Identification and partial characterization of human platelet C1q binding sites.
We recently showed that human blood platelets bind the complement component, C1q, and mAb directed against lymphoblastoid C1q receptors in a specific and saturable manner. To identify and further characterize platelet C1q binding sites, human platelets were washed, solubilized in Triton X-100 and either subjected to SDS-PAGE and Western blotting by using monoclonal (II1/D1) and polyclonal antibodies recognizing C1qR on lymphoblastoid cells, or applied to a C1q-Sepharose affinity column under low ionic strength conditions (20 mM NaCl). Adherent proteins were eluted with buffer containing 300 mM NaCl. Western blotting with monoclonal and polyclonal antibodies directed against C1qR showed exclusive reactivity with a 67,000 m.w. protein possessing intrachain disulfide bonds. SDS-PAGE of C1q-Sepharose eluates also revealed the presence of a 67,000 protein which was accompanied to varying degrees by a 94,000 constituent. Because similar m.w., 125I-labeled proteins were recovered from C1q-Sepharose columns to which lysed, surface-labeled platelets had been applied, both 94,000 and 67,000 components appear to be platelet membrane constituents. The 94,000 and 67,000 species, however, appear to be antigenically distinct. The 94,000 protein was immunoprecipitated by polyclonal antibodies against platelet membrane glycoprotein IIIa but not polyclonal antibodies against C1qR, whereas the 67,000 protein was immunoprecipitated exclusively by the polyclonal anti-C1qR antibody. The 67,000 protein thus appears to represent platelet C1q binding sites resembling C1qR on lymphoblastoid cells.
Peerschke
E I
EI
Department of Pathology, State University of New York, Stony Brook 11794.
Ghebrehiwet
B
B
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D002352:Carrier Proteins; D002846:Chromatography, Affinity; D003166:Complement Activating Enzymes / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D008970:Molecular Weight; D011233:Precipitin Tests; D011951:Receptors, Complement / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; C087299:C1QBP protein, human; D002352:Carrier Proteins; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011951:Receptors, Complement; C032402:complement 1q receptor; D003166:Complement Activating Enzymes