JBC Papers in Press. Published on April 23, 2013 as Manuscript M113.464750 The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M113.464750 Mechanism of batroxobin binding to fibrinogen and fibrin BATROXOBIN BINDS FIBRIN WITH HIGHER AFFINITY AND PROMOTES CLOT EXPANSION TO A GREATER EXTENT THAN THROMBIN Trang T. Vu1,2, Alan R. Stafford1,3, Beverly A. Leslie1,3, Paul Y. Kim1,3, James C. Fredenburgh1,3 and Jeffrey I. Weitz1,2,3,4† Thrombosis and Atherosclerosis Research Institute1 and the Departments of Medical Sciences,2 Medicine,3 and Biochemistry and Biomedical Sciences,4 McMaster University, Hamilton, Ontario, Canada. Running title: Mechanism of batroxobin binding to fibrinogen and fibrin To whom correspondence should be addressed: Dr. Jeffrey Weitz, Thrombosis and Atherosclerosis Research Institute, 237 Barton St. E., Hamilton, Ontario L8L 2X2, Canada Phone: 905-574-8550 Fax: (905) 575-2646 E-mail: [email protected] Downloaded from Keywords: Batroxobin, thrombin, fibrin(ogen), fibrinopeptides Background: Snake venom protease γA/γ'-fibrin(ogen) than γA/γA-fibrin(ogen). batroxobin clots fibrinogen in a manner In contrast, batroxobin binds both http://www.jbc.org/ distinct from thrombin. fibrin(ogen) isoforms with similar high Results: Batroxobin binds fibrin(ogen) with affinity (Kd values of about 0.5 μM) even higher affinity than thrombin and promotes though it does not interact with the γ'-chain. greater clot expansion. The batroxobin binding sites on Conclusion: Batroxobin’s distinctive fibrin(ogen) only partially overlap with by guest on July 22, 2019 interaction with fibrin(ogen) may contribute to those of thrombin because thrombin its unique pattern of fibrinopeptide release. attenuates, but does not abrogate, the Significance: Clinically, batroxobin is used as interaction of γA/γA-fibrinogen with a defibrinogenating agent, but its capacity to batroxobin. Furthermore, although both promote clot expansion may promote thrombin and batroxobin bind to the microvascular thrombosis. central E region of fibrinogen with a Kd value of 2-5 μM, the α17-53 and Bβ1-42 SUMMARY regions bind thrombin, but not batroxobin. Batroxobin is a thrombin-like serine Once bound to fibrin, the capacity of protease from the venom of Bothrops atrox batroxobin to promote fibrin accretion is moojeni that clots fibrinogen. In contrast to 18-fold greater than that of thrombin; a thrombin, which releases fibrinopeptide A finding that may explain the microvascular and B from the NH2 terminal domains of thrombosis that complicates envenomation the Aα- and Bβ- chains of fibrinogen, by Bothrops atrox moojeni. Therefore, respectively, batroxobin only releases batroxobin binds fibrin(ogen) in a manner fibrinopeptide A. Because the mechanism distinct from thrombin, which may responsible for these differences is contribute to its higher affinity interaction, unknown, we compared the interactions of selective fibrinopeptide A release, and batroxobin and thrombin with the prothrombotic properties. predominant γA/γA isoform of fibrin(ogen) Fibrinogen is important for optimal and the γA/γ' variant with an extended γ- primary and secondary hemostasis because of chain. Thrombin binds to the γ'-chain and its critical role in platelet aggregation and forms a higher affinity interaction with fibrin clot formation (1). Fibrinogen is a dimeric glycoprotein composed of two pairs of 1 Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Mechanism of batroxobin binding to fibrinogen and fibrin Aα-, Bβ- and γ-chains connected by numerous fVIII and fV (10,17). The higher affinity, disulfide bonds (2). The NH2-termini of the bivalent interaction of thrombin with γA/γ'- six chains form the central E domain, which is fibrin affords thrombin more protection from connected by the coiled-coil regions to the fluid-phase inhibitors than the univalent peripheral D-domains formed by the COOH- interaction of thrombin with γA/γA-fibrin (18). termini (2). There are two circulating isoforms Batroxobin is a serine protease isolated of fibrinogen that differ with respect to their γ- from Bothrops atrox moojeni venom that clots chains. The predominant fibrinogen isoform is fibrinogen (19). In contrast to thrombin, a homodimer that contains two γA-chains fibrinogen is the sole substrate for batroxobin, consisting of 411 residues and is designated and batroxobin only releases FpA and is not γA/γA-fibrinogen. About 10-15% of circulating inhibited by antithrombin or heparin cofactor fibrinogen is heterodimeric, containing one II (20). Because of these properties, γA-chain and one variant γ'-chain that batroxobin is often used in clinical possesses a 16-residue anionic extension at its laboratories to determine whether prolonged COOH-terminus. This minor fibrinogen thrombin clotting times are the result of population is designated γA/γ'-fibrinogen (3-5). heparin contamination or abnormal fibrinogen Epidemiological studies suggest that elevated molecules (21). In addition, batroxobin has Downloaded from levels of circulating γA/γ'-fibrinogen are been used clinically for prevention