Roles of Low Specificity and Cofactor Interaction Sites on Thrombin during Factor XIII Activation
2003; Elsevier BV; Volume: 278; Issue: 34 Linguagem: Inglês
10.1074/jbc.m305364200
ISSN1083-351X
AutoresHelen Philippou, James B. Rance, Timothy Myles, Scott W. Hall, Robert A. S. Ariëns, Peter J. Grant, Lawrence Leung, David A. Lane,
Tópico(s)Platelet Disorders and Treatments
ResumoFactor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin. Factor XIII is activated by thrombin, and this reaction is enhanced by the presence of fibrin(ogen). Using a substrate-based screening assay for factor XIII activity complemented by kinetic analysis of activation peptide cleavage, we show by using thrombin mutants of surface-exposed residues that Arg-178, Arg-180, Asp-183, Glu-229, Arg-233, and Trp-50 of thrombin are necessary for direct activation of factor XIII. These residues define a low specificity site known to be important also for both protein C activation and for inhibition of thrombin by antithrombin. The enhancing effect of fibrinogen occurs as a consequence of its conversion to fibrin and subsequent polymerization. Surface residues of thrombin further involved in high specificity fibrin-enhanced factor XIII activation were identified as His-66, Tyr-71, and Asn-74. These residues represent a distinct interaction site on thrombin (within exosite I) also employed by thrombomodulin in its cofactor-enhanced activation of protein C. In competition experiments, thrombomodulin inhibited fibrin-enhanced factor XIII activation. Based upon these and prior published results, we propose that the polymerization process forms a fibrin cofactor that acts to approximate thrombin and factor XIII bound to separate and complementary domains of fibrinogen. This enables enhanced factor XIII activation to be localized around the fibrin clot. We also conclude that proximity to and competition for cofactor interaction sites primarily directs the fate of thrombin. Following initiation of coagulation, a series of carefully regulated serine proteinase reactions take place resulting in the generation of thrombin and the formation of an insoluble fibrin clot. Thrombin is a serine proteinase responsible for proteolytic cleavage of multiple substrates involved in the coagulation pathway (1Esmon C.T. Science. 1987; 235: 1348-1352Crossref PubMed Scopus (568) Google Scholar, 2Mann K.G. Lundblad R.L. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Haemostasis and Thrombosis. 2nd Ed. Churchill Livingstone, New York1987: 148-161Google Scholar). One of the main roles of thrombin is the conversion of fibrinogen to an insoluble fibrin clot. This process is initiated by proteolytic cleavage of two pairs of fibrinopeptides, FPA 1The abbreviations used are: FPA and FPB, fibrinopeptides A and B, respectively; HPLC, high pressure liquid chromatography; WT, wild-type. and FPB, from the Aα- and Bβ-chains of fibrinogen, respectively. FPA is cleaved first, leading to spontaneous polymerization of the fibrin monomers. This is shortly followed by proteolysis of the Bβ-chain, which is associated with lateral aggregation of the fibrin protofibrils to produce thicker fiber bundles (3Weisel J.W. Biophys. J. 1986; 50: 1079-1093Abstract Full Text PDF PubMed Scopus (164) Google Scholar, 4Weisel J.W. Veklich Y. Gorkun O.V. J. Mol. Biol. 1993; 232: 285-297Crossref PubMed Scopus (130) Google Scholar). To ensure a more stable clot structure, activated factor XIII (factor XIIIa), a transglutaminase, covalently cross-links specific glutamine and lysine side chains of the protofibrils, resulting in increased resistance of the clot to chemical, physical, and proteolytic insults (5Ichinose A. Bloom A.L. Forbes C.D. Thomas D.P. Tuddenham E.G.D. Haemostasis and Thrombosis. Churchill Livingstone, New York1994: 531-546Google Scholar, 6Lorand L Ann. N. Y. Acad. Sci. 2001; 936: 291-311Crossref PubMed Scopus (200) Google Scholar). Factor XIII is a heterologous tetramer with a molecular mass of 324,000 Daltons. It consists of two A-subunits that contain the active site of the transglutaminase and two B-subunits that serve a carrier function for the hydrophobic A-subunit in the aqueous environment of human plasma (5Ichinose A. Bloom A.L. Forbes C.D. Thomas D.P. Tuddenham E.G.D. Haemostasis and Thrombosis. Churchill Livingstone, New York1994: 531-546Google Scholar, 6Lorand L Ann. N. Y. Acad. Sci. 2001; 936: 291-311Crossref PubMed Scopus (200) Google Scholar). Thrombin activates factor XIII by cleavage of a 37 amino acid activation peptide from the factor XIII A-subunits (7Takagi T. Doolittle R.F Biochemistry. 