Enhanced Thrombin Sensitivity of a Factor VIII-Heparin Cofactor II Hybrid
1996; Elsevier BV; Volume: 271; Issue: 35 Linguagem: Inglês
10.1074/jbc.271.35.20985
ISSN1083-351X
AutoresJan Voorberg, Gunny van Stempvoort, Johanna M. Klaasse Bos, Koen Mertens, Jan A. van Mourik, Marie-José S.H. Donath,
Tópico(s)Blood properties and coagulation
ResumoGeneration of thrombin at a site of vascular injury is a key event in the arrest of bleeding. In addition to the conversion of fibrinogen into the insoluble fibrin, thrombin can initiate a number of positive and negative feedback mechanisms that either sustain or down-regulate clot formation. We have modulated the thrombin sensitivity of human blood coagulation factor VIII, an essential cofactor in the intrinsic pathway of blood coagulation. We have substituted an acidic region of factor VIII corresponding to amino acid sequence Asp712-Ala736 by amino acid sequence Ile51-Leu80 of the thrombin inhibitor heparin cofactor II. Functional analysis of the resulting factor VIII-heparin cofactor II hybrid, termed des-(868-1562)-factor VIII-HCII, revealed an increase in procoagulant activity as measured in a one-stage clotting assay. Incubation of purified des-(868-1562)-factor VIII-HCII with different amounts of thrombin showed that this protein was more readily activated by thrombin when compared with des-(868-1562)-factor VIII, a control protein lacking amino acid sequence Ile51-Leu80 of heparin cofactor II. This was manifested by an increase in the second order rate constant of activation by thrombin for des-(868-1562)-factor VIII-HCII (12.0 ± 0.48 × 106−1 s−1) compared with des-(868-1562)-factor VIII (1.77 ± 0.21 × 106−1 s−1). Our data suggest that amino acid sequence Ile51-Leu80 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin which results in accelerated clot formation. Generation of thrombin at a site of vascular injury is a key event in the arrest of bleeding. In addition to the conversion of fibrinogen into the insoluble fibrin, thrombin can initiate a number of positive and negative feedback mechanisms that either sustain or down-regulate clot formation. We have modulated the thrombin sensitivity of human blood coagulation factor VIII, an essential cofactor in the intrinsic pathway of blood coagulation. We have substituted an acidic region of factor VIII corresponding to amino acid sequence Asp712-Ala736 by amino acid sequence Ile51-Leu80 of the thrombin inhibitor heparin cofactor II. Functional analysis of the resulting factor VIII-heparin cofactor II hybrid, termed des-(868-1562)-factor VIII-HCII, revealed an increase in procoagulant activity as measured in a one-stage clotting assay. Incubation of purified des-(868-1562)-factor VIII-HCII with different amounts of thrombin showed that this protein was more readily activated by thrombin when compared with des-(868-1562)-factor VIII, a control protein lacking amino acid sequence Ile51-Leu80 of heparin cofactor II. This was manifested by an increase in the second order rate constant of activation by thrombin for des-(868-1562)-factor VIII-HCII (12.0 ± 0.48 × 106−1 s−1) compared with des-(868-1562)-factor VIII (1.77 ± 0.21 × 106−1 s−1). Our data suggest that amino acid sequence Ile51-Leu80 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin which results in accelerated clot formation. INTRODUCTIONSelective interaction of thrombin with a number of blood coagulation factors is essential to ascertain control over both pro- and anticoagulant pathways (1Huber R. Carell R.W. Biochemistry. 1989; 28: 8951-8966Google Scholar, 2Kane W.H. Davie E.W. Blood. 1988; 71: 539-555Google Scholar, 3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10364-10370Google Scholar, 4Gailani D. Broze Jr., G.J. Science. 1991; 253: 909-912Google Scholar, 5Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1992; 89: 351-353Google Scholar, 6Esmon C.T. Thromb. Haemostasis. 