Activation of the complement system plays a key role in inflammation associated with vascular injury. Recently, platelet P-selectin was shown to activate C3 via the alternative pathway of human complement. As platelets also posses binding sites for C1q, the recognition unit of the classical complement pathway, the present study examined classical pathway activation on platelets.Complement activation was assessed by either a solid phase enzyme-linked immunosorbent assay (ELISA) or flow cytometry.Using the ELISA approach, 2- to 10-fold increases (P < 0.001) in C1q and C4d deposition were demonstrated on adherent platelets following exposure (60 min 37 degrees C) to diluted (1/10) human plasma or serum. Similar results were obtained by flow cytometry using activated platelets in suspension. C1q and C4d deposition on platelets was accompanied by an approximately 4-fold increase in fluid phase C4d and C3a generation. Consistent with activation of the classical complement pathway, C4 cleavage failed to occur in serum depleted of C1q but was unchanged in factor B deficient serum. C4 activation was enhanced by platelet stimulation using chemical (SFLLRN peptide) or mechanical (shear) means, and decreased following platelet exposure to plasmin. These treatments were accompanied by changes in platelet surface gC1qR/p33 expression, a cellular C1q binding protein. In purified systems, recombinant gC1qR/p33 supported C4 activation, in a C1q dependent manner.These data provide the first evidence for C1q dependent classical complement pathway activation on platelets, and support a role for gC1qR/p33 in this process. However, monoclonal antibodies (mAb) to gC1qR/p33 produced only modest (20% +/- 8%, mean +/- SD, n = 5) reductions in C4 activation on platelets. Thus, further studies are required to investigate the involvement of additional platelet membrane constituents in classical complement pathway activation.