and associated with an increased risk of treatment of thrombosis because of its cardiovascular disease (6,7). capacity to lower circulating fibrinogen levels Thrombin is the protease that converts (22-25). fibrinogen to insoluble fibrin by releasing Although it is well established that 1 http://www.jbc.org/ fibrinopeptides (Fp) A and B from the NH2- batroxobin induces defibrinogenation, the termini of the Aα- and Bβ-chains, respectively interaction of batroxobin with fibrinogen has (8). Fibrin polymerization occurs when the not been studied nor is it known whether, like newly exposed NH2-termini on one fibrin thrombin, batroxobin binds to fibrin and monomer bind to pre-existing, complementary triggers thrombus expansion. The potential by guest on July 22, 2019 sites on the D-domains of adjacent fibrin procoagulant activity of batroxobin is monomers (2,8,9). During this process, some important because patients envenomated by thrombin remains bound to the fibrin clot (10). Bothrops atrox moojeni often experience Thrombin possesses two anion binding sites thrombotic complications (26,27). To address that flank the active site and mediate substrate these questions, we (a) characterized the binding and catalysis (11,12). Exosite I binding of batroxobin to γA/γA- and γA/γ'- mediates thrombin binding to the Aα- and Bβ- fibrinogen to determine whether its fibrinogen chains in the central E-region of fibrin(ogen), binding sites overlap with those of thrombin whereas exosite 2 is responsible for because both enzymes release FpA; (b) thrombin’s interaction with the unique COOH- assessed the binding of batroxobin to γA/γA- terminus of the γ'-chain of a second and γA/γ'-fibrin clots to determine whether the fibrin(ogen) molecule (13,14). Consequently, protease remains bound to the product thrombin binds γA/γA-fibrin solely via exosite following catalysis; and (c) measured the 1 with a Kd value of 2 to 4 μM. In contrast, procoagulant activity of fibrin-bound both exosites are engaged when thrombin batroxobin to determine whether like fibrin- binds to γA/γ'-fibrin(ogen), which results in a bound thrombin, batroxobin promotes clot ~20-fold higher affinity interaction than that expansion. with γA/γA-fibrin (Kd value of 80 to 200 nM) (15,16). Because fibrin-bound thrombin EXPERIMENTAL PROCEDURES retains activity and is protected from inhibition by antithrombin and heparin Materials cofactor II, the fibrin clot serves as a reservoir of thrombin that promotes thrombus expansion Reagents—Human α–thrombin, fXIII and by locally activating platelets and factor (f) XI, plasminogen-free fibrinogen were from 2 Mechanism of batroxobin binding to fibrinogen and fibrin Enzyme Research Laboratories (South Bend, Modification of proteins and peptides— IN). Fibrinogen was rendered fXIII-free and Batroxobin and thrombin were radiolabeled by 125 125 γA/γA- and γA/γ'-fibrinogen were separated by reaction with I-YPR, prepared using Na I fractionation on DEAE-Sepharose (GE (McMaster University Nuclear Reactor, Healthcare) as previously described (18,28). Hamilton, ON) and Iodo-beads (Pierce, Batroxobin from the venom of Bothrops atrox Rockford, IL) as described (18,30). 125I-YPR- moojeni was from Pentapharm (Basel, batroxobin and 125I-YPR-thrombin Switzerland). A 20-amino acid analog of the concentrations were 1-2 and 6-7 μM, COOH-terminus of the γ'-chain of fibrinogen respectively, as determined by absorbance at with a Cys at the NH2-terminus, termed γ'- 280 nm, and had specific radioactivities of peptide (CVRPEHPAETEYDSLYPEDDL), ~500,000-700,000 cpm/μg. Unfractionated and a γ'-peptide-directed IgG from sheep were fibrinogen (0.5 mg in phosphate buffered prepared by Bachem Bioscience, Inc. (King of saline) was radiolabeled with 1 mCi Na125I Prussia, PA). The two Tyr residues of the γ'- using 1 Iodo-bead for 5 min at 23°C. The peptide were modified with phosphate groups sample was then subjected to gel filtration on to mimic the sulfated Tyr residues found in the a PD-10 column (GE Healthcare). The labeled native γ'-chain and to increase the affinity for fibrinogen was over 90% clottable and had a thrombin (29). The γ'-peptide-directed IgG specific radioactivity of 925,000 cpm/μg. Downloaded from was subjected to affinity chromatography FPR-batroxobin, FPR-thrombin, and biotin using immobilized γ'-peptide as described conjugated γ'- and α17-53-peptides were previously (30). The α17-53-peptide prepared as described (33). (CGPRVVERHQSAAKDSDWPFASDEDW http://www.jbc.org/ NYKAPSGCRM) with an NH2-terminal Cys Methods residue was synthesized by Life(pro)tein (Plainfield, New Jersey). Chromogenic Characterization of thrombin and substrates H-D-Phe-Pip-Arg-p-nitroaniline (S- batroxobin—The integrity of thrombin and 2238) and pyroGlu-Pro-Arg-nitroaniline (CS- batroxobin was assessed by SDS-PAGE by guest on July 22, 2019 21(66)) were from Chromogenix (Milano, analysis on 4-15% polyacrylamide gradient Italy) and Aniaria (Neuville-sur-Oise, France), gels (Bio-Rad, Mississauga, Ontario) under respectively. D-Phe-Pro-Arg-chloromethyl non-reducing conditions.