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Introduction: Many stroke patients do not fully respond to intravenous rt-PA, possibly due to factors that increase clot resistance or impede reperfusion. GPIb receptors on the platelet surface initiate vessel occlusion through an interaction with subendothelium-bound von Willebrand Factor and the interaction transduces signals resulting in further activation. An increase in GPIb availability may result in macro or micro-vascular thrombosis. We report the effect of rt-PA on a potential compensatory conformational response of platelet GPIb.Methods: 5 subjects with large artery stroke and treated with rt-PA within 4.5 hours of onset were enrolled after consent. Blood was obtained immediately before and 1.3 hours post rt-PA bolus. Blood was incubated with 3 monoclonal antibodies (mABs: AN51, 6D1 and SZ2) and control antibody. AN51 and 6D1 are conformation sensitive and SZ2 is relatively less conformation sensitive. Flow cytometry was performed to assess fluorescent intensity of platelets pre and post-treatment and corrected for non-specific staining with control IgG. Blood from healthy donors subjected to appropriate concentrations of rt-PA in vitro was tested for conformational changes with AN51 and 6D1.Results: Compared to pre-treatment, 4 of 5 patients showed increased GPIb or binding with AN51 and 6D1 post treatment, with an overall median percentage increase with AN51 of 40.6% and 60.3% with 6D1. SZ2 showed a smaller increase (30.7%). The 1 subject who showed no change had the only hemorrhagic transformation. Blood from healthy donors showed an increase of 21.4% with AN51 and 41.3% with 6D1 after rt-PA.Conclusion: These results indicate for the first time rt-PA induced conformation changes on platelet GPIb in acute ischemic stroke in vivo despite that GPIb is not the direct target. These changes were also seen with rt-PA in vitro. It is possible that up-regulation of GPIb receptors plays a protective role in preventing hemorrhages but also may mediate increased propensity for clot resistance or downstream thrombosis. If confirmed in a larger ongoing sample, measuring GPIb binding may provide a rational approach to post-rt-PA antiplatelet therapy and identify a risk for hemorrhagic transformation.