HD1, a Thrombin-directed Aptamer, Binds Exosite 1 on Prothrombin with High Affinity and Inhibits Its Activation by Prothrombinase

Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (Kd values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the kcat for prothrombin activation by prothrombinase and a 6-fold increase in the Km, highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir54–65(SO-3). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin. Incorporation of prothrombin into the prothrombinase complex is essential for rapid thrombin generation at sites of vascular injury. Prothrombin binds directly to anionic phospholipid membrane surfaces where it interacts with the enzyme, factor Xa, and its cofactor, factor Va. We demonstrate that HD1, a thrombin-directed aptamer, binds prothrombin and thrombin with similar affinities (Kd values of 86 and 34 nm, respectively) and attenuates prothrombin activation by prothrombinase by over 90% without altering the activation pathway. HD1-mediated inhibition of prothrombin activation by prothrombinase is factor Va-dependent because (a) the inhibitory activity of HD1 is lost if factor Va is omitted from the prothrombinase complex and (b) prothrombin binding to immobilized HD1 is reduced by factor Va. These data suggest that HD1 competes with factor Va for prothrombin binding. Kinetic analyses reveal that HD1 produces a 2-fold reduction in the kcat for prothrombin activation by prothrombinase and a 6-fold increase in the Km, highlighting the contribution of the factor Va-prothrombin interaction to prothrombin activation. As a high affinity, prothrombin exosite 1-directed ligand, HD1 inhibits prothrombin activation more efficiently than Hir54–65(SO-3). These findings suggest that exosite 1 on prothrombin exists as a proexosite only for ligands whose primary target is thrombin rather than prothrombin. HD1, a thrombin-directed aptamer, binds exosite 1 on prothrombin with high affinity and inhibits its activation by prothrombinase.Journal of Biological ChemistryVol. 290Issue 8PreviewVOLUME 281 (2006) PAGES 37477–37485 Full-Text PDF Open Access Thrombin is the most versatile component of the hemostatic system, mediating procoagulant, anticoagulant, and anti-fibrinolytic pathways (1Di Cera E. Chest. 2003; 124: 11S-17SAbstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). The diverse activities of thrombin are regulated, at least in part, by electropositive exosites flanking its active site (2Lane D.A. Philippou H. Huntington J.A. Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (271) Google Scholar). Exosite 1 binds ligands that interact with the active site of thrombin, including fibrinogen, heparin cofactor II, and protease-activated receptor, the major thrombin receptor on cells (2Lane D.A. Philippou H. Huntington J.A. Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (271) Google Scholar). In contrast, exosite 2, which binds ligands such as heparin (3Fortenberry Y.M. Whinna H.C. Gentry H.R. Myles T. Leung L.L. Church F.C. J. Biol. Chem. 2004; 279: 43237-43244Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 4De Cristofaro R. De C ia E. Landolfi R. Circulation. 1998; 98: 1297-1301Crossref PubMed Scopus (29) Google Scholar) and platelet glycoprotein Ibα (5Adam F. Bouton M.C. Huisse M.G. Jandrot-Perrus M. Trends Mol. Med. 2003; 9: 461-464Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 7De Cristofaro R. De C ia E. Rutella S. Weitz J.I. J. Biol. Chem. 2000; 275: 3887-3895Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar), serves to tether thrombin for subsequent interactions with substrates or inhibitors. Prothrombin, the precursor of thrombin, lacks an active site and has immature or inaccessible exosites (8Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Kaczmarek E. Kaminski M. McDonagh J. Biochim. Biophys. Acta. 1987; 914: 275-282Crossref PubMed Scopus (9) Google Scholar). Because exosite 1 on prothrombin exhibits reduced affinity for certain ligands, it has been designated proexosite 1 (8Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). This proexosite gains functional activity during prothrombin conversion to thrombin, as evidenced by fluorescent ligand binding studies (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Thus, Anderson and Bock (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) reported that fluorescein-hirudin54–65(SO-3) (f-Hir54–65(SO-3)), 2The abbreviations used are: Hir54–65(SO-3), Tyr63-sulfated COOH-terminal hirudin peptide amino acids 54–65; FITC, fluorescein isothiocyanate; PCPS, l-α-phosphatidyl-choline and l-α-phosphatidyl-l-serine; pre1, prethrombin 1; pre2, prethrombin 2; mIIa, meizothrombin; mIIa(-F1), meizothrombin des F1; F1, prothrombin fragment 1; F2, prothrombin fragment 2; b-, biotin-; f-, fluorescein-; DAPA, dansylarginine