1974; 13: 750-756Crossref PubMed Scopus (184) Google Scholar). Consequently, the carrier B-subunits dissociate from the activated A-subunits to completely unmask the active site (8Lorand L. Gray A.J. Brown K. Credo R.B. Curtis C.G. Domanik R.A. Stenberg P. Biochem. Biophys. Res. Commun. 1974; 56: 914-922Crossref PubMed Scopus (77) Google Scholar). Activation of factor XIII is closely controlled by the presence of its substrate fibrin(ogen). The presence of fibrin(ogen) accelerates activation of factor XIII ∼80-fold (9Janus T.J. Lewis S.D. Lorand L. Shafer J.A. Biochemistry. 1983; 22: 6269-6272Crossref PubMed Scopus (83) Google Scholar). This acceleration is caused by an enhancing effect of fibrin on both thrombin cleavage of the factor XIII activation peptide and dissociation of the factor XIII A- and B-subunits. It has been shown that residues 242–424 in the αC-domain of fibrinogen regulate the dissociation of the B-chains from the thrombin-cleaved A-subunits of factor XIII (10Credo R.B. Curtis C.G. Lorand L. Biochemistry. 1981; 20: 3770-3778Crossref PubMed Scopus (73) Google Scholar). The enhancement effect of fibrinogen on factor XIII activation can be readily inhibited by specific inhibitors of fibrin polymerization (11Ariens R.A.S. Philippou H. Nagaswami C. Weisel J.W. Lane D.A. Grant P.J. Blood. 2000; 96: 988-995Crossref PubMed Google Scholar, 12Greenberg C.S. Miraglia C.C. Blood. 1985; 66: 466-469Crossref PubMed Google Scholar). However, the process by which polymerizing fibrin enhances the activation of factor XIII is poorly understood. Approximately 10% total fibrinogen exists as a variant known as γ′. This fibrinogen variant occurs by alternative splicing of mRNA resulting in the deletion of four amino acids from the C terminus of γA with a substitution of an additional 20 amino acid residues (13Fornace A.J. Cummings D.E. Comeau C.M. Kant J.A. Crabtree G.R. J. Biol. Chem. 1984; 259: 12826-12830Abstract Full Text PDF PubMed Google Scholar). The γ′-region is thought to bind factor XIII zymogen and thrombin (14Meh D.A. Siebenlist K.R. Mosesson M.W. J. Biol. Chem. 1996; 271: 23121-23125Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar) but the functional consequences of this for factor XIII activation are as yet uncertain (15Siebenlist K.R. Meh D.A. Mosesson M.W. Biochemistry. 1996; 35: 10448-10453Crossref PubMed Scopus (137) Google Scholar). In addition to activating factor XIII and proteolytic cleavage of FPA and FPB from fibrinogen, thrombin is also responsible for numerous other proteolytic interactions. It activates factors V, VIII, XI, and thrombin-activatable fibrinolysis inhibitor (2Mann K.G. Lundblad R.L. Colman R.W. Hirsh J. Marder V.J. Salzman E.W. Haemostasis and Thrombosis. 2nd Ed. Churchill Livingstone, New York1987: 148-161Google Scholar, 16Bajzar L. Morser J. Nesheim M. J. Biol. Chem. 1996; 28: 16603-16608Abstract Full Text Full Text PDF Scopus (601) Google Scholar). Furthermore, thrombin cleaves the protease (activated) receptors, resulting in platelet activation and aggregation (17Coughlin S.R. Nature. 