SummaryC-reactive protein (CRP) is a strong predictor for acute cardiovascular events. Several endothelial prothrombotic effects of CRP have been recently reported. This study examined the effect of CRP on bovine aortic endothelial cell (EC) activation and capacity to recruit human platelets under flow conditions using the cone and plate(let) analyser method. Human recombinant CRP promoted platelet adhesion in a dose-and timedependent manner, with a maximal effect at 20 lg/ml (increase of 174% over baseline, P < 0AE01). Similar effects were observed following incubation of EC with sera of transgenic mice that express human CRP (10 lg/ml). Antiintercellular adhesion molecule-1 neutralising monoclonal antibody and nitric oxide donor, sodium nitroprusside, blocked the effect of CRP, reducing adhesion from 202% to 128% (P < 0AE05) and 114% (P ¼ 0AE02) respectively. The pro-adhesive effect of CRP was abolished by calphostin C (a protein kinase C inhibitor), whereas the extracellular signal-regulated kinase antagonist, PD98059, did not have any effect. CRP promoted P-selectin expression on the EC surface and blockade of P-selectin reversed CRPinduced platelet adhesion. In conclusion, CRP promoted platelet adhesion to EC. Our results emphasise the possible role of CRP in linking inflammation and thrombosis and provide a potential mechanism for the high incidence of vascular events associated with high CRP levels.Keywords: C-reactive protein, platelets, endothelium, adhesion, cone and platelet analyser.
Materials and methods
Cells and reagentsBovine aortic endothelial cells (BAEC) were maintained in an incubator at 37°C, 5% CO 2 and a humidified atmosphere. Cells were grown in Dulbecco's modified Eagles medium supplemented with 10% fetal calf serum, l-glutamine and 1% penicillin-streptomycin in four-well tissue culture plates (NUNC, Rochester, NY, USA) until they reached 80% confluence, prior to being utilised in the platelet adhesion assay. The following reagents were used in the study: monoclonal antibodies (mAbs) raised against intercellular adhesion molecule-1 (ICAM-1) and P-selectin (Dako, A/S, Glostrup, Denmark), sodium nitroprusside (SNP), PD98059 and calphostin C (Calbiochem-Novabiochem Corp., San Diego, CA, USA), media and tissue culture supplements (Biological Industries, Beit Haemek, Israel).
Platelet isolation and labellingThis study conformed to principles of Declaration of Helsinki. Whole fresh blood was collected from healthy, young volunteers (n ¼ 17), by venipuncture and stabilised in a vacutainer with a final concentration of 3AE8% sodium citrate. After resting for 30 min, platelet-rich plasma was prepared by centrifugation at 165 g for 12 min and transferred to a separate test tube. The platelets were labelled by incubation with calcein-AM (30 min, room temperature; Molecular Probes, Eugene, OR, USA), after which they were reconstituted with autologous packed cells immediately prior to the cone and platelet analyser (CPA) assay.
Platelet adhesion assayPlatelet adhesion to EC under physiological shear...
