Flow cytometry was used to determine whether activated platelets and platelet-derived microparticles can be detected directly in whole blood after a hemostatic insult. Two different in vivo models of platelet activation were examined: (1) a standardized bleeding time, and (2) cardiopulmonary bypass. Platelets and microplatelets were identified with a biotinylated anti-glycoprotein (GP)lb antibody and a fluorophore, phycoerythrin-streptavidin. Microparticles were distinguished from platelets by light scatter. Activated platelets were detected with three fluorescein-labeled monoclonal antibodies (MoAbs): (1) PAC1, which binds to the activated form of GPIIb-IIIa; (2) 9F9, a newly developed antibody that is specific for fibrinogen bound to the surface of activated platelets; and (3) S12, which binds to an alpha- granule membrane protein expressed on the platelet surface after granule secretion. In nine normal subjects, bleeding times ranged from 4.5 to 7.5 minutes. Over this time, there was a progressive increase in the amount of PAC1, 9F9, and S12 bound to platelets in blood emerging from the bleeding time wound. With all three antibodies, platelet activation was apparent as early as 30 seconds after the incision (P less than .03). Activation was accompanied by a progressive decrease in the concentration of platelets in blood from the wound, while the concentration of microparticles increased slightly. In nine patients undergoing open heart surgery, 1 hour of cardiopulmonary bypass caused a 2.2-fold increase in the relative proportion of microparticles in circulating blood (P less than .001). Moreover, bypass caused platelet activation as evidenced by a mean two- to threefold increase in PAC1 binding to platelets. Although this increase was significant (P less than .02), PAC1 binding exceeded the normal range for unstimulated control platelets in only 5 of 9 patients, and 9F9 and S12 binding exceeded the normal range in only two patients. Taken together, these studies demonstrate that it is now feasible using flow cytometry to evaluate the extent of platelet activation and the presence of platelet- derived microparticles in the circulation of humans.

Normal primary platelet aggregation requires agonist-mediated activation of membrane GPIIb-IIIa, binding of fibrinogen to GPIIb-IIIa, and cellular events after ligand binding. PAC1 monoclonal antibody distinguishes between resting and activated states of GPIIb-IIIa, and other antibodies preferentially recognize GPIIb (PMI-1) or IIIa (anti- LIBS1) after the binding of fibrinogen or fibrinogen-mimetic peptides, such as GRGDSP. Using these antibodies and platelet flow cytometry, we studied two distinct persistent platelet aggregation abnormalities. Platelets from a thrombasthenic variant, which contained near-normal amounts of GPIIb-IIIa, failed to aggregate or bind PAC1 in response to agonists. In addition, GRGDSP, which binds to normal GPIIb-IIIa without prior cell activation, failed to increase the binding of PMI-1 or anti- LIBS1 to the thrombasthenic platelets, suggesting a primary defect in ligand binding. Chromatography of detergent-solubilized platelets on a KYGRGDS affinity column confirmed that the patient's GPIIb-IIIa lacked the fibrinogen binding site. In another patient with myelofibrosis and defective aggregation, PAC1 failed to bind to adenosine diphosphate- stimulated platelets, but did bind when protein kinase C was directly activated with phorbol myristate acetate. Furthermore, the binding of PMI-1 and anti-LIBS1 increased in response to GRGDSP, confirming a defect in agonist-mediated fibrinogen receptor activation rather than in fibrinogen binding or events distal to binding. These studies indicate that this immunochemical approach is useful in classification of clinical abnormalities of platelet aggregation as defects in either (a) fibrinogen receptor activation, (b) fibrinogen binding, or (c) postoccupancy events.

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The erythrocytes from a patient with Scott syndrome, a bleeding disorder characterized by an isolated defect in expression of platelet procoagulant activity, have been studied. When incubated with the calcium ionophore A23187, Scott syndrome red blood cells (RBCs) expressed less than 10% of the prothrombinase (enzyme complex of coagulation factors Va and Xa) activity of A23187-treated RBCs obtained from normal controls. Consistent with the results from enzyme assay, the ionophore-treated Scott syndrome erythrocytes exhibited diminished membrane vesiculation and decreased exposure of membrane binding sites for factor Va compared with identically treated controls. When examined by scanning electron microscopy, untreated Scott syndrome RBCs were indistinguishable from normal cells. After incubation with A23187, however, the morphology of Scott syndrome RBCs contrasted markedly from normal erythrocytes. Whereas the Ca2+ ionophore induced marked echinocytosis and spiculation of normal RBCs, Scott syndrome RBCs remained mostly discoid under these conditions, with only an occasional echinocyte-like cell observed. These aberrant responses to intracellular Ca2+ were also observed for resealed ghosts prepared from Scott syndrome erythrocytes, indicating that they are related to a defect in the membrane or membrane-associated cytoskeleton. The finding that the erythrocytes of this patient share many of the membrane abnormalities reported previously for Scott syndrome platelets suggests that this defect is common to both cell lines and involves a membrane component required for vesicle formation and for expression of prothrombinase sites.