2000; 407: 258-264Crossref PubMed Scopus (2156) Google Scholar). These functions of thrombin are all procoagulant (ultimately promoting clot formation). However, thrombin is also able to behave as an anticoagulant by activating protein C when bound to thrombomodulin. Activated protein C cleaves and thereby inhibits both activated factors V and VIII. Additionally, thrombin is directly inhibited by formation of an irreversible complex with antithrombin. As substrates of thrombin participate in either procoagulant or anticoagulant functions, thrombin is critical for regulating hemostasis. The unique specificity of thrombin toward its substrates is thought to arise by combination of insertion loops flanking the upper and lower faces of the active site (Leu-46/Asn-57 and Leu-144/Gly-155), which occlude and restrict the active site and two exosites (I and II) on opposite faces of the active cleft. Binding of substrates, cofactors, and inhibitors to either exosite is important for overcoming restricted access to the active site. The sodium-binding site defined by a cylindrical channel shaped by three β-strands appears to be important for the procoagulant (fast) form of thrombin (18Di Cera E. Guinto E.R. Vindigni A. Dang Q.D. Ayala Y.M. Wuyi M. Tulinsky A. J. Biol. Chem. 1995; 270: 22089-22092Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). The aim of this study was to determine how thrombin specifically recognizes factor XIII in preference to its many other substrates and how activation can be enhanced by fibrin. For this purpose, we have utilized a library of 53 thrombin mutants encompassing a total of 78 surface exposed, charged, and polar residues mutated to alanine. This library has previously been used to identify residues of thrombin involved in the interaction with many of its substrates including protein C, thrombin-activatable fibrinolysis inhibitor, fibrinogen (19Hall S.W. Nagashima M. Zhao L. Morser J. Leung L.L. J. Biol. Chem. 1999; 274: 25510-25516Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), antithrombin (20Tsiang M. Jain A.K. Gibbs C.S. J. Biol. Chem. 1997; 272: 12024-12029Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), factor V (21Myles T. Yun T.H. Hall S.W. Leung L.L. J. Biol. Chem. 2001; 276: 25143-25149Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), and factor VIII (22Myles T. Yun T.H. Leung L.L. Blood. 2002; 100: 2820-2826Crossref PubMed Scopus (60) Google Scholar). Here, we have used the library to investigate which residues of thrombin are involved in direct factor XIII activation and in the fibrin-enhanced reaction. Knowledge of these residues has enabled us to draw general conclusions about how the activities of thrombin are directed. A library of 53 thrombin mutants encompassing a total of 78 surface exposed charged and polar residues mutated to alanine was prepared and checked for amidolytic activity as described previously (Table I) (20Tsiang M. Jain A.K. Gibbs C.S. J. Biol. Chem. 1997; 272: 12024-12029Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 23Tsiang M. Jain A.K. Dunn K.E. Rojas M.E. Leung L.L. Gibbs C.S. J. Biol. Chem. 1995; 270: 16854-16863Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). These mutant thrombins were screened for their ability to activate factor XIII using a modification of a 5-(biotinamido)pentylamine incorporation assay (24Song Y.C. Sheng D. Taubenfeld S.M. Matsueda G.R. Anal. Biochem. 1994; 223: 88-92Crossref PubMed Scopus (37) Google Scholar). Factor XIII was purified from plasma donor pools using previously described methods (11Ariens R.A.S. Philippou H. Nagaswami C. Weisel J.W. Lane D.A. Grant P.J. Blood. 2000; 96: 988-995Crossref PubMed Google Scholar). To screen for factor XIII activation in the absence of fibrin(ogen), we first coated microtiter plates with 10 μg/ml N,N-dimethyl casein (Sigma) for 16 h. Factor XIII (6.7 nm) and 36 nm human thrombin (American Diagnostica, Greenwich, CT) were added and incubated in activation buffer (40 mm Tris-HCl, pH 8.3, 140 mm NaCl, 0.1 mm dithiothreitol, 5 mm CaCl2, 0.02% (w/v) NaN3) containing 0.1 mm 5-(biotinamido)pentylamine (Perbio Science UK Limited, Cheshire, United Kingdom) for 60 min at room temperature. The reaction was stopped by the addition of 200 mm EDTA, pH 8.3. The amount of factor XIII that was activated by thrombin was determined by measuring the amount of 5-(biotinamido)pentylamine cross-linked to the N,N-dimethylcasein using an alkaline phosphatase streptavidin conjugate (Sigma) followed by incubation with p-nitrophenol substrate (Sigma) and detection of the signal at 405 nm using a microtiter plate reader (Labsystems, Hampshire, United Kingdom).Table IEffect of thrombin mutants on the direct activation of factor XIIIMutationaResidues numbered consecutively from the start of the thrombin B-chain, 1-259. Residues in the A-chain are indicated by a lowercase “a,” 1a-36a.