1993; 70: 29-34Google Scholar, 7Stubbs M.T. Bode W. Thromb. Res. 1993; 69: 1-58Google Scholar). Determination of the three-dimensional structure of thrombin has greatly contributed to our current knowledge on the action of this serine protease (8Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 11: 3467-3475Google Scholar, 9Skrzypczak E. Rydel T. Tulinsky A. Fenton J.W. Mann K.G. J. Mol. Biol. 1989; 206: 755-757Google Scholar). Studies on the three-dimensional structure of a complex of thrombin and hirudin, an inhibitor derived from the leech Hirudo medicinalis, revealed that a positively charged area, the so-called anion binding exosite I of thrombin, interacts strongly with a stretch of negatively charged amino acids present at the carboxyl terminus of hirudin (10Grütter M.G. Priestle J.P. Rahuel J. Grossenbacher H. Bode W. Hofsteenge J. Stone S.R. EMBO J. 1990; 9: 2361-2365Google Scholar, 11Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton II, J.W. Science. 1990; 249: 277-280Google Scholar). Similar areas of negatively charged amino acids are present in the thrombin receptor, thrombomodulin and heparin cofactor II and have been shown to interact with anion binding exosite I of thrombin (12Liu L.W. Vu T.K.H. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Google Scholar, 13Vu T-K. Wheaton V.I. Hung D.T. Charo I. Coughlin S.R. Nature. 1991; 353: 674-677Google Scholar, 14Mathews I.I. Padmanabhan K.P. Ganesh V. Tulinsky A. Ishii M. Chen J. Turck C.W. Coughlin S.R. Fenton II, J.W. Biochemistry. 1994; 33: 3266-3279Google Scholar, 15Tsiang M. Lentz S.R. Dittman W.A. Wen D. Scarpati E.M. Sadler J.E. Biochemistry. 1990; 29: 10602-10612Google Scholar). Studies using synthetic peptides corresponding to the negatively charged amino acids of the proteins mentioned above have shown that the affinity of these peptides for thrombin varies considerably (15Tsiang M. Lentz S.R. Dittman W.A. Wen D. Scarpati E.M. Sadler J.E. Biochemistry. 1990; 29: 10602-10612Google Scholar, 16Hortin G.L. Benutto B.M. Biochem. Biophys. Res. Commun. 1990; 169: 437-442Google Scholar). These observations suggest that the ability of thrombin to interact with different components of the hemostatic system is, at least in part, determined by the affinity of anion binding exosite I of thrombin for stretches of negatively charged amino acids present on its substrates. Modulation of the pro- and anticoagulant activities of thrombin may be accomplished by simply exchanging negatively charged areas between the different substrates of this serine protease.Factor VIII is an essential cofactor for factor IXa in the conversion of factor X to Xa in the intrinsic pathway of blood coagulation (3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10364-10370Google Scholar). Molecular cloning of the factor VIII cDNA revealed that factor VIII consists of a series of homologous domains which can be represented as follows: A1-A2-B-A3-C1-C2 (17Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. Amphlett G.W. Foster B. Coe M.L. Knutson G.J. Fass D.N. Hewick R.M. Nature. 1984; 312: 342-347Google Scholar, 18Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblatt F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R. Capon D.J. Nature. 1984; 312: 337-342Google Scholar). Proteolytic processing of factor VIII at amino acid position Arg1648 occurs during biosynthesis of factor VIII (19Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Google Scholar). Consequently, factor VIII circulates in plasma as a metal ion-linked heterodimer consisting of a heavy chain (A1-A2-B) and light chain (A3-C1-C2). Activation of factor VIII by thrombin proceeds through limited proteolysis at amino acid positions Arg372, Arg740, and Arg1689 (19Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Google Scholar, 20Pitmann D.D. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2429-2433Google Scholar). Consequently, activated factor VIII consists of a heterotrimer of the separate A1 and A2-domains together with a thrombin cleaved light chain (21Lollar P. Parker C.G. Biochemistry. 1989; 28: 666-674Google Scholar, 22Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Google Scholar). We have shown previously that an acidic region corresponding to amino acid sequence Asp713-Arg740 of factor VIII is involved in the activation of factor VIII by thrombin (23Mertens K. Donath M.J.S.H. Van Leen R.W. De Keyzer-Nellen M.J.M. Verbeet M. Ph Klaasse Bos J.M. Leyte A. Van Mourik J.A. Br. J. Haematol. 