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 preparedfrom nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfusedinto the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAbdirected against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence,and (iii) analyze platelet function with mAbs. 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. Comparedwith platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negativeplatelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectinwas fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulatingPKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participationin platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) bindingof mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derivedmicroparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, butcontinue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for trackingof platelets and measurement of platelet function in vivo.

> BACKGROUND: One of the variables to determine the quality of platelets (PLTs) in vitro is measurement of CD62P expression. Different protocols are in use, however, making comparison of results virtually impossible. It was our aim to develop a uniform CD62P protocol that would yield comparable results in various laboratories.

> Summary.  Background and objectives: Low response to antiplatelet therapy may be a risk factor for the development of ischemic complications in patients with non-ST segment elevation acute coronary syndrome (NSTE ACS) undergoing coronary stenting. Methods: We prospectively studied the platelet response to both clopidogrel and aspirin in 106 NSTE ACS consecutive patients undergoing percutaneous coronary intervention (PCI) with stenting. A single post-treatment blood sample was obtained just before PCI and analyzed by platelet aggregometry using both ADP and arachidonic acid (AA) as agonists to explore the responses to clopidogrel and aspirin, respectively. Patients were divided into quartiles according to the ADP or AA induced maximal intensity of platelet aggregation. Patients of the highest quartile (quartile 4) were defined as the ‘low-responders’. Results: Twelve recurrent cardiovascular (CV) events occurred during the 1-month follow-up. Clinical outcome was significantly associated with platelet response to clopidogrel [Quartile 4 vs. 1, 2, 3: OR (95% CI) 22.4 (4.6–109)]. Low platelet response to aspirin was significantly correlated with clopidogrel low response ( P  = 0.003) but contributed less to CV events [OR (95%CI): 5.76 (1.54–35.61)]. Conclusions: A post-treatment ADP-induced platelet aggregation performed just before PCI identifies low responders to dual antiplatelet therapy with an increased risk of recurrent CV events.