platelets, while thrombin treatment shifted the fluorescence signal to negative values. Thus, MoAb RPM.9 appears to bind to a fragment NH2-terminal to the thrombin cleavage site, which is released from thrombin activated platelets.