% ActivityMutationaResidues numbered consecutively from the start of the thrombin B-chain, 1-259. Residues in the A-chain are indicated by a lowercase “a,” 1a-36a.% ActivityMean ± S.D.Mean ± S.D.Wild-type100 ± 0R73A140 ± 1.7S4aA/E6aA/D8aA77 ± 3.4N74A84 ± 3.3K17aA/K18aA/S19aA74 ± 2.5K77A117 ± 7.8K23aA82 ± 2.1E82A/K83A99 ± 0.9R26aA89 ± 1.6R89A/R93A/E94A78 ± 7.5E27aA80 ± 1.1R98A88 ± 7.5E30aA/D34aA90 ± 6.3K106A130 ± 6.6E3A/D6A102 ± 13K107A131 ± 6.2R20A108 ± 16K106A/K107A133 ± 7.1K21A90 ± 10.4D113A146 ± 3.5S22A88 ± 5.8Y114A146 ± 13.8Q24A78 ± 13D122A/R123A/E124A91 ± 26.4E25A71 ± 1S128A/R123A/Q131A96 ± 12.2S22A/Q24A/E25A89 ± 2.6K145A/T147A/W148A120 ± 4.4D35A86 ± 0.4T149A/N151A96 ± 3R36A90 ± 2.8K154A92 ± 4.3W50A53 ± 6.2S158A98 ± 3.2D51A89 ± 3.3E169A/K174A/D175A94 ± 0.1K52A90 ± 4.1R178A/R180A/D183A41 ± 0.5*N57A/D58A70 ± 6.4D193A/K196A93 ± 5.9R62A105 ± 6.7N216A/N217A65 ± 2K65A82 ± 4.7E229A6.9 ± 2.5*H66A80 ± 18R233A39 ± 3.6*R68A109 ± 5.1D234A132 ± 13T69A112 ± 1.3R245A/K248A/Q251A107 ± 2R70E127 ± 0R245A98 ± 6.8Y71A117 ± 15K248A106 ± 2.2a Residues numbered consecutively from the start of the thrombin B-chain, 1-259. Residues in the A-chain are indicated by a lowercase “a,” 1a-36a. Open table in a new tab To assess the activity of thrombin mutants for either fibrinogen-enhanced or fibrin-enhanced activation of factor XIII, microtiter plates were coated with either (i) 0.12 nm fibrinogen (Calbiochem and Merck Biosciences Ltd, Nottingham, United Kingdom) for 1 h or (ii) 0.12 nm fibrinogen for 1 h, which was preincubated with 46 nm thrombin for 20 min in the presence of 5 mm CaCl2 at 37 °C to form fibrin. Residual thrombin was removed by thorough washing using 40 mm Tris-HCl, 750 mm NaCl, pH 8.3, followed by a final wash with 40 mm Tris-HCl, 140 mm NaCl, pH 8.3. For both fibrinogen-enhanced and fibrin-enhanced factor XIII activation, 6.7 nm factor XIII and 18 nm thrombin were incubated in activation buffer containing 0.1 mm 5-(biotinamido)pentylamine for 8 min at room temperature. The reaction was stopped by the addition of 200 mm EDTA, pH 8.3. The amount of factor XIII that was activated by thrombin was determined by measuring the amount of 5-(biotinamido)pentylamine cross-linked to the fibrin(ogen) using alkaline phosphatase streptavidin conjugate followed by incubation with p-nitrophenol substrate (and measuring at 405 nm). When activation of factor XIII by thrombin was performed in the presence of thrombomodulin, increasing molar concentrations of rabbit thrombomodulin (Hematologic Technologies Inc.) were preincubated with 1.8 nm human thrombin prior to the addition of 6.7 nm factor XIII (in the absence and presence of fibrin, see above). Factor XIII activation peptide cleavage was determined by reverse-phase HPLC using a Pepmap 0.46 × 250 mm C18 column (Applied Biosystems, Warrington, United Kingdom) and the AKTA basic chromatography system (Amersham Biosciences). Application and elution buffers for HPLC were phosphate- and acetonitrile-based as previously described (11Ariens R.A.S. Philippou H. Nagaswami C. Weisel J.W. Lane D.A. Grant P.J. Blood. 2000; 96: 988-995Crossref PubMed Google Scholar) with elution using an 8.5–40% acetonitrile gradient. Purified factor XIII was dialyzed against reaction buffer (9.47 mm sodium phosphate, 137 mm NaCl, 2.5 mm KCl, 0.1% polyethylene glycol, pH 7.4) prior to use. In the absence of fibrinogen, 1.8 μm factor XIII was incubated at 37 °C with 4.1 nm of the thrombin mutant under investigation. 