CHARACTERIZATION OF HUMAN PLATELET ClQ BINDING SITES

Abstract

We have recently shown that platelets possess specific binding sites for Clq, a subcomponent of the first component of complement, Cl, and that occupancy of these receptor sites correlates with the previously described inhibitory effect of Clq on collagen-induced platelet aggregation. To further characterize platelet Clq receptors, washed platelets were solubilized in 5 mM sodium phosphate buffer, pH 7.5 containing 10 mM EDTA, 150 mM NaCl, 10 mM EACA, 0.5 mM PMSF, and 1% Triton X-100. After dialysis against 5 mM phosphate buffer pH 7.5 containing 10 mM EDTA, 20 mM NaCl, 10 mM EACA,0.5 mM PMSF and 0.1% Triton X-100, the lysate was passed over Clq-Sepharose-4B affinity columns. A single protein peak eluted with buffer containing 300 mM NaCl. This peak was composed of two predominant molecular weight species (85-95K, 60-66K) as assessed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. When 125-iodine surface labeled platelets were solubilized and applied to Clq-Sepharose affinity resins, the same two molecular weight species eluted and could be visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. Immunoabsorption studies performed under nondenaturing conditions using protein A and the IIl/Dl monoclonal antibody, which binds specifically to platelets and inhibits platelet-Clq interactions, revealed that the 85-95K molecular weight component was preferentially absorbed, but incomplete immunoabsorption of the 60-66K molecular weight constituent was also noted. Affinity purified Clq binding sites sedimented as a single peak during 5-40% sucrose density ultracentrifugation with an S value of approximately 2.4. In addition, both the 85-95K and the 60-66K molecular weight species coeluted in the void volume of Sephadex G-100 columns. The data suggest that the 85-95K and 60-66K molecular weight species represent platelet membrane Clq binding sites, and that these sites may form weak, noncovalently associated complexes.