1993; 85: 133-145Google Scholar, 24Donath, M. J. S. H., 1995, Activation and Limited Proteolysis of Human Blood Coagulation Factor VIII. Ph.D. thesis, University of Amsterdam.Google Scholar). Here, we have replaced amino acid sequence Asp712-Ala736 of factor VIII by amino acid sequence Ile51-Leu80 of heparin cofactor II, a potent inhibitor of thrombin (25Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Google Scholar). Functional importance of amino acid sequence Ile50-Leu80 of heparin cofactor II in the inhibition of thrombin has been suggested by analysis of heparin cofactor II derivatives that lack this particular amino acid sequence (26Van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Google Scholar). The factor VIII-heparin cofactor II hybrid constructed, designated des-(868-1562)-factor VIII-HCII, was expressed in mouse fibroblast C127 cells, and analysis of the properties of the resulting protein suggests that amino acid sequence Ile50-Ser81 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin.RESULTS AND DISCUSSIONWe have shown previously that efficient activation of blood coagulation factor VIII by thrombin is dependent on the presence of amino acid sequence Asp713-Arg740 of the factor VIII heavy chain (23Mertens K. Donath M.J.S.H. Van Leen R.W. De Keyzer-Nellen M.J.M. Verbeet M. Ph Klaasse Bos J.M. Leyte A. Van Mourik J.A. Br. J. Haematol. 1993; 85: 133-145Google Scholar). Here, we have replaced amino acid sequence Asp713-Ala736 of des-(868-1562)-factor VIII by amino acid sequence Ile51-Leu80 of heparin cofactor II (Fig. 1A). The modified factor VIII cDNA was expressed in C127 cells, and the properties of the resulting hybrid protein were compared with des-(868-1562)-factor VIII, a B-domain deleted factor VIII which has been previously characterized (23Mertens K. Donath M.J.S.H. Van Leen R.W. De Keyzer-Nellen M.J.M. Verbeet M. Ph Klaasse Bos J.M. Leyte A. Van Mourik J.A. Br. J. Haematol. 1993; 85: 133-145Google Scholar) (Fig. 1A). Characterization of the conditioned medium of transfected cells expressing des-(868-1562)-factor VIII-HCII revealed that the factor VIII-heparin cofactor hybrid had normal cofactor activity (data not shown). Also the amount of factor VIII antigen in the conditioned medium was similar for both des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII (data not shown). Inspection of the procoagulant activity as measured in a one-stage clotting assay showed that limited amounts of des-(868-1562)-factor VIII-HCII sufficed to effectively reduce the clotting time of plasma deficient of factor VIII (Fig. 1B). This observation indicates that amino acid sequence Ile51-Leu80 of heparin cofactor II endows factor VIII with improved functional properties.To determine the molecular basis of the enhanced procoagulant activity of des-(868-1562)-factor VIII-HCII, we have immunopurified and characterized this hybrid protein. First, the subunit composition of the purified material was analyzed by SDS-PAGE followed by immunoblotting (Fig. 2). Monoclonal antibody CLB-CAg 69 directed against amino acid sequence Lys1673-Arg1689 of the factor VIII light chain (30Leyte A. Mertens K. Distel B. Evers R.F. De Keyzer-Nellen M.J.M. Groenen-Van Dooren M.M.C.L. De Bruin J. Pannekoek H. Van Mourik J.A. Verbeet M. Ph Biochem. J. 1989; 263: 187-194Google Scholar), reacted with the 200- and 80-kDa species of both des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII while monoclonal antibody MAS530 directed against the heavy chain of factor VIII reacted with the 200- and 120-kDa species of these proteins (Fig. 2). These data indicate that in addition to a small amount of single chain protein both des-(868-1562)-factor VIII and des-(868-1562)-factor VIII-HCII consist predominantly of a heterodimer composed of a light chain of 80 kDa and a heavy chain of 120 kDa, which contains a residual portion of the B-domain. Monoclonal antibody CLB-CAg 9 did not react with des-(868-1562)-factor VIII-HCII, since amino acid sequence Asp712-Ala736, which constitutes the epitope of this monoclonal antibody (30Leyte A. Mertens K. Distel B. Evers R.F. De Keyzer-Nellen M.J.M. Groenen-Van Dooren M.M.C.L. De Bruin