100-μl samples were then removed at five intervals between 1 and 220 min (dependent on the expected activity of the mutant in question). The reactions were stopped by the addition of 10 μl of 3 m perchloric acid, the precipitate was removed by centrifugation, and 100 μl of sample was loaded onto the C18 column. To investigate factor XIII activation peptide release in the presence of fibrinogen, 1.8 μm factor XIII and 3.1 μm fibrinogen were incubated at 37 °C with 1.83 nm of the thrombin mutant in question. The reaction mixture was immediately aliquoted into 100-μl volumes to avoid subsampling difficulties following fibrin formation. Reactions were stopped at five intervals between 1 and 160 min by the addition of 10 μl of 3 m perchloric acid. The precipitate was removed by centrifugation, and 100 μl of the reaction was loaded onto the C18 column. The relative amounts of activation peptides released were determined by measurement of the areas under the respective peaks using Unicorn Analysis software (Amersham Biosciences). Catalytic efficiencies were calculated by fitting of the data from time course experiments to Equation 1 (9Janus T.J. Lewis S.D. Lorand L. Shafer J.A. Biochemistry. 1983; 22: 6269-6272Crossref PubMed Scopus (83) Google Scholar), kcat/Km=-ln(1-[AP]/[APf])[E]×t(Eq. 1) where [AP] is the concentration of activation peptide at a given time, [APf] is the concentration at full activation, [E] is the total concentration of thrombin, and t is time. Data fitting was performed using Enzfitter software (Biosoft, Cambridge, United Kingdom). This equation was used to fit all of the activation peptide release curves, although it is recognized that this is an approximation in the cases of fibrinogen-enhanced factor XIII activation peptide release and for FPB release. Purified fibrinogen fragment D was also studied as an enhancer of factor XIII activation. The fragment D preparation (Calbiochem) was first characterized with respect to its ability to bind factor XIII as it is sensitive to proteolytic cleavage at its C terminus where a factor XIII binding site is located. Binding analysis of factor XIII to both fragment D and fibrinogen was carried out with surface plasmon resonance using a BIAcore 3000 system (BIAcore UK, Stevenage, United Kingdom). Fragment D at 75 μg/ml and fibrinogen at 50 μg/ml were covalently coupled to an activated carboxymethyl dextran-coated biosensor chip (CM5) using the manufacturer's recommended protocol for kinetic analysis. The conditions employed were similar to those employed elsewhere (25Tsurupa G. Tsonev L. Medved L. Biochemistry. 2002; 41: 6449-6459Crossref PubMed Scopus (61) Google Scholar) for the study of tissue-type plasminogen activator and plasminogen binding to the αC-domain of fibrinogen. However, the running buffer contained 5 mm EDTA rather than 0.1 mm phenylmethylsulfonyl fluoride, and the chip was regenerated using buffer containing 750 mm NaCl. The association and dissociation data were recorded after subtraction of signal in the reference cell, which was activated and blocked in the absence of ligand. Each data set was generated using factor XIII in a range of concentrations between 7.8 and 125 nm and was fitted to the 1:1 (Langmuir) binding model using the BIAevaluation software supplied with the equipment. Fibrinogen and fragment D were found to be able to bind factor XIII with K Ds of 210 ± 69 nm (n = 6) and 198 ± 54 nm (n = 6), respectively. In the reactions to study enhancement of factor XIII activation, a 2-fold molar excess of the isolated fragment D was used (as fibrinogen contains two D-domains). Specifically, 1.8 μm factor XIII and either 2.25 μm fibrinogen or 4.5 μm fragment D were incubated at 37 °C with 1.83 nm wild-type (WT) thrombin. The reaction mixture was immediately aliquoted into 100-μl volumes. The reactions were then stopped at five intervals (between 1 and 16 min for fibrinogen and between 1 and 80 min for fragment D) by the addition of 10 μl of 3 m perchloric acid followed by HPLC, as explained above. The 53-variant thrombins were screened for their abilities to activate factor XIII in the absence of fibrinogen using the casein-coated microtiter plate assay with incorporation of pentylamine used as the reporter for factor XIIIa