In Vivo Tracking of Platelets: Circulating Degranulated Platelets Rapidly Lose Surface P-Selectin but Continue to Circulate and Function

To examine the hypothesis that surface P-selectin-positive degranulated platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from non-human primates baboons were labeled with PKH2 a lipophilic fluorescent dye, thrombin-activated, washed, and re-infused into the same baboons. Three color whole blood flow cytometry was used to simultaneously 1 identify platelets with a monoclonal antibody directed against GPIIb-IIIa integrin alpha sub IIb Beta sub 3, 2 distinguish infused platelets by their PKH2 fluorescence, and 3 analyze platelet function with monoclonal antibodies. Two hours after infusion of autologous thrombin-activated platelets P-selectin-positive, PKH2-labeled, 95 - 1 mean - SEM, n 5 of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin pre-infusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 hours after infusion and only slightly less 48 hours after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 - 16.7 ngmL increase in the plasma concentration of soluble P selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their a participation in platelet aggregates emerging from a bleeding time wound, b binding to Dacron in an arteriovenous shunt, c binding of monoclonal antibody PAC1 directed against the fibrinogen binding site on GPIIb-IIIa, and d generation of procoagulant platelet-derived microparticles.