Normal circulating platelets do not adhere to intact, undisturbed endothelium. Studies have shown, however, that platelets will adhere to virally infected or thrombin-stimulated human umbilical vein endothelial cells. Using a novel platelet/endothelial cell adhesion assay we studied the interaction of thrombin-activated platelets to human saphenous vein endothelial cells (HSVEC), and its mechanism(s). Biotinylated platelets were exposed to Hepes-Tyrode buffer, 10E5 or PAC-1 [monoclonal antibodies (Mabs) blocking GPIIb-IIIa], AK4 (Mab blocking P-selectin), 6D1 (Mab blocking vWf binding to GPIb), RGDS (small peptide blocking the fibrinogen binding site), or EDTA (dissociates GPIIb-IIIa complex) and then activated with thrombin. The platelets were subsequently exposed to thrombin-stimulated monolayer HSVEC. Phycoerythrin-streptavidin was added to the wells to fluorescently label the platelets, followed by formaldehyde fixation and washing to remove nonadherent platelets. Adhesion of platelets to HSVEC was assessed using a fluorescent multiwell plate reader. Antibodies which blocked the GPIIb-IIIa receptor and agents which competitively bound the receptor all significantly inhibited activated platelet adhesion to the activated HSVEC. We have found that thrombin significantly increases platelet/HSVEC adhesion, and this event is mediated via the integrin GPIIb-IIIa (fibrinogen receptor). These GPIIb-IIIa receptor blocking Mabs and RGDS may be useful adjuncts for improving patency following angiographic intervention and/or vein grafting in patients with high risk of thrombosis. The assay we have developed is a valuable and relatively simple method for assessing platelet/endothelial cell adhesion and activation. (C) 1996 Academic Press, Inc.

Very few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 microliters of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting alpha granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of S12, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of S12, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

In this study, the question of whether glycoprotein Ib (GPIb) mediates both high and moderate affinity pathways of alpha-thrombin-induced platelet activation was examined. Flow cytometric studies, using a panel of monoclonal antibodies (MoAbs), showed that Serratia marcescens protease treatment removed greater than 97% of the glycocalicin portion of GPIb but did not affect the changes in the expression of GPIX or GMP- 140 that were induced by high concentrations of alpha-thrombin (10 nmol/L). However, Serratia treatment almost completely abolished the increase in platelet surface GMP-140 induced by low concentrations of alpha-thrombin (0.5 nmol/L) and diminished the downregulation of platelet surface GPIX by 60.9% +/- 5.6% (mean +/- SEM, n = 3). When present in 20-fold molar excess, an MoAb directed against the alpha- thrombin/von Willebrand factor (vWf) binding domains of GPIb completely blocked the ristocetin-dependent binding of vWf to platelets but inhibited only to about 50% the binding of alpha-thrombin and the activation-dependent binding of vWf. In platelets treated with Serratia marcescens protease to remove GPIb, a concentration of this MoAb 16,000- fold in excess of the maximum possible remaining copies of GPIb failed to inhibit platelet activation by alpha-thrombin. These studies demonstrate that activation of intact platelets by alpha-thrombin proceeds by both GPIb-dependent and GPIb-independent mechanisms.