Long Range Communication between Exosites 1 and 2 Modulates Thrombin Function

Abstract

Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), γ′-peptide (an analog of the COOH terminus of the γ′-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to γA/γA-fibrin, an interaction mediated solely by exosite 1. When γA/γA-fibrinogen was clotted with thrombin in the presence of HD22, γ′-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for γA/γA-fibrin 6-fold and accelerated the dissociation of thrombin from preformed γA/γA-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with γA/γA-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54–65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized γ′-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation. Although exosites 1 and 2 regulate thrombin activity by binding substrates and cofactors and by allosterically modulating the active site, it is unclear whether there is direct allosteric linkage between the two exosites. To begin to address this, we first titrated a thrombin variant fluorescently labeled at exosite 1 with exosite 2 ligands, HD22 (a DNA aptamer), γ′-peptide (an analog of the COOH terminus of the γ′-chain of fibrinogen) or heparin. Concentration-dependent and saturable changes in fluorescence were elicited, supporting inter-exosite linkage. To explore the functional consequences of this phenomenon, we evaluated the capacity of exosite 2 ligands to inhibit thrombin binding to γA/γA-fibrin, an interaction mediated solely by exosite 1. When γA/γA-fibrinogen was clotted with thrombin in the presence of HD22, γ′-peptide, or prothrombin fragment 2 there was a dose-dependent and saturable decrease in thrombin binding to the resultant fibrin clots. Furthermore, HD22 reduced the affinity of thrombin for γA/γA-fibrin 6-fold and accelerated the dissociation of thrombin from preformed γA/γA-fibrin clots. Similar responses were obtained when surface plasmon resonance was used to monitor the interaction of thrombin with γA/γA-fibrinogen or fibrin. There is bidirectional communication between the exosites, because exosite 1 ligands, HD1 (a DNA aptamer) or hirudin-(54–65) (an analog of the COOH terminus of hirudin), inhibited the exosite 2-mediated interaction of thrombin with immobilized γ′-peptide. These findings provide evidence for long range allosteric linkage between exosites 1 and 2 on thrombin, revealing further complexity to the mechanisms of thrombin regulation. Long range communication between exosites 1 and 2 modulates thrombin function.Journal of Biological ChemistryVol. 290Issue 8PreviewVOLUME 284 (2009) PAGES 25620–25629 Full-Text PDF Open Access As the central effector of hemostasis, thrombin is engaged in procoagulant, anticoagulant, and fibrinolytic processes. These seemingly contrasting roles are regulated, at least in part, by thrombin's interactions with other factors in the blood and vasculature. The binding of ligands to thrombin is promoted by exosites 1 and 2, which are positively charged domains that flank the active site. These exosites facilitate the binding of substrates or cofactors and align them for optimal interaction with the active site (1Lane D.A. Philippou H. Huntington J.A. Blood. 2005; 106: 2605-2612Crossref PubMed Scopus (274) Google Scholar). Exosite 1 is predominantly used to gain access to the active site by substrates such as fibrinogen (2Meh D.A. Siebenlist K.R. Mosesson M.W. J. Biol. Chem. 1996; 271: 23121-23125Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar), factors V (3Myles T. Yun T.H. Hall S.W. Leung L.L. J. Biol. Chem. 2001; 276: 25143-25149Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar) and VIII (4Esmon C.T. Lollar P. J. Biol. Chem. 1996; 271: 13882-13887Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), and the protease-activated receptors (PARs) 2The abbreviations used are: PARprotease-activated receptor5-IAF5-iodoacetamidofluoresceinF1prothrombin fragment 1F2prothrombin fragment 2SPRsurface plasmon resonanceHBSHepes-buffered salineFPRckd-Phe-Pro-Arg chloromethyl ketoneYPRckd-Tyr-Pro-Arg chloromethyl ketoneRUresponse unit(s). on platelets (5Ayala Y.M. Cantwell A.M. Rose T. Bush L.A. Arosio D. Di Cera E. Proteins. 2001; 45: 107-116Crossref PubMed Scopus (102) Google Scholar). Effectors that modulate thrombin activity, including thrombomodulin (6Ye J. Liu L.W. Esmon C.T. Johnson A.E. J. Biol. Chem. 1992; 267: 11023-11028Abstract Full Text PDF PubMed Google Scholar), hirudin (7Naski M.C. Fenton 2nd, J.W. Maraganore J.M. Olson S.T. Shafer J.A. J. Biol. Chem. 1990; 265: 13484-13489Abstract Full Text PDF PubMed Google Scholar), and heparin cofactor II (8Baglin T.P. Carrell R.W. Church F.C. Esmon C.T. Huntington J.A. Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 11079-11084Crossref PubMed Scopus (186) Google Scholar), also utilize exosite 1. Thrombomodulin alters the specificity of thrombin by hindering access of other substrates to exosite 1 (9Esmon C.T. Esmon N.L. Harris K.W. J. Biol. Chem. 1982; 257: 7944-7947Abstract Full Text PDF PubMed Google Scholar) and by providing new binding sites for protein C and thrombin-activable fibrinolysis inhibitor, thereby promoting anticoagulant and T. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are mediated by exosite 2, which as a that thrombin for in other heparin exosite 2 Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar) and thrombin by and heparin cofactor II S.T. J. Biol. Chem. 1982; 257: Full Text PDF PubMed Google T. PubMed Scopus Google Scholar). Exosite 2 also is used by on platelets to thrombin for of J.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google E. Hall S.W. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google L.A. Di Cera E. Biol. PubMed Scopus Google Scholar). protease-activated prothrombin fragment 1 prothrombin fragment 2 surface plasmon resonance chloromethyl chloromethyl unit(s). Although the of the exosites is to substrates and cofactors with thrombin, there is evidence that the exosites also as allosteric of thrombin when from or are to exosite 1 on thrombin, an surface of the active site thereby providing access to substrates Di Cera E. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). The binding of a thrombomodulin fragment to exosite 1 was to the of an active site J. Esmon N.L. Esmon C.T. Johnson A.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar), which the of protein C and thrombin-activable fibrinolysis in an allosteric exosite from heparin cofactor II or fibrinogen decrease the of protein C J. Biol. Chem. Full Text PDF PubMed Google Scholar). the binding of ligands to exosite 1 alters the of L.W. Esmon C.T. J. Biol. Chem. Full Text PDF PubMed Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are to exosite because prothrombin fragment 2 a of exosite 2 and the at which thrombin fibrinogen to fibrin J. Biol. Chem. Full Text PDF PubMed Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and is inhibited by Olson S.T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Esmon C.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). of the that are allosteric in to the active site of thrombin a in fluorescence when exosite 2 is J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. 1992; 267: Full Text PDF PubMed Google Scholar). Although there is evidence for allosteric of the active site by the exosites, it unclear whether there is direct allosteric between the exosites. between exosites 1 and 2 by L.W. J. Johnson A.E. Esmon C.T. J. Biol. Chem. Full Text PDF PubMed Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google M.C. 45: PubMed Scopus Google Scholar), by Olson S.T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the was to and ligands to this we the of exosite ligands on the fluorescence of a thrombin variant that was labeled in exosite 1. we the of ligands on thrombin binding to fibrin. To the that thrombin γA/γA-fibrinogen exosite 1 (2Meh D.A. Siebenlist K.R. Mosesson M.W. J. Biol. Chem. 1996; 271: 23121-23125Abstract Full Text Full Text PDF PubMed Scopus (117) Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), exosite 2 this was J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). used fibrin and surface plasmon resonance to the of exosite ligands on thrombin binding to were to the of ligands on the of thrombin dissociation from preformed fibrin clots. we whether exosite ligands modulate the binding of thrombin to an exosite and were obtained from was a from from was from HD1 and HD22 the exosite and and a variant of HD22, were by the and at the were to for and on for an analog of the of the γ′-chain of the γ′-peptide was from To of the γ′-peptide were with in of J. PubMed Scopus Google Scholar). and were obtained from analog of of protein hirudin was from substrates and were from was from and were from was from was from fibrinogen by on and was by as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was from prothrombin as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with the The was with and to inhibit and 1 and to a on a at a of the with a was a of and to prothrombin fragment 1 from from the at at The of and was by on or were by at and at The of and were at of Esmon C.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar) and L.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), at in was to a of and in the for at a with to were was a of and an of at was by with as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of was to γ′-peptide and in of and the was for 1 at was used to the γ′-peptide from were and the γ′-peptide were at The binding of exosite and ligands to was by of in was at with a in a and a was at an of and of of and an at in an the fluorescence was titrated with to of heparin γ′-peptide HD1 HD22 or hirudin-(54–65) the fluorescence to between the fluorescence a were obtained from and for of the were by to the binding is the of the titrated is the of is the fluorescence and is the dissociation of HD22 γ′-peptide or were to a of and in at of and was to to a of 2 γA/γA-fibrinogen and 2 in a of and were to for at was as by As a was used in of fibrin were by at for of were and for a to the of The of was by from the of in the of the The of to in the of fibrinogen was of of exosite were by of a and the at was To the that active thrombin from of active thrombin, the of exosite ligands on binding of active thrombin to γA/γA-fibrin also was 2 was clotted with in the presence of 1 to and in the presence of of HD22 for at the fibrin was by and of were and to by the of of activity was in a at on the activity of thrombin, the of thrombin was and used to the of the The of HD22 on thrombin binding was as of of γA/γA-fibrinogen and 2 was was by of in the or presence of The of was as of of γA/γA-fibrinogen were by of a to The interaction of with γA/γA-fibrinogen or γA/γA-fibrin in the or presence of exosite ligands was by a or were to of a an were in and were with 1 in between or was at a of in at and of γA/γA-fibrinogen or of was To immobilized γA/γA-fibrinogen to γA/γA-fibrin, was in of To 2 was at a of for 1 in the presence of HD22, γ′-peptide, or with was used to monitor dissociation and of the were for and by the from the was immobilized a by of of a in at a of by of of 1 were with of and 1 at a of to γ′-peptide was to the at a of to As a a was binding HD22, γ′-peptide, HD1 or and 1 or 2 were the were for of and by the from the The of or that in the presence of exosite was by by the in the of were by The of exosite ligands on the of of from fibrin was as J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and in with were to a was by of for at fibrin to the were and for were in of with or 2 or were from for and to the The of in the at was to the and were to a with a J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the ligands on thrombin activity were a fibrinogen To of a were 2 and HD22, γ′-peptide, or in a of an of γA/γA-fibrinogen and to of 