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

摘要

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 ng/mL 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.