activity. Only three thrombin mutants showed <50% WT activity (see results marked by asterisk in Table I). These were a triple mutant of residues Arg-178/Arg-180/Asp-183 and mutants of Glu-229 and Arg-233 that had 41, 6.9, and 39% activity, respectively. Mutant W50A showed 53% activity (see Table I). These three variants, E229A, R233A, and R178A/R180A/D183A, together with W50A were assessed further using increasing amounts of thrombin to confirm the role of these residues in the activation of factor XIII (data not shown). Each mutant required 4–8-fold increases in concentration to achieve the activity attained by WT thrombin. The above results in Table I, obtained with the microtiter plate-screening assay, employed an arbitrary cutoff of 50% WT activity to select the thrombin mutants with reduced ability to activate factor XIII. To obtain a more reliable and quantitative assessment of activity reduction, the kinetic parameter k cat/K m was determined for WT and mutant thrombins. This was achieved by determination of factor XIII activation peptide release using HPLC analysis. As factor XIII was isolated from normal pooled plasma, its activation peptide was heterogeneous, containing the polymorphic forms Val-34 and Leu-34 (see Fig. 1A). Rather than sum the relative amounts of each form, their individual relative release rates were determined for each thrombin preparation (see time courses of release in Figs. 1, C and D). The results of catalytic efficiency determinations are illustrated in Fig. 2. The k cat/K m values obtained for the activation of Val-34 and Leu-34 forms of the factor XIII using WT thrombin (0.18 ± 0.008 and 0.33 ± 0.049 μm–1 s–1, respectively) conform to prior reports using plasma with human thrombin as an activator (11Ariens R.A.S. Philippou H. Nagaswami C. Weisel J.W. Lane D.A. Grant P.J. Blood. 2000; 96: 988-995Crossref PubMed Google Scholar). Each thrombin mutant with <50% WT activity selected from Table I for kinetic analysis had greatly reduced k cat/K m with the R233A mutant being particularly affected (k cat/K m of 0.011 ± 0.0008 and 0.018 ± 0.001 μm–1 s–1 for cleavage of the Val-34 and Leu-34 forms of activation peptide, respectively) (Fig. 2). The position of the mutated residues on the surface of thrombin (see below) together with their limited number suggests that activation of factor XIII in the absence of cofactor is due primarily to direct (and inefficient) interactions of factor XIII within the long extended active site cleft between the S1 specificity pocket and exosite II.Fig. 2k cat /K m determinations for cleavage of the factor XIII activation peptide by WT thrombin and mutants. The mutants identified in Table I as having reduced ability to activate factor XIII (in the absence of fibrinogen) were investigated in solution with factor XIII and kinetic parameter k cat/K m determined from HPLC analysis of the activation peptides. The results for the two polymorphic forms (Val-34 and Leu-34) are depicted in separate panels.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Preliminary screening experiments for fibrinogen-enhanced factor XIII activation used microtiter plates coated with fibrinogen and utilizing pentylamine incorporation into the resultant fibrin by activated factor XIII as a reporter. The rate of factor XIII activation was greatly enhanced by the presence of fibrinogen, requiring only 8 min and 50% less thrombin to achieve the equivalent amount of factor XIII activity generated in1hin the absence of fibrinogen. More thrombin residues were found to influence fibrinogen-enhanced factor XIII activation when compared with direct factor XIII activation alone (Table II). Thrombin variants with 50% or less activities compared with WT were H66A, Y71A, N74A (which map to exosite I of thrombin), and R89A/R93A/E94A (which map to exosite II) in addition to the previously characterized mutants W50A, R178A/R180A/D183A, E229A, and R233A. Dose-response experiments for these mutant thrombins showed that 4–8-fold increased amounts of the mutants were required to attain