USE OF A MONOCLONAL ANTIBODY TO MEASURE THE SURFACE EXPRESSION OF THROMBOSPONDIN FOLLOWING PLATELET ACTIVATION

A radiolabelled monoclonal antibody (mAb) against native thrombospondin (TSP) has been used to quantitatively assess the surface exposure of intracellular TSP following platelet stimulation. This mAb, designated 5G11, was purified from ascitic fluid by ammonium sulfate precipitation followed by chromatogloghy on DEAE Trisacryl. The isolated IgG were labelled with I by the chloramine T method (sp.act. 200-500 cpm/ ng). The specificity of the mAb was established by immunoblot-ting and crossed immunoelectrophoresis using platelet protein extracts. When the labelled IgG (20 μg/ml) were incubated with resting platelets in Tyrode's buffer binding was of the order of 2,000 molecules per platelet. Binding was increased 2 fold and 5-7 fold respectively upon ADP- and thrombin-(or ionophore A23187) stimulation. Unactivated platelets from 2 patients with the Gray Platelet Syndrome bound baseline levels of 5G11, but binding did not increase after platelet activation. In the presence of saturating concentrations of mAb 5G11, an average of 30,000 molecules of IgG were bound by normal platelets stimulated by thrombin. This binding was strongly reduced in the presence of EDTA. It was not significantly affected by AP-2, an anti-GP IIb-IIIa monoclonal antibody which inhibited by more than 85% the binding of plasma fibrinogen but which did not inhibit the surface expression of platelet fibrinogen. It was decreased but not prevented by the presence of an excess of rabbit anti-fibrinogen Fab fragments during the stimulation, while binding at the lower end of the normal range was observed on two different occasions using platelets isolated from an afibrinogenemic patient lacking platelet fibrinogen. These results suggest that while platelet fibrinogen may contribute to the surface organization of TSP other component(s) are required for the full expression of TSP on the platelet surface.