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It is now well established that monocytes adhere to endothelial cells activated by oxidized low-density lipoproteins (LDL). However, the adhesive receptors on endothelial cells involved in binding monocytes, following an insult by oxidized LDL, remains to be elucidated. In this study we have looked at the effect of native or oxidized LDL on the expression of P-selectin. Native LDL (N-LDL) was oxidized by incubation with either endothelial cells (EC-LDL) or copper (Cu-LDL), or in culture medium as a control (C-LDL). Expression of P-selectin was assayed with an anti-P-selectin (CD62) monoclonal antibody (LYP20). Results show that EC-LDL and Cu-LDL, but not N-LDL or C-LDL, induce the expression of P-selectin by human umbilical-vein endothelial cells (HUVECs). Induction of P-selectin by low concentrations (20 micrograms/ml) of LDL is directly related to the state of oxidation of the LDL particles. In addition, high concentrations (100 micrograms/ml) of N-LDL also activate HUVECs by inducing P-selectin expression. This expression was sustained for a period of over 1 h on LDL-activated endothelial cells, in contrast with thrombin- or histamine-activated endothelial cells, whose P-selectin levels fall within 15-20 min after induction. E-selectin, in contrast with P-selectin, could not be induced by endothelial cells treated with low or high concentrations of oxidized LDL. Results in this study show that P-selectin expressed by oxidized-LDL-treated endothelial cells are involved in mediating the adhesion of a monocytic cell line (U937) or monocytes in peripheral-blood mononuclear cells. An anti-P-selectin monoclonal antibody (LYP20) inhibited the binding of U937 cells and monocytes. These results strongly suggest that P-selectin is involved in the early stages of atherogenesis.

Adhesion of platelets to collagen, exposed on the subendothelium as a result of vessel wall injury, is a vital step in the formation of a haemostatic plug. Glycoprotein CD36, also known as GPIIIb/GPIV, is one of the platelet glycoproteins known to interact with collagen. The aim of this work was to identify structural/functional sites on CD36 interacting with collagen. Eight peptides, corresponding to sites presumed to be hydrophylic, were synthesized by Fmoc (Fluorenylmethoxycarbonyl) chemistry. Peptides were tested for their ability to inhibit platelet aggregation induced by type I collagen. Peptide E5 (WLNETGTIGDEKA; 415-427), but not the other peptides, inhibited aggregation and secretion of washed platelets induced by collagen but had no effect on thrombin or ADP induced aggregation. Moreover, peptide E5 was shown to interfere with the binding of (125)I-labelled CD36 to collagen. Peptide E5 had little or no effect on collagen-induced platelet aggregation performed in platelet rich plasma (PRP), as previously described in the cases of monoclonal antibodies directed against α(2)or β(1). These results indicate that peptide E5 represents a site on CD36 that interacts with collagen and is involved in platelet functions.

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