WT thrombin activity, confirming the validity of the screening assay (results not shown).Table IIThrombin mutants with reduced ability to activate factor XIII in the presence of fibrinogen and preformed fibrinMutation% ActivityFibrinogen-enhanced factor XIII activationFibrin-enhanced factor XIII activationMean ± S.D.Mean ± S.D.Wild-type100 ± 4.0100 ± 4.0W50A16 ± 1.321 ± 3.7H66A20 ± 4.119 ± 2.4Y71A34 ± 1.536 ± 5.3N74A44 ± 1.449 ± 8.8R89A/R93A/E94A36 ± 8.568 ± 9.3R178A/R180A/D183A23 ± 3.026 ± 5.0E229A7.4 ± 1.329 ± 0.9R233A27 ± 1.922 ± 5.1 Open table in a new tab All of the mutants that resulted in reduced fibrinogen-enhanced activation of factor XIII were studied further in experiments in which factor XIII activation was carried out on the surface of preformed fibrin. Fibrinogen coated onto microtiter plates was preincubated with WT thrombin to form fibrin. Thrombin was subsequently removed by washing with 40 mm Tris-HCl, 750 mm NaCl, pH 8.3. Complete removal of thrombin was confirmed by the absence of activation of factor XIII in the blank sample. Fibrin-enhanced factor XIII activation by the mutants was then assessed. The results (Table II) show that mutants with impaired direct activation of factor XIII (Trp-50, Arg-178/Arg-180/Asp-183, Glu-229, and Arg-233) have reduced activity in this assay as might be expected because of their essential role in factor XIII activation. Furthermore, mutation of residues His-66, Tyr-71, and Asn-74 as well as reducing fibrinogen-enhanced factor XIII activation directly reduces fibrin-enhanced factor XIII activation (19 ± 2.4, 36 ± 5.3, and 49 ± 8.8% (mean ± S.D.), respectively of WT thrombin activity) (Table II). The mutation of residues Arg-89, Arg-93, and Glu-94 affects fibrinogen-enhanced activation of factor XIII (36 ± 8.5% WT thrombin activity) more than fibrin-enhanced factor XIII activation (68 ± 9.3% of WT activity). This probably reflects their important role in fibrinopeptide cleavage. Thrombin mutants H66A, Y71A, and N74A therefore influenced both fibrinogen- and fibrin-enhanced factor XIII activation in the microtiter plate assays and were subsequently assessed further by HPLC analysis of the activation peptide release in the presence of fibrinogen. The activation experiments generated activation peptides from both factor XIII (Val-34 and Leu-34 forms of the activation peptide) and fibrinogen (FPA and FPB). These peptides were completely resolved on HPLC (Fig. 1B) and estimates for the catalytic efficiencies for each of these cleavage reactions were obtained (Table III). The presence of fibrinogen enhanced the release of the Val-34 and Leu-34 factor XIII activation peptides by WT thrombin to k cat/K m of 2.29 ± 0.21 and 2.84 ± 0.38 μm–1 s–1, respectively. Each of the three mutants, H66A, Y71A, and N74A, had reduced enhancement effects, and Y71A was particularly reduced with k cat/K m values of 0.038 ± 0.002 and 0.11 ± 0.016 μm–1 s–1 for cleavage of the Val-34 and Leu-34 activation peptides, respectively. These data confirm reduced fibrin-enhanced factor XIII activation of the three mutants. However, it is evident from Table III that the mutants also had impaired ability to convert fibrinogen to fibrin. The k cat/K m for WT thrombin cleavage of FPA is 4.54 ± 0.58 μm–1 s–1 and is lower for all of the mutants falling to 0.023 ± 0.0004 μm–1 s–1 for Y71A. There was a parallel reduction in the catalytic efficiencies associated with FPB release (Table III). These data show that residues His-66, Tyr-71, and Asn-74 have a role in FPA and FPB cleavage as well as fibrin-enhanced FXIII activation.Table IIIKinetic evaluation (kcat/Km) of activation peptide cleavage during the fibrinogen-enhanced activation of factor XIII by WT and mutant thrombinsMutationk cat/KmVal-34Leu-34FPAFPBμM-1 s-1Wild-type2.29 ± 0.212.84 ± 0.384.54 ± 0.581.38 ± 0.007N74A0.94 ± 0.301.49 ± 0.831.78 ± 0.610.62 ± 0.25H66A0.33 ± 0.050.58 ± 0.160.53 ± 0.100.15 ± 0.002Y71A0.038 ± 0.0020.11 ± 0.0160.023 ± 0.0040.011 ± 0.003 Open table in a new t
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