Olson, University or college of Illinois at Chicago. binding. Mutation of K98 to A in factor IXa results in enhanced reactivity with all inhibitors examined, while almost completely abrogating the enhancing effects of enoxaparin. The results implicate K98 and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite. Factor IXa (fIXa)1 is usually a vitamin K-dependent blood coagulation factor that is essential for the amplification or consolidation phase of blood coagulation (1,2). As with other blood coagulation factors (namely factors VIIa, Xa and thrombin) fIXa is usually a member of the serine protease family and shares a high degree of homology with trypsin. Despite this homology, the blood coagulation enzymes differ drastically from trypsin in that their activities are profoundly modulated by the binding of various protein and non-protein cofactors. In the case of fIXa, the ability of activated factor VIII (fVIIIa), anionic phospholipid and ionic calcium to enhance the procoagulant activity of fIXa is usually well documented (3C6); resulting in a 109-fold increase in activity of fIXa. The molecular details of this conversion have not been defined in total and are the subject of intense investigation by numerous groups. The major inhibitor of fIXa in plasma is usually antithrombin, whose reactivity with fIXa essentially requires heparin (7C9). Heparin is known to bind to antithrombin and sterically alter its conformation to allow this serpin to react with its target (10C13). Heparin also binds to fIXa (14) allowing long chains of heparin to additionally catalyze the conversation of fIXa with antithrombin via the formation of bridged complexes where heparin functions as a template. Recently, we have shown that low molecular excess weight heparin binding to fIXa enhances reactivity of fIXa with the Kunitz-type inhibitor BPTI (15), suggesting that oligosaccharide binding can also allosterically modulate the fIXa active site region. In this study we examine in greater detail the ability of heparin to modulate fIXa reactivity towards several isolated Kunitz-type inhibitor domains. We show that this modulatory effect of heparin can be completely abrogated by mutating a single amino acid residue in the 99-loop region of the extended fIXa active site cleft outside of the heparin binding exosite. Experimental Procedures Materials Factor IXa, factor VIIa, factor XIa and the factor X activator from Russells Viper venom (RVV-X) were purchased from Haematologic Technologies Inc. (Essex Junction, VT). Recombinant soluble tissue factor (the extracellular domain name of tissue factor) was expressed and purified from bacteria as previously explained (16). Factor Xa was prepared from plasma-derived factor X as previously explained (17). Enoxaparin (Lovenox?) was purchased from RU.521 (RU320521) Aventis Pharmaceuticals (Bridgewater, NJ). Purified heparin-derived oligosaccharides of 6, 10, 14 and 18 saccharide models (H6, H10, H14 and H18) were prepared and characterized essentially as explained (18C20) and were a generous gift of Dr. Steven T. Olson, University or college of Illinois at Chicago. Bovine serum albumin (Portion V, fatty acid free) was from Calbiochem (La Jolla, CA), and ethylene glycol was from Fisher Scientific. The chromogenic substrate CBS 31.39 (CH3SO2-d-LGR-strain BL21(DE3). Transformed bacterial cells were first produced to log phase at 37C in TB media made up of 50 g/ml Carbenicillin. Protein expression was induced by addition of IPTG to 0.5 mM (0.1 mM for TFPI-K1) and the cells were allowed to continue growing for 4 h at 37C. Inclusion bodies were isolated essentially as explained (25) and solubilized with 6 M Guanidine HCl made up of 20 mM DTT, 50 mM Tris-HCl pH 8.0 and 1 mM EDTA to obtain a total protein concentration of roughly 20 mg/ml. The solution was then clarified by centrifugation (16,000 g for 30 min) and oxidative refolding (26) of each protein preparation was performed by quick dilution into 20 vols of buffer made up of 50 mM Tris-HCl pH 8.0, 1 M guanidine HCl, 1 mM EDTA, 2.5 mM oxidized glutathione (Sigma) and 1 mM DTT. The diluted protein answer was incubated at room heat for 6 h with slow stirring for completion of protein refolding followed by exhaustive dialysis into an appropriate buffer for ion-exchange chromatography. Construction and expression of wild-type and mutant fIX The coding sequence for wild-type fIX in pBR322 (27) was a nice gift.As with other blood coagulation factors (namely factors VIIa, Xa and thrombin) fIXa is a member of the serine protease family and