J. Pannekoek H. Van Mourik J.A. Verbeet M. Ph Biochem. J. 1989; 263: 187-194Google Scholar), has been replaced by amino acid sequence Ile51-Leu80 of heparin cofactor II (Fig. 2).Fig. 2Subunit composition of des-(868-1562) factor VIII-HCII and des-(868-1562)-factor VIII. Purified proteins were analyzed by SDS-PAGE (7.5%) and following immunoblotting proteins were visualized with anti-factor VIII antibodies. Lanes 1 and 4, CLB-CAg 69, directed against amino acid sequence Lys1673-Arg1689 of the light chain of factor VIII; lanes 2 and 5, MAS 530 (Seralab, Sussex, United Kingdom) directed against the heavy chain of factor VIII; lanes 3 and 6, CLB-CAg 9, directed against amino acid sequence Ser710-Arg740 of factor VIII. A molecular weight marker is shown at the right of the figure. The single chain (sc), heavy chain (hc), and light chain (lc) of factor VIII are indicated at the left of the figure.View Large Image Figure ViewerDownload (PPT)Previous studies from our laboratory have shown that factor VIII-del(713-1637) lacks procoagulant activity in a one-stage clotting assay and is fully activated only in the presence of relatively high concentrations of thrombin (23Mertens K. Donath M.J.S.H. Van Leen R.W. De Keyzer-Nellen M.J.M. Verbeet M. Ph Klaasse Bos J.M. Leyte A. Van Mourik J.A. Br. J. Haematol. 1993; 85: 133-145Google Scholar). The increased procoagulant activity of des-(868-1562)-factor VIII-HCII in a one-stage clotting assay suggests that this molecule is more readily activated by thrombin than des-(868-1562)-factor VIII. To study this issue in more detail the sensitivity of both des-(868-1652)-factor VIII-HCII and des-(868-1562)-factor VIII toward thrombin was determined. Activation of des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII by thrombin was monitored by probing the ability of the activated factor VIII to function as a cofactor in the factor IXa-dependent conversion of factor X into Xa (27Donath M.J.S.H. De Laaf R.T.M. Biessels P.T.M. Lenting P.J. Van de Loo J-W. Van Mourik J.A. Voorberg J. Mertens K. Biochem. J. 1995; 312: 49-55Google Scholar). Addition of various amounts of thrombin allows for determination of the thrombin sensitivity of both des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII (Fig. 3). Inspection of the pattern obtained when 0.1 n thrombin was used for activation of both proteins revealed that des-(868-1562)-factor VIII-HCII is more readily activated then des-(868-1562)-factor VIII at this particular concentration of thrombin (Fig. 3). A similar pattern is observed when 0.5 and 1.0 n thrombin were used to activate the two different proteins (Fig. 3). These results provide evidence for an increased thrombin sensitivity of des-(868-1562)-factor VIII-HCII compared with des-(868-1562)-factor VIII.Fig. 3Activation of factor VIII by thrombin. Purified des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII were incubated with various concentrations of thrombin and the activation of factor VIII was monitored by probing the ability of factor VIII as a cofactor for the factor IXa-dependent conversion of factor X to Xa. Under our experimental conditions the rate of factor Xa generation was found to be dependent on the amount of thrombin added to the reaction mixture. Representative examples of factor Xa generation curves for three different concentrations of thrombin are given (0.1, 0.5, and 1.0 n thrombin) for both des-(868-1562)-factor VIII-HCII (•) and des-(868-1562)-factor VIII (∘). The solid lines represent fits to Equation 1, which is given under “Experimental Procedures.”View Large Image Figure ViewerDownload (PPT)In order to define the increased thrombin sensitivity in a quantitative manner, we have determined the second order rate constant of activation by thrombin for both des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII using a previously established method (24Donath, M. J. S. H., 1995, Activation and Limited Proteolysis of Human Blood Coagulation Factor VIII. Ph.D. thesis, University of Amsterdam.Google Scholar). Factor Xa generation curves obtained at different concentrations of thrombin were used to calculate the first order rate constant of activation for both