Characterization of the Mechanism of Interaction between C-Reactive Protein and GPIIb/IIIa.

C-reactive protein (CRP) is a well established marker for inflammation, and a good predictor of coronary heart disease. It is also known to interact with the platelet FcγRIIa and to enhance the inhibition of platelet aggregation by aspirin by an unknown mechanism. CRP has also recently been demonstrated to compete for PAC-1 binding in collagen stimulated platelets, suggesting that CRP interacts with GPIIb/IIIa. In order to study the mechanism of interaction with platelets directly, we carried out platelet adhesion assays. We coated plates with recombinant CRP which was in the native pentameric form as confirmed by size exclusion chromatography. Platelets adhered to immobilized CRP and to immobilized fibrinogen to a similar extent. Adhesion to CRP and fibrinogen was inhibited by GPIIb/IIIa antagonists but not by antibody to the FcγRIIa (IV.3). Platelet adhesion to CRP was increased 5-fold when platelets were treated with 1 mM MnCl2. Adhesion of MnCl2 treated platelets was also inhibited by GPIIb/IIIa antagonists. When viewed by confocal microscopy, the adherent platelets displayed pseudopodia, but did not spread fully. Treatment of platelets with MnCl2 stimulated lamellipodia formation and full platelet spreading on CRP. Analysis of the exposed residues on the surface of the CRP crystal structure identified two RGD-like sequences, RQD and DGK. The peptides KRQDN and VDGKP derived from CRP inhibited platelet adhesion to fibrinogen suggesting that these sequences could be responsible for the interaction with GPIIb/IIIa. Our data demonstrates that CRP in the native pentameric form interacts with GPIIb/IIIa and that it has an increased affinity for the activated conformation of GPIIb/IIIa. This interaction is likely to be due to an interaction with the surface exposed KRQDN and VDGKP CRP sequences. We have previously demonstrated that CRP acts as a GPIIb/IIIa antagonist when in solution. Furthermore, pentameric CRP cannot support platelet adhesion under shear conditions. Thus it is likely that elevated circulating levels of soluble CRP reduces the overall thrombotic potential.