α2β1 Integrin Is Not Recognized by Rhodocytin but Is the Specific, High Affinity Target of Rhodocetin, an RGD-independent Disintegrin and Potent Inhibitor of Cell Adhesion to Collagen

Abstract

We have recombinantly expressed a soluble form of human α 2β1 integrin that lacks the membrane-anchoring transmembrane domains as well as the cytoplasmic tails of both integrin subunits. This soluble α2β1 integrin binds to its collagen ligands the same way as the wild-type α2β 1 integrin. Furthermore, like the wild-type form, it can be activated by manganese ions and an integrin-activating antibody. However, it does not bind to rhodocytin, a postulated agonist of α2β 1 integrin from the snake venom of Calloselasma rhodostoma, which elicits platelet aggregation. Taking advantage of the recombi. nantly expressed, soluble α2β1 integrin, an inhibition assay was established in which samples can be tested for their capability to inhibit binding of soluble α2β1 integrin to immobilized collagen. Thus, by scrutinizing the C. rhodostoma snake venom in this protein-protein interaction assay, we found a component of the snake venom that inhibits the interaction of soluble α2β1 integrin to type I collagen efficiently. N-terminal sequences identified this inhibitor as rhodocetin, a recently published antagonist of collagen-induced platelet aggregation. We could demonstrate that its inhibitory effect bases on its strong and specific binding to α2β1 integrin, proving that rhodocetin is a disintegrin. Standing apart from the growing group of RGD-dependent snake venom disintegrins, rhodocetin interacts with α2β1 integrin in an RGD-independent manner. Furthermore, its native conformation, which is stabilized by disulfide bridges, is indispensibly required for its inhibitory activity. Rhodocetin does not contain any major collagenous structure despite its high affinity to α2β1 integrin, which binds to collagenous molecules much more avidly than to noncollagenous ligands, such as laminin. Blocking α2β1 integrin as the major collagen receptor on platelets, rhodocetin is responsible for hampering collagen-induced, α2β1 integrin-mediated platelet activation, leading to hemorrhages and bleeding disorders of the snakebite victim. Moreover, having a widespread tissue distribution, α2β1 integrin also mediates cell adhesion, spreading, and migration. We showed that rhodocetin is able to inhibit α2β1 integrin-mediated adhesion of fibrosarcoma cells to type I collagen completely.