HUMAN MICROVASCULAR ENDOTHELIAL-CELLS ADHERE TO THROMBOSPONDIN-1 VIA AN RGD CSVTCG DOMAIN-INDEPENDENT MECHANISM/

Thrombospondin-1 (TSP-1), a 450-kDa glycoprotein secreted by platelets and endothelial cells at sites of tissue injury or inflammation, play s an important role in angiogenesis, inflammation, and vascular occlus ive skin diseases, Many of the physiologic and pathologic activities o f TSP-1 are dependent upon its interactions with endothelial cells. To better understand the basis of these activities, we examined the mech anisms mediating the binding of human dermal microvascular endothelial cells (HDMEC) to immobilized TSP-1. HDMEC bound to but did not spread on TSP-1 in a concentration-dependent manner. Monoclonal antibodies ( MoAbs) which recognize two purported TSP-1 binding proteins, CD36 and the alpha v integrin chain, or TSP-1-derived peptides CGRGDS and CSVTC G, alone or in combination with heparin, did not inhibit HDMEC adhesio n to immobilized TSP-1. Furthermore, CSVTCG-ovalbumin conjugates faile d to support HDMEC adhesion. Although RGD-containing peptides immobili zed on plastic wells Supported HDMEC binding, they also induced cell s preading not characteristic of cell binding to TSP-1 and binding was i nhibited by free RGD peptide, Two MoAbs against different domains of T SP-1 (A4.1 and C6.1) failed to block HDMEC binding to TSP-1, but both MoAbs inhibited G361 human melanoma cell binding to TSP-1 by 60%. Acid treatment of TSP-1 almost completely abrogated its ability to Support HDMEC binding, while acid treatment inhibited G361 binding by 50%. Ho wever, either antibody completely abrogated G-361 cell binding to acid -treated TSP-1. These data demonstrate that HDMEC bind to immobilized TSP-1 in an RGD- and CSVTCG-independent manner via an acid labile epit ope(s) which is recognized via a receptor or receptors distinct from C D36 or alpha v beta 3 integrin receptor.