N-(3-ethyl-1,5-pentanediyl)amide; Chz-Th, chromozym thrombin. the exosite 1-binding COOH terminus of hirudin, binds thrombin with an affinity 130-fold higher than that for prothrombin (Kd values of 25 nm and 3.2 μm, respectively). Prothrombin activation intermediates display intermediate affinities for f-Hir54–65(SO-3) that increase with the extent of activation (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). Diminished affinity of other thrombin ligands for proexosite 1 on prothrombin also has been observed (13Fischer B.E. Schlokat U. Himmelspach M. Dorner F. Protein Eng. 1998; 11: 715-721Crossref PubMed Scopus (12) Google Scholar, 14Wu Q. Picard V. Aiach M. Sadler J.E. J. Biol. Chem. 1994; 269: 3725-3730Abstract Full Text PDF PubMed Google Scholar). In contrast to the progressive maturation of proexosite 1, exosite 2 displays more abrupt development. Exosite 2 is not accessible until fragment 2 (F2) is released from prothrombin. Thus, prethrombin 2 (pre2) and thrombin have similar affinities for heparin, whereas meizothrombin (mIIa) and meizothrombin des F1 [mIIa(-F1)], which retain the F2 domain, do not bind heparin (15Schoen P. Lindhout T. J. Biol. Chem. 1987; 262: 11268-11274Abstract Full Text PDF PubMed Google Scholar). Understanding the functional maturation of the exosites on thrombin has increased in importance with emerging evidence that the exosites serve not only as binding domains but also as allosteric regulators of thrombin activity (16Mengwasser K.E. Bush L.A. Shih P. Cantwell A.M. Di Cera E. J. Biol. Chem. 2005; 280: 26997-27003Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 18Fredenburgh J.C. Stafford A.R. Weitz J.I. J. Biol. Chem. 1997; 272: 25493-25499Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Numerous studies reveal that ligand binding to either exosite can modify the activity of thrombin. Thus, peptide (16Mengwasser K.E. Bush L.A. Shih P. Cantwell A.M. Di Cera E. J. Biol. Chem. 2005; 280: 26997-27003Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 19Hortin G.L. Trimpe B.L. J. Biol. Chem. 1991; 266: 6866-6871Abstract Full Text PDF PubMed Google Scholar), glycosaminoglycan (20Pike R.N. Buckle A.M. Le Bonniec B.F. Church F.C. FEBS J. 2005; 272: PubMed Scopus Google Scholar, M. F. S. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), and Leung PubMed Scopus Google Scholar) ligands have been to thrombin. a of These which into binding for ligands Biophys. Chem. 2005; PubMed Scopus Google Scholar, P. J. J. Mol. Biol. 1994; PubMed Scopus Google Scholar). In the of thrombin, HD1 binds to exosite 1 J.A. PubMed Scopus Google Scholar), whereas binds to exosite 2 H. Weitz J.I. Stafford A.R. P. Med. Biol. 2003; PubMed Scopus Google Scholar). Because of high affinity and HD1 and serve as to the of the exosites on thrombin. The of the complex has been Sadler J.E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), as has the of the complex J. A. Protein 1994; PubMed Scopus Google Scholar). These suggest that the exosite 1-directed ligands bind to but Thus, interacts with the of exosite 1, whereas HD1 binds to binding sites on thrombin, it is that HD1 and do not interactions with prothrombin and its used HD1 and to the functional maturation of exosite 1 and the exosite aptamer, to exosite 2 thrombin, and factor from Va and dansylarginine from and from from HD1 the exosite 1 and and a of and or by the and at of the to by to for by on for Leung PubMed Scopus Google Scholar). a from from and from a factor a from and its Tyr63-sulfated COOH-terminal Hir54–65(SO-3), from of thrombin from whereas and from from and l-α-phosphatidyl-choline from from and a of PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). used to the of Scopus Google Scholar). The at in with FITC, of peptide in of and of in to a of 2 the in and the for at of 1 to the The to a that with and with and the fluorescent which by the with a in a of the at nm, and on D.A. 11: PubMed Scopus Google Scholar), of into Hir54–65(SO-3). of Prothrombin of the prothrombin activation intermediates of PubMed Scopus Google Scholar, L.A. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The of PubMed Scopus Google Scholar) on and by prothrombin with nm thrombin in for 2 at The by the of nm and the of thrombin activity The to on a by with factor 1 in with nm factor for at The by the of 1 and 1 and inhibition of the thrombin or factor activity with or and factor The to on a and by prothrombin or pre1, with The in the of to Prothrombin or in with in 25 in the of for at The to on a Protein at nm the and for pre1, pre2, mIIa, and mIIa(-F1), A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the an The of of the prothrombin intermediates by and at of exosites 1 and 2 HD1 and or in 2 1 and to a at with a and a at an of nm at and of nm, with at nm and a of nm, a the to with of prothrombin or thrombin the to