des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII. Secondary plots of the first order rate constants against the concentration of thrombin yielded a second order rate constant of activation for des-(868-1562)-factor VIII-HCII (12.0 ± 0.48 × 106−1 s−1) and des-(868-1562)-factor VIII (1.77 ± 0.21 × 106−1 s−1) (Fig. 4). The data obtained show that des-(868-1562)-factor VIII-HCII is approximately 7-fold more readily activated by thrombin than des-(868-1562)-factor VIII. Our results suggest that des-(868-1562)-factor VIII-HCII may more efficiently compete for the limited amounts of amounts of thrombin generated at a site of vascular injury than des-(868-1562)-factor VIII. This property may render des-(868-1562)-factor VIII more effective in treatment of patients with hemophilia A than currently available factor VIII preparations. Our study implies that exchange of acidic regions of other substrates of thrombin may be utilized to selectively modulate the action of this serine protease under physiological and pathophysiological conditions.Fig. 4Second order rate constant of activation of des-(868-1562)-factor VIII-HCII by thrombin. The first order rate constant (k) of activation of des-(868-1562)-factor VIII-HCII and des-(868-1562)-factor VIII was determined at different concentrations of thrombin. For des-(868-1562)-factor VIII, k was determined in triplicate at 0.1, 0.5, 1.0, and 2.5 n of thrombin. For des-(868-1562)-factor VIII-HCII thrombin concentrations of 0.1, 0.2, 0.5, and 1.0 n were analyzed in triplicate. From the slope of a plot of k against the concentration of thrombin, the apparent second order rate constant of activation by thrombin for both des-(868-1562)-factor VIII-HCII (•) and des-(868-1562)-factor VIII (∘) was calculated. Residual activation of factor VIII in the absence of thrombin is most likely explained by limited amounts of non-acetylated factor X in the acetylated factor X preparation, which may cause feedback activation of factor VIII.View Large Image Figure ViewerDownload (PPT) INTRODUCTIONSelective interaction of thrombin with a number of blood coagulation factors is essential to ascertain control over both pro- and anticoagulant pathways (1Huber R. Carell R.W. Biochemistry. 1989; 28: 8951-8966Google Scholar, 2Kane W.H. Davie E.W. Blood. 1988; 71: 539-555Google Scholar, 3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10364-10370Google Scholar, 4Gailani D. Broze Jr., G.J. Science. 1991; 253: 909-912Google Scholar, 5Coughlin S.R. Vu T.K.H. Hung D.T. Wheaton V.I. J. Clin. Invest. 1992; 89: 351-353Google Scholar, 6Esmon C.T. Thromb. Haemostasis. 1993; 70: 29-34Google Scholar, 7Stubbs M.T. Bode W. Thromb. Res. 1993; 69: 1-58Google Scholar). Determination of the three-dimensional structure of thrombin has greatly contributed to our current knowledge on the action of this serine protease (8Bode W. Mayr I. Baumann U. Huber R. Stone S.R. Hofsteenge J. EMBO J. 1989; 11: 3467-3475Google Scholar, 9Skrzypczak E. Rydel T. Tulinsky A. Fenton J.W. Mann K.G. J. Mol. Biol. 1989; 206: 755-757Google Scholar). Studies on the three-dimensional structure of a complex of thrombin and hirudin, an inhibitor derived from the leech Hirudo medicinalis, revealed that a positively charged area, the so-called anion binding exosite I of thrombin, interacts strongly with a stretch of negatively charged amino acids present at the carboxyl terminus of hirudin (10Grütter M.G. Priestle J.P. Rahuel J. Grossenbacher H. Bode W. Hofsteenge J. Stone S.R. EMBO J. 1990; 9: 2361-2365Google Scholar, 11Rydel T.J. Ravichandran K.G. Tulinsky A. Bode W. Huber R. Roitsch C. Fenton II, J.W. Science. 1990; 249: 277-280Google Scholar). Similar areas of negatively charged amino acids are present in the thrombin receptor, thrombomodulin and heparin cofactor II and have been shown to interact with anion binding exosite I of thrombin (12Liu L.W. Vu T.K.H. Esmon C.T. Coughlin S.R. J. Biol. Chem. 1991; 266: 16977-16980Google Scholar, 13Vu T-K. Wheaton V.I. Hung D.T. Charo I. Coughlin S.R. Nature. 1991; 353: 674-677Google Scholar, 14Mathews I.I. Padmanabhan K.P. Ganesh V. Tulinsky A. Ishii M. Chen J. Turck C.W. Coughlin S.R. Fenton II, J.W. Biochemistry. 