shares a high degree of homology with trypsin. inhibitor is usually sterically hindered by the 99-loop of factor IXa, specifically residue K98. RU.521 (RU320521) Slow-binding kinetic studies support the formation of a poor initial enzyme-inhibitor complex between factor IXa and basic pancreatic trypsin inhibitor that is facilitated by enoxaparin binding. Mutation of K98 to A in factor IXa results in enhanced reactivity with all inhibitors examined, while almost completely abrogating the enhancing effects of enoxaparin. The results implicate K98 and the 99-loop of factor IXa in defining enzyme inhibitor specificity. More importantly, these results demonstrate the ability of factor IXa to be allosterically modulated by occupation of the heparin-binding exosite. Factor IXa (fIXa)1 is usually a vitamin K-dependent blood coagulation factor that is essential for the amplification or consolidation phase of blood coagulation (1,2). As with other blood coagulation factors (namely factors VIIa, Xa and thrombin) fIXa is usually a member of the serine protease family and shares a high degree of homology with trypsin. Despite this homology, the blood coagulation enzymes differ drastically from trypsin in that their activities are profoundly modulated by the binding of various protein and non-protein cofactors. In the case of fIXa, the ability of activated factor VIII (fVIIIa), anionic phospholipid and ionic calcium to enhance the procoagulant activity of fIXa is usually well documented (3C6); resulting in a 109-fold increase in activity of fIXa. The molecular details of this conversion have not been defined in total and are the subject of intense investigation by numerous groups. The major inhibitor of fIXa in RU.521 (RU320521) plasma is usually antithrombin, whose reactivity with fIXa essentially requires heparin (7C9). Heparin is known to bind to antithrombin and sterically alter its conformation to allow this serpin to react with its target (10C13). Heparin also binds to fIXa (14) allowing long chains of heparin to additionally catalyze the conversation of fIXa with antithrombin via the formation of bridged complexes where heparin functions as a template. Recently, we have shown that low molecular excess weight heparin binding to fIXa enhances reactivity of fIXa with the Kunitz-type inhibitor BPTI (15), suggesting that oligosaccharide binding can also allosterically modulate the fIXa active site region. In this study we examine in greater detail the ability of IKK-beta heparin to modulate fIXa reactivity towards several isolated Kunitz-type inhibitor domains. We show that this modulatory effect of heparin can be completely abrogated by mutating a single amino acid residue in the 99-loop region of the extended fIXa active site cleft outside of the heparin binding exosite. Experimental Procedures Materials Factor IXa, factor VIIa, factor XIa and the factor X activator from Russells Viper venom (RVV-X) were purchased from Haematologic Technologies Inc. (Essex Junction, VT). Recombinant soluble tissue factor (the extracellular domain name of tissue factor) was expressed and purified from bacteria as previously explained (16). Factor Xa was prepared from plasma-derived factor X as previously explained (17). Enoxaparin (Lovenox?) was purchased from Aventis Pharmaceuticals (Bridgewater, NJ). Purified heparin-derived oligosaccharides of 6, 10, 14 and 18 saccharide models (H6, H10, H14 and H18) were prepared and characterized essentially as explained (18C20) and were a generous gift of Dr. Steven T. Olson, University or college of Illinois at Chicago. Bovine RU.521 (RU320521) serum albumin (Portion V, fatty acid free) was from Calbiochem (La Jolla, CA), and ethylene glycol was from Fisher Scientific. The chromogenic substrate CBS 31.39 (CH3SO2-d-LGR-strain BL21(DE3). Transformed bacterial cells were first produced to log phase at 37C in TB media made up of 50 g/ml Carbenicillin. Protein expression was induced by addition of IPTG to 0.5 mM (0.1 mM for TFPI-K1) and the cells were allowed to continue growing for 4 h at 37C. Inclusion bodies were isolated essentially as explained (25) and solubilized with 6 M Guanidine HCl made up of 20 mM DTT, 50 mM Tris-HCl pH 8.0 and 1 mM EDTA to obtain a total protein concentration of roughly 20 mg/ml. The solution was then clarified by centrifugation (16,000 g for 30 min) and oxidative refolding (26) of each protein preparation was performed by quick dilution into 20 vols of buffer made up of 50 mM Tris-HCl pH 8.0, 1 M guanidine HCl, 1 mM EDTA, 2.5 mM oxidized glutathione (Sigma).
