Biochemistry 1995,34, zyxwvu 8701 -8707 8701 Alteration of the Substrate and Inhibitor Specificities of Blood Coagulation Factor VIIa: Importance of Amino Acid Residue K192 zyx + Pierre F. Neuenschwander and James H. Monissey" Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 Received February zyxwvutsr 8, 1995; Revised Manuscript Received May zyxwv 2, zyxw 1995@ ABSTRACT: Initiation of blood coagulation occurs when the plasma serine protease factor VIIa (fVIIa) binds to its cell-surface receptor/cofactor, tissue factor (TF). This binding interaction mediates a large enhancement in both the proteolytic activity and the amidolytic activity (hydrolysis of small peptidyl- amide substrates) of fVIIa. This necessitates local changes in the catalytic center of fVIIa of which little is understood. Studies with thrombin and activated protein C have demonstrated that residue (chymotrypsinogen numbering system) near the active site of these proteases is an important determinant for substrate and inhibitor specificity. By homology, residue 192 in fVIIa is K, bringing into question the potential role of this residue in fVIIa. We have prepared two mutants of fVIIa in which K'92 has been replaced by either Q (as in factors IX and X) or E (as in thrombin). Both mutants were found to be defective in clotting: fVIIK192Q was zyxwvut 44% active, while fVIIK192E was completely ineffective. This defect was attributable to proportional decreases in specificity constants for activation of factor X. Although both mutant enzymes were catalytically competent with respect to amidolytic activity, the selectivity of fVIIaK192E was greatly altered. Inhibition of both mutants by the TF pathway inhibitor (TFPI) and bovine pancreatic trypsin inhibitor (BPTI) was also drastically altered. Neither mutant was inhibited by TFPI, while fVIIaK192Q was inhibited by BPTI better than wild-type fVIIa. In contrast, fVIIaK192E was poorly inhibited by BPTI and made more refractory to inhibition when bound to TF. These results suggest a potential role for KI9* in governing the substrate and inhibitor specificities of fVIIa. It is generally accepted that under normal hemostatic conditions the blood coagulation enzyme cascade is triggered when damage to vascular endothelium exposes cellular tissue factor (TF),' an integral membrane protein and the receptor/ cofactor for the plasma serine protease NIIa [reviewed by Carson and Brozna (1993)l. Since NIIa in the absence of TF is a relatively inefficient protease, the fVIIa*TFcomplex can be considered a two-subunit enzyme where TF acts as the regulatory subunit and fVIIa acts as the catalytic subunit. Upon binding to TF, the enzymatic activity of fVIIa is dramatically enhanced, and it is the NIIa*TF complex which subsequently catalyzes the activation of fVII, fIX, and zyxwvu fX, thus simultaneously amplifying and propagating the coagula- tion response. As implied by the original proposals of an enzyme cascade for describing blood coagulation (Davie & Ratnoff, 1964; Macfarlane, 1964), the amplificative nature of linked enzyme ~ + This research was supported in part by NIH Grant R01 HL47014 (to J.H.M.) and Grant F32 HL08710 (to P.F.N.). * To whom all correspondence should be addressed at: Oklahoma Medical Research Foundation, 825 NE 13th St., Oklahoma City, OK 73104. Tel: (405) 271-7892. Fax: (405) 271-3137. @ Abstract published in Advance ACS Abstracts, June 15, 1995. Abbreviations: TF, tissue factor; TFPCPS, TF relipidated in phospholipid vesicles composed of 80% phosphatidylcholine (PC) and 20% phosphatidylserine (PS); sTF, soluble TF1-219-the extracellular domain of TF; fVII, blood-clotting factor VI1 (single chain); fVIIa, activated fVII (two chain); fVIIK192Q and fVIIK192E, mutated fVII where amino acid residue Ki9* has been changed to Q or E; fVIIaK192Q and fVIIaK192E, activated fVIIK192Q and fVIIK192E; fIX, blood- clotting factor IX; zyxwvutsrqp fX, blood-clotting factor X; fXa, activated fX; BPTI, bovine pancreatic trypsin inhibitor; TFPI, TF pathway inhibitor; PCR, polymerase chain reaction. Standard single-letter amino acid abbrevia- tions are used throughout. 0006-2960/95/0434-8701$09.00/0 0 reactions essentially requires that the proteases involved be highly specific to prevent unwanted activation and enable precise control of the system. It is not surprising, therefore, that the blood-clotting serine proteases all share the property of exhibiting extremely narrow substrate (and inhibitor) specificity. However, unlike the digestive enzymes trypsin and chymotrypsin-the classical representatives of the serine protease family of enzymes-whose specificities and mech- anisms of action have been intensively studied, the molecular bases of the specificities of the larger and more complex blood-clotting serine proteases remain largely unresolved. The ability of TF to affect the kinetics of substrate hydrolysis catalyzed by WIIa is well documented (Zur & Nemerson, 1980; Bach et al., 1986; Bom & Bertina, 1990; Bom et al., 1990) and generally involves a large increase in the specificity constant (k,,JK,,,) for substrate hydrolysis. While the Ca*+-binding y-carboxyglutamic acid-rich domain of NIIa has been suggested to be important for recognition of natural substrates (Martin et al., 1993), there are sugges- tions in the literature that TF may also play a role in direct macromolecular substrate recognition in the WIIa.TF com- plex (Ruf et al., 1992). However, this has yet to be conclusively demonstrated. Unlike other cofactor-regulated enzymes in blood coagulation, fVIIa shows enhanced activity upon binding to TF which is not limited to hydrolysis of native macromolecular protein substrates but also includes hydrolysis of small synthetic peptidyl-amide substrates (Ruf et al., 1991). The enhancement observed with amide substrates appears to be substrate-dependent and can take the form of a decrease in K,, an increase in k,,,, or both (Higashi et al., 1992; Butenas et al., 1993, 1994; Neuen- schwander et al., 1993). Thus, binding of NIIa to TF seems 1995 American Chemical Society