Activation of the platelets with TRAP-6 (which stimulates PAR-1) and the PMNs with C5a (which stimulates the C5a receptor and up-regulates surface CD11b) triggered formation of PPC. PPC formation by 75C100 %. Using plasma protein-depleted blood and blood from a C3-deficient patient, we corroborated the dependence on C3, obtaining comparable results after reconstitution with purified C3. By analogy with platelets, PMPs isolated from human serum were found to expose C3(H2O) and bind to PMNs. This conversation was also blocked by the anti-C3(H2O) and anti-CD11b monoclonal antibodies, indicating that C3(H2O) and CD11b are involved in tethering PMPs to PMNs. We confirmed the direct interaction between C3(H2O) and CD11b by quartz crystal microbalance analysis using purified native SRPKIN-1 C3 and recombinant CD11b/CD18 and by flow cytometry using PMP and recombinant CD11b. Transfectants expressing CD11b/CD18 were also shown to specifically adhere to surface-bound C3(H2O). We have identified contact-activated C3(H2O) as a novel ligand for CD11b/CD18 that mediates PPC formation and the binding of PMPs to PMNs. Given the various roles of C3 in thrombotic reactions, this finding is likely to have important pathophysiological implications. platelet-leukocyte complexes (PLC) are formed at least in part as a result of tethering via platelet-exposed P-selectin and its ligand P-selectin glycoprotein ligand-1 (PSGL-1) on the leukocytes, in a manner resembling the initial phase of leukocyte rolling onto SRPKIN-1 activated endothelial cells. The P-selectin-PSGL-1 interactions constitute a primary attachment of platelets to leukocytes (23), but cell adhesion molecules (CAM) form more stable bonds via integrins at a later stage (24). In the case of PLC formation, blocking experiments using receptor-specific monoclonal antibodies (mAbs) have indicated that the integrin CD11b/CD18 (complement receptor 3 [CR3]; Mac-1) is involved (25, 26). Glycoprotein Ib (GPIb) (25C27), junctional adhesion molecule C (JAM-C) (28), fibrinogen (29), and CD40L (30), among others, have been suggested as counter-ligands of CD11b/CD18 on platelets. However, given that CD11b/CD18 is an important complement SRPKIN-1 receptor, it is possible that platelet-bound C3 acts as a ligand of CD11b/CD18, thereby contributing to the formation of PPCs. We and others have reported that complement activation can be triggered by platelet activation (7, 9, 31). For instance, the classical pathway of complement can be elicited by chondroitin sulfate released from activated platelets (31). Moreover, the involvement of P-selectin and properdin in triggering alternative pathway activation has also been suggested (7, 10). Binding of complement components such as C1q, C4, C3, or C9 to activated platelets has been shown in a number of studies (7, 9, 32), but we have recently demonstrated that under physiological conditions, this binding is not a result of the proteolytic activation of complement (8). Analyses of the bound C3 molecules by flow cytometry and Western blotting showed that they consist of intact – and -chains and that, unlike C3b, the -chain of C3 still contained the C3a portion of the molecule. However, unlike native C3, the reactivity to conformational epitopes and the cleavage pattern and reactivity to complement receptors indicated that the bound C3 was instead in the form of C3(H2O). C3(H2O) is generated by the hydrolysis of the internal thiol ester bond in native C3 without convertase-elicited proteolytic cleavage of the molecule. Like C3b, C3(H2O) is cleaved by factor I in the -chain and is inactivated with respect to convertase formation, yielding iC3(H2O). C3(H2O) and iC3(H2O) are known to interact with C3 receptors such as CR1(CD35) (33), CR2 (CD21) (34), and a CR3 (CD11b/CD18)-like molecule from (35), and we have confirmed that the platelet-bound C3(H2O)/iC3(H2O) binds to soluble CR1 (CD35) (8). In a previous study, we showed that PPC formation is, to a substantial degree, dependent on platelet-mediated complement activation and C5a receptor stimulation (31), occurring as the result SRPKIN-1 of the up-regulation of CD11b/CD18 on the leukocyte surface. The fact that activated platelets in whole blood also expose an activated form of C3 (i. e. C3(H2O) (8) suggests that C3 may be directly involved in the formation of PPCs. Our previous studies have Rabbit Polyclonal to RHOB indicated that the platelet-bound C3(H2O) is partially cleaved by factor I into iC3(H2O), the equivalent of iC3b, which is a ligand of CR3 (CD11b/CD18) (36). Here, we have identified C3(H2O)/iC3(H2O) as a novel ligand of CD11b/CD18 and have shown that C3 alone, in the absence of any proteolytic activation, can support the formation of PPC. The dependence of PPC formation on C3(H2O)/iC3(H2O) and CD11b/CD18 was corroborated by the robust inhibition achieved with anti-C3a and anti-CD11b mAbs. In addition, platelet microparticles (PMPs) were shown to expose C3(H2O) SRPKIN-1 in a fashion similar to that shown by activated platelets, and the interaction of PMPs with PMNs was found to be similar in nature. Materials.