1994; 33: 3266-3279Google Scholar, 15Tsiang M. Lentz S.R. Dittman W.A. Wen D. Scarpati E.M. Sadler J.E. Biochemistry. 1990; 29: 10602-10612Google Scholar). Studies using synthetic peptides corresponding to the negatively charged amino acids of the proteins mentioned above have shown that the affinity of these peptides for thrombin varies considerably (15Tsiang M. Lentz S.R. Dittman W.A. Wen D. Scarpati E.M. Sadler J.E. Biochemistry. 1990; 29: 10602-10612Google Scholar, 16Hortin G.L. Benutto B.M. Biochem. Biophys. Res. Commun. 1990; 169: 437-442Google Scholar). These observations suggest that the ability of thrombin to interact with different components of the hemostatic system is, at least in part, determined by the affinity of anion binding exosite I of thrombin for stretches of negatively charged amino acids present on its substrates. Modulation of the pro- and anticoagulant activities of thrombin may be accomplished by simply exchanging negatively charged areas between the different substrates of this serine protease.Factor VIII is an essential cofactor for factor IXa in the conversion of factor X to Xa in the intrinsic pathway of blood coagulation (3Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10364-10370Google Scholar). Molecular cloning of the factor VIII cDNA revealed that factor VIII consists of a series of homologous domains which can be represented as follows: A1-A2-B-A3-C1-C2 (17Toole J.J. Knopf J.L. Wozney J.M. Sultzman L.A. Buecker J.L. Pittman D.D. Kaufman R.J. Brown E. Shoemaker C. Orr E.C. Amphlett G.W. Foster B. Coe M.L. Knutson G.J. Fass D.N. Hewick R.M. Nature. 1984; 312: 342-347Google Scholar, 18Vehar G.A. Keyt B. Eaton D. Rodriguez H. O'Brien D.P. Rotblatt F. Oppermann H. Keck R. Wood W.I. Harkins R.N. Tuddenham E.G.D. Lawn R. Capon D.J. Nature. 1984; 312: 337-342Google Scholar). Proteolytic processing of factor VIII at amino acid position Arg1648 occurs during biosynthesis of factor VIII (19Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Google Scholar). Consequently, factor VIII circulates in plasma as a metal ion-linked heterodimer consisting of a heavy chain (A1-A2-B) and light chain (A3-C1-C2). Activation of factor VIII by thrombin proceeds through limited proteolysis at amino acid positions Arg372, Arg740, and Arg1689 (19Eaton D. Rodriguez H. Vehar G.A. Biochemistry. 1986; 25: 505-512Google Scholar, 20Pitmann D.D. Kaufman R.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 2429-2433Google Scholar). Consequently, activated factor VIII consists of a heterotrimer of the separate A1 and A2-domains together with a thrombin cleaved light chain (21Lollar P. Parker C.G. Biochemistry. 1989; 28: 666-674Google Scholar, 22Fay P.J. Haidaris P.J. Smudzin T.M. J. Biol. Chem. 1991; 266: 8957-8962Google Scholar). We have shown previously that an acidic region corresponding to amino acid sequence Asp713-Arg740 of factor VIII is involved in the activation of factor VIII by thrombin (23Mertens K. Donath M.J.S.H. Van Leen R.W. De Keyzer-Nellen M.J.M. Verbeet M. Ph Klaasse Bos J.M. Leyte A. Van Mourik J.A. Br. J. Haematol. 1993; 85: 133-145Google Scholar, 24Donath, M. J. S. H., 1995, Activation and Limited Proteolysis of Human Blood Coagulation Factor VIII. Ph.D. thesis, University of Amsterdam.Google Scholar). Here, we have replaced amino acid sequence Asp712-Ala736 of factor VIII by amino acid sequence Ile51-Leu80 of heparin cofactor II, a potent inhibitor of thrombin (25Tollefsen D.M. Thromb. Haemostasis. 1995; 74: 1209-1214Google Scholar). Functional importance of amino acid sequence Ile50-Leu80 of heparin cofactor II in the inhibition of thrombin has been suggested by analysis of heparin cofactor II derivatives that lack this particular amino acid sequence (26Van Deerlin V.M.D. Tollefsen D.M. J. Biol. Chem. 1991; 266: 20223-20231Google Scholar). The factor VIII-heparin cofactor II hybrid constructed, designated des-(868-1562)-factor VIII-HCII, was expressed in mouse fibroblast C127 cells, and analysis of the properties of the resulting protein suggests that amino acid sequence Ile50-Ser81 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin.
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