CONTORTROSTATIN, A HOMODIMERIC SNAKE-VENOM DISINTEGRIN, IS A POTENT INHIBITOR OF OSTEOCLAST ATTACHMENT

Disintegrins are small disulfide-rich proteins containing an Arg-Gly-A sp (RGD) sequence near their carboxyl terminus, These polypeptides inh ibit binding of adhesion molecules to their receptors (integrins) on t he surface of cells, Osteoclasts express integrins, heterodimeric cell surface adhesion receptors, that have been shown to be involved in in teractions with the extracellular matrix (ECM), including attachment t o bone and bone resorption, It has recently been shown that disintegri ns effectively inhibit attachment of osteoclasts to components of the ECM and also disrupt osteoclast-mediated bone resorption, Here we char acterize the effects of contortrostatin (CTS), a novel homodimeric sna ke venom disintegrin, on osteoclast attachment, Plastic dishes coated with CTS were able to support osteoclast attachment with a high affini ty (EC50,CTS = 86 +/- 6.7 nM) similar to that of vitronectin (VTN; EC5 0,VTN = 80 +/- 20 nM), Further, CTS was observed to inhibit completely osteoclast attachment to fetal bovine serum (FBS; IC50,FBS = 0.36 +/- 0.04 nM) and VTN (IC50,VTN = 0.85 +/- 0.13 nM), We used monoclonal an tibodies directed against the beta(1) (monoclonal antibody [MAb] CD29) and beta(3) (MAb F11) integrin subunits to explore the mechanism of o steoclast attachment to immobilized CTS, Only MAb F11 inhibited attach ment to immobilized CTS (IC50 = 0.41 +/- 0.12 mu g/ml), suggesting tha t binding to CTS is mediated in part by a beta(3) integrin, presumably the alpha(v) beta(3) VTN receptor, In further support of an integrin- mediated mechanism, binding of osteoclasts to CTS is inhibited by the RGD peptide, GRGDSP, CTS was also more potent (IC50,FBS = 0.36 +/- 0.0 4 nM) at inhibiting osteoclast attachment to FBS-coated wells than the monomeric snake venom disintegrin echistatin (IC50,FBS = 8.9 +/- 1.5 nM) or VTN (IC50,FBS = 97.5 +/- 25.5 nM), Taken together, these data s uggest that the snake venom disintegrin CTS is a potent inhibitor of b eta(3) integrin-mediated osteoclast attachment, presumably involving t he VTN receptor (an alpha(v) beta(3) integrin), Further studies of the mechanism of CTS-osteoclast interactions may aid in the design of pep tide mimetics to act as antiresorptive agents for the treatment of ost eoporosis and other skeletal pathology.