the nm fluorescent the a the values from as a of the the data by to the where is the of the is the of aptamer, is the and is the to the of to from thrombin or prothrombin. in of to the and Prothrombin or thrombin to of or nm, the the with of Hir54–65(SO-3). nm to in a similar where prothrombin or nm thrombin to nm and the with of HD1 nm used as an to the interaction of HD1 with prothrombin. HD1 immobilized on a which is with increase the cells with 1 at a of a the cells at for which the cells with at for 2 factor Va competes with for prothrombin a nm prothrombin into a in the or of nm factor and at values or for the values over a of prothrombin to HD1 in the of factor Va as a of that in its Prothrombin used to the of HD1 or on prothrombin activation by prothrombinase a of a J.C. Stafford A.R. J. V. Weitz J.I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (29) Google Scholar). of of PCPS, nm factor Xa, and nm factor in for at 1 prothrombin with HD1 or Hir54–65(SO-3), in from to μm, for in a of in a the of the prothrombinase to to by of of a peptide and thrombin by of at nm for a of as by used to thrombin on the activity of thrombin of Chz-Th, as in a thrombin of prothrombin activation In the of HD1 on prothrombin activation by factor in the of factor Va or 1 prothrombin either with nm factor and PCPS, nm factor and nm factor or with 2 nm factor and nm factor Va. As a the of HD1 on prothrombin activation also 1 prothrombin with in the or of HD1, and thrombin generation as the of HD1 on the of prothrombin activation by prothrombin from to with prothrombinase nm factor Xa, nm factor and in the or of 25 HD1 for to of thrombin prothrombin and by to the where is the and is the of prothrombin activation kcat by by the factor of Prothrombin the of HD1 on the prothrombin activation activation intermediates in the or of HD1 by Prothrombin with in the or of The by the of the prothrombinase of 2 nm factor Xa, nm factor and in into for 2 and to and the at least The are as the of Exosite 1 to Thrombin and of the of thrombin in complex with or with HD1 that exosite 1-directed ligands interact with but domains on thrombin Sadler J.E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. A. Protein 1994; PubMed Scopus Google Scholar). ligands interact with the domains on prothrombin as do on thrombin is to the of to from prothrombin or thrombin. As in the by nm thrombin is to a nm This increase in is HD1 is not with binding. The also to the complex is with Hir54–65(SO-3), that the site on thrombin with that of HD1 In the the of thrombin to f-Hir54–65(SO-3) in a reduction in the complex is with HD1, the but not to These findings suggest that the site on thrombin only with the prothrombin in of thrombin. As in the of prothrombin to in a increase in with Hir54–65(SO-3), the but not In the by prothrombin is to is in the complex is with HD1, that the site on prothrombin not with the The of to the in to not These data suggest that prothrombin binds exosite 1 ligands and that the site on prothrombin only with the of to Prothrombin the in that with prothrombin is similar in to that which it binds thrombin, the affinity of for prothrombin and thrombin. The of nm and with prothrombin or thrombin, and the in The of prothrombin to in a increase in and a with a of 86 nm Thus, the affinity of for prothrombin is higher than that of and is with the of nm reported for 2 of P. R. P. J. Bock P.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to the affinity of for prothrombin As in 1, binds mIIa, mIIa(-F1), pre1, and with values of 86 and nm, binds thrombin with a of 34 Thus, binds prothrombin and its activation intermediates with an affinity similar to that for thrombin. These findings suggest that the site on prothrombin during prothrombin conversion to values of and for prothrombin activation intermediates and that values not des that values not Open in a f-Hir54–65(SO-3) in of with (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), binds prothrombin with an affinity than that for thrombin (Kd values of and nm, These data suggest the site on the site on prothrombin during prothrombin conversion to thrombin that its affinity for the A. Chem. 1994; PubMed Scopus (29) Google Scholar) and functional H.C. J.A. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) data that F2 binds exosite exosite 2 only accessible on thrombin and pre2, the affinity of an exosite aptamer, for prothrombin and thrombin. As binds only to and thrombin, with values of and nm, Thus, data the that exosite 2 is only accessible on prothrombin of HD1 on Prothrombin by 1 on prothrombin to prothrombin activation by prothrombinase by at least in part, the interaction of prothrombin with factor Va P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). Because HD1 binds exosite 1 of prothrombin with high affinity, to HD1 attenuates prothrombin activation by with factor Va for prothrombin binding. The of prothrombin activation by prothrombinase in the or of HD1 attenuates prothrombin activation by prothrombinase in a and the by with an of nm, a with the of for prothrombin. to the by which HD1 inhibits prothrombin by factor in the or of of the prothrombinase complex omitted from the prothrombinase HD1 prothrombin activation by over with factor Va of either or nm, values of and nm, In contrast, HD1 on prothrombin activation factor Va omitted from the prothrombinase These suggest that the inhibitory of HD1 on prothrombin activation is factor with other exosite 1 ligands P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). In of HD1 on prothrombin activation by not a factor of prothrombin. is the intermediate during prothrombin activation M. R. J. PubMed Scopus Google Scholar, S. Church J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar). generation is and factor whereas is the intermediate that is in the of HD1 the extent of prothrombin by prothrombinase in the or of HD1, and of the to or not to from prothrombin. In the of HD1, is the major and prothrombin activation is by HD1 is prothrombin activation also mIIa, but prothrombin activation at conversion of the fragment to in the of HD1 the inhibitory of HD1 on thrombin. Thus, HD1 attenuates prothrombin activation by prothrombinase but to have on the prothrombin activation pathway. In of the of prothrombin is that factor Va the kcat for factor of whereas the of the J. H.C. J. Biol. Chem. Full Text PDF PubMed Google Scholar, M. 2005; PubMed Scopus (28) Google Scholar). Because HD1 attenuates prothrombin activation by prothrombinase in a factor Va-dependent the of HD1 on the of activation. Prothrombin, in from to μm, with prothrombinase in the or of 25 HD1, and the of thrombin generation In the of HD1, the and kcat values are nm and values with the reported of nm and kcat of M. J. Biol. Chem. 2005; 280: Full Text Full Text PDF PubMed Scopus Google Scholar). 25 HD1, the at a a of nm and a kcat of Thus, the is in the of HD1 than it is in its a 6-fold increase in and 2-fold reduction in These findings HD1 produces over 90% inhibition of prothrombin activation by prothrombinase of HD1 on the Va HD1 competes with factor Va for prothrombin a In prothrombin binding to immobilized in the or of 1 factor Va. factor Va is prothrombin binding to immobilized is reduced by not These findings the that HD1 the factor Va-prothrombin prothrombin activation by prothrombinase is essential for a of thrombin at sites of vascular S. J. 2003; PubMed Scopus Google Scholar). activation to prothrombin into the prothrombinase is not only by prothrombin interaction with M. R. J. PubMed Scopus Google Scholar), but also by prothrombin binding to factor Va S. Church J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar). is that exosite 1 on prothrombin at least in part, its interaction with factor Va P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). exosite 1 is to as an immature proexosite on prothrombin on the that binds prothrombin with an affinity than that for thrombin and attenuates prothrombin activation (8Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, A.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). data with HD1 the of proexosite 1 on prothrombin and on exosite maturation during prothrombin conversion to thrombin. We demonstrate Hir54–65(SO-3), HD1 (a) binds prothrombin and thrombin with similar high affinity, (b) inhibits prothrombin activation by prothrombinase in a factor Va-dependent and competes with factor Va for prothrombin binding. HD1 into the and of exosite 1 on prothrombin. The inhibitory of HD1 and can by in binding interactions with prothrombin and thrombin. of and HD1 to Prothrombin and of the of the and that the exosite 1-directed ligands bind to but of exosite 1 Sadler J.E. A. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. A. Protein 1994; PubMed Scopus Google Scholar). because competes with for binding to prothrombin and thrombin. These findings can in the of and HD1 to for binding with ligand binding. Thus, exosite 1 are in J. A. Protein 1994; PubMed Scopus Google Scholar, M.G. J.C. B.F. Protein 1997; PubMed Scopus Google Scholar), and HD1 from its binding is that directly competes for the that bind with a HD1 from prothrombin or thrombin. Thus, HD1 displays a with as for binding M. K.E. Leung J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, Hir54–65(SO-3), which with thrombin J. A. Protein 1994; PubMed Scopus Google Scholar), has a The that HD1 only competes with that the of the ligands but the mediating the interaction of HD1 with thrombin or prothrombin only a of that binding. Thus, studies in the of exosite 1 of prothrombin. The interactions of HD1 and with prothrombin of the functional maturation