Isolation and Characterization of EMS16, a C-Lectin Type Protein from Echis multisquamatus Venom, a Potent and Selective Inhibitor of the R21 Integrin ² Cezary Marcinkiewicz,* ,‡ Roy R. Lobb, § Mariola M. Marcinkiewicz, | James L. Daniel, ⊥ J. Bryan Smith, ⊥ Carol Dangelmaier, ⊥ Paul H. Weinreb, § Dorothy A. Beacham, # and Stefan Niewiarowski ‡,| Sol Sherry Thrombosis Research Center, Department of Pharmacology, and Department of Physiology, Temple UniVersity, School of Medicine, 3400 North Broad Street, Philadelphia, PennsylVania 19140, Biogen Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, and Cardeza Foundation for Hematologic Research, Thomas Jefferson UniVersity, Philadelphia PennsylVania 19107 ReceiVed February 24, 2000; ReVised Manuscript ReceiVed June 14, 2000 ABSTRACT: We have isolated and characterized EMS16, a potent and selective inhibitor of the R21 integrin, from Echis multisquamatus venom. It belongs to the family of C-lectin type of proteins (CLPs), and its amino acid sequence is homologous with other members of this protein family occurring in snake venoms. EMS16 (M r ∼ 33K) is a heterodimer composed of two distinct subunits linked by S-S bonds. K562 cells transfected with R2 integrin selectively adhere to immobilized EMS16, but not to two other snake venom- derived CLPs, echicetin and alboaggregin B. EMS16 inhibits adhesion of R21-expressing cells to immobilized collagen I at picomolar concentrations, and the platelet/collagen I interaction in solution at nanomolar concentrations. EMS16 inhibits binding of isolated, recombinant I domain of R2 integrin to collagen in an ELISA assay, but not the interaction of isolated I domain of R1 integrin with collagen IV. Studies with monoclonal antibodies suggested that EMS16 binds to the R2 subunit of the integrin. EMS16 inhibits collagen-induced platelet aggregation, but has no effect on aggregation induced by other agonists such as ADP, thromboxane analogue (U46619), TRAP, or convulxin. EMS16 also inhibits collagen- induced, but not convulxin-induced, platelet cytosolic Ca 2+ mobilization. In addition, EMS16 inhibits HUVEC migration in collagen I gel. In conclusion, we report a new, potent viper venom-derived inhibitor of R21 integrin, which does not belong to the disintegrin family. A collagen receptor, the R21 integrin, is expressed on platelets, endothelial, many epithelial, and some tumor cells. Its R2 subunit contains an I domain, a ∼200 amino acid extracellular region homologous with the von Willebrand factor (vWF) 1 A domain. A homologous domain is also found in other R subunits of integrins such as R1, RM, and RL(1-3). Ligand binding of these integrins is frequently mediated through the I domain and in most cases is metal dependent (4). Together with R11, the R21 integrin belongs to a subset of integrins that binds to collagen I, collagen IV, and laminin, but with different affinities (5, 6). In view of the importance of R21 in physiology and disease, this integrin is now the focus of research in many labora- tories. R21 expressed on endothelial cells plays a significant role in angiogenesis and neovascularization (7). Thus, inhibitors of this integrin may be important in drug develop- ment for tumor therapy. Viper venoms contain a number of proteins showing antagonistic effects on adhesive receptors expressed on the cell surfaces. These proteins belong to two classes: disin- tegrins and C-lectin proteins (CLPs). Short, RGD-containing disintegrins include echistatin, which inhibits three integrins (RIIb3, Rv3, R51), and eristostatin, which is selective to RIIb3(8-10). The heterodimeric disintegrins are another class, which are more specific for leukocyte integrins. Two of them, EC3 and EMF10, characterized recently (11, 12), inhibit R41/R47 and R51, respectively, with a high degree of selectivity. The metalloproteinase-disintegrin (jararhagin) inhibits collagen-induced platelet aggregation mediated by the R21 integrin(13). C-lectin proteins repre- sent a separate, large family of snake venom proteins. The protototype of CLPs is the mannose binding protein. The snake venom CLPs demonstrate several biological activities; however, there has been no report of their anti-integrin activity. Echicetin (14), agkicetin (15), flavocetins (16), and tokaracetin (17) bind to the GPIb/IX complex and inhibit ² Aided in part by an Initial Investigatorship from the American Heart Association South-Eastern Pennsylvania Chapter (C.M.), W. W. Smith Charitable Trust Research Grant (C.M.), NIH FIRST award (D.A.B.), American Heart Association (D.A.B. and S.N.), American Diabetes Association (S.N.), and Barra Foundation (S.N.). * Corresponding author. Tel: 215-707-4419; Fax: 215-707-2783; e-mail: cmarcink@nimbus.temple.edu. ‡ Sol Sherry Thrombosis Research Center, Temple University. § Biogen Inc. | Department of Physiology, Temple University. ⊥ Department of Pharmacology, Temple University. # Cardeza Foundation for Hematologic Research, Thomas Jefferson University. 1 Abbreviations: AP, alkaline phosphatase; BSA, bovine serum albumin; CLP, C-lectin protein; CMFDA, 5-chloromethylfluorescein diacetate; GST, glutathione-S-transferase; HBSS, Hanks balanced salt solution; HUVEC, human umbilical vein endothelial cells; mab, monoclonal antibody; MIDAS, metal ion-dependent adhesion site; PBS, phosphate-buffered saline; PRP, platelet rich plasma; TBS, Tris-buffered saline; TFA, trifluoroacetic acid; TRAP, thrombin receptor activating peptide; VCAM-1, vascular cell adhesion molecule 1; vWF, von Willebrand factor. 9859 Biochemistry 2000, 39, 9859-9867 10.1021/bi000428a CCC: $19.00 © 2000 American Chemical Society Published on Web 07/18/2000