BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 226, 94–100 (1996) ARTICLE NO. 1316 Flow Cytometric Analysis of the Platelet Surface Area and Surface Density of Glycoprotein IIb-IIIa of Unactivated Human Platelets of Various Sizes V. Leytin, 1 H. Shapiro, I. Novikov,* and J. Radnay Department of Hematology, Meir Hospital, Kfar-Saba, Israel; and *Department of Clinical Epidemiology, Sheba Hospital, Tel-Hashomer, Israel Received June 22, 1996 Large (L) activated platelets exhibit greater aggregability and express more activated glycoprotein IIb- IIIa (GPIIb-IIIa) per cell and per unit surface area than small (S) activated platelets. We studied the binding of CD61 monoclonal antibody to GPIIb-IIIa on resting platelets and developed a new method for determining platelet surface area by flow cytometry. Using this method, we found that resting L platelets contain two times more GPIIb-IIIa per cell than S platelets but the same amount of GPIIb-IIIa per unit surface area. The data suggest that the greater aggregability of L platelets is likely to be due to increased activation and/ or expression of GPIIb-IIIa rather than to elevated density of unactivated GPIIb-IIIa on resting L platelets.  1996 Academic Press, Inc. Platelet aggregation plays a key role in haemostasis and thrombosis. This fundamental reaction is mediated by the binding of fibrinogen to the trans-membrane aIIbb3 integrin, the platelet GPIIb-IIIa complex (1-5). GPIIb-IIIa complexes do not bind fibrinogen on resting platelets. Activation of platelets by various agonists, regardless of the initiating stimulus, induces a common event - conformational changes resulting in the conversion of unactivated GPIIb-IIIa into activated GPIIb-IIIa complexes which serve as fibrinogen receptors (1-5). The binding of fibrinogen to these receptors on adjoining platelets results in the cross-linking of platelets into an aggregate (6). Human platelets are markedly heterogeneous in size and manifest size-dependent functional heterogeneity. L platelets are more sensitive to aggregating agents, more rapidly recruited into aggregates in vitro (7-9) and more effective in arresting bleeding than S ones (7,10). Using flow cytometry and FITC-PAC1, a monoclonal antibody specific for activated GPIIb-IIIa, Frojmovic et al. demonstrated that after stimulation, L platelets contain 3-4 times more activated GPIIb-IIIa per cell than S platelets (11). In addition, L platelets expressed 2-3 times more activated GPIIb-IIIa per unit SA than S platelets, as determined by direct microscopic SA measurements of L and S platelets isolated by a cell sorter (11). Flow cytometric analysis of resting platelets showed that L platelets contained more unactivated GPIIb-IIIa per cell than S platelets (12-15). However, there are no data on the densities of GPIIb-IIIa on the platelet surface in these subpopulations since SA of S and L platelets have not been determined. Here we study the distribution of GPIIb-IIIa on resting S, M and L platelets using FITC- 1 Dr. V. Leytin c/o H. Shapiro, Fax: 972-9-900369 Abbreviations: GPIIb-IIIa: glycoprotein IIb-IIIa; PRP: platelet-rich plasma; S, M, L, T: small, medium, large, total platelets, respectively; FS: forward scatter; SS: side scatter; FITC: fluorescein isothiocyanate; FL: fluorescence of platelet-bound FITC-antibody; SA, V: surface area and volume of platelets, respectively; D: density of GPIIb-IIIa on the platelet surface (amount of GPIIb-IIIa per unit SA). 0006-291X/96 $18.00 Copyright  1996 by Academic Press, Inc. All rights of reproduction in any form reserved. 94