of exosite 1 in prothrombin. Exosite 1 on contrast to Hir54–65(SO-3), HD1 binds prothrombin and prothrombin intermediates with affinities similar to that for thrombin. These findings suggest that the that the site in exosite 1 during prothrombin conversion to thrombin. In contrast, the site maturation during prothrombin activation. of the to maturation of the exosite 1 because the affinity of for the of prothrombin is higher than for pre1, and (11Anderson P.J. Bock P.E. J. Biol. Chem. 2003; 278: 44489-44495Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 12Anderson P.J. Nesset A. Bock P.E. J. Biol. Chem. 2003; 278: 44482-44488Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). data suggest that the of a proexosite on prothrombin to it a proexosite in of its affinity for thrombin such as fibrinogen, it not serve as a proexosite for also not a proexosite in the of factor whose primary is to bind prothrombin rather than thrombin. In as as of (8Anderson P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: 16428-16434Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar), that exosite 1 is an to the Va Prothrombin with that HD1 inhibits prothrombin activation by prothrombinase in a factor Va-dependent and that factor Va competes with prothrombin for HD1 binding. data the importance of exosite 1 in the Va a by with other exosite 1-directed ligands, such as P.J. Nesset A. Dharmawardana K.R. Bock P.E. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, A.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar) or a from the of PubMed Scopus Google Scholar). HD1 attenuates prothrombin it not it because exosite 1 is not the binding site for factor Va. Thus, of the S. J.A. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar), F2 S. 1997; PubMed Scopus Google Scholar), and the M. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar) and 1 of F1 H. H. J. J. 1997; PubMed Scopus Google Scholar) also to the Va the that prothrombin with factor the inhibitory of HD1 on prothrombin activation by prothrombinase that exosite 1 on prothrombin an in the Va The contribution of factor Va and to factor activity prothrombin is studies that factor Va which prothrombin intermediate is by factor S. Church J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. Full Text PDF PubMed Google Scholar). that the activation whereas factor Va the of thrombin of the M. R. J. PubMed Scopus Google Scholar, S. Church J. Biol. Chem. 1987; 262: Full Text PDF PubMed Google Scholar, R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus (29) Google Scholar, R. PubMed Scopus Google Scholar). that HD1 the of prothrombin activation by without the activation which prothrombin is to thrombin, that exosite 1 on prothrombin is a major for its into the prothrombinase complex but not to factor of prothrombin. of Exosite 1 on Prothrombin in by is of exosite 1-directed ligands that has been to prothrombin activation by and prothrombin activation by the factor Va but not by prothrombinase PubMed Scopus Google Scholar, Google Scholar). a of a of factor binds prothrombin with a of nm and inhibits prothrombinase activity with an of M. J. Biol. Chem. 2004; 279: Full Text Full Text PDF PubMed Scopus Google Scholar). of ligands bind exosite 1 on inhibits prothrombin activation by prothrombinase as as HD1 inhibits prothrombin activation by prothrombinase by over Kinetic that HD1 the of the more than the This is with the that exosite 1 on prothrombin is a major site for factor Va and is for prothrombin into the prothrombinase The of prothrombin activation observed in the of HD1 factor Va interactions with factor prothrombin that are of exosite 1 S. 1997; PubMed Scopus Google Scholar). Because of its high affinity for HD1 a to the contribution of the Va interaction to prothrombin activation by data suggest that the that prothrombin a proexosite 1 to and bind prothrombin more than thrombin, is not the with also bind prothrombin more than or because binds and with high affinity H.C. J.A. J. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar) and is a more of prothrombin activation by prothrombinase than A.R. J. Biol. Chem. 2003; 278: Full Text Full Text PDF PubMed Scopus Google Scholar). exosite 1 on prothrombin is for binding ligands, such as factor that have a In contrast, serves as a proexosite for thrombin-directed ligands or HD1 as an because of its to directly thrombin activity Leung PubMed Scopus Google Scholar, M. K.E. Leung J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). data suggest that HD1 has Thus, in to thrombin HD1 also attenuates thrombin generation by prothrombin activation by at the the and pathways of prothrombinase is an target for thrombin the is to thrombin activity to an than that thrombin data suggest that an that binds prothrombin over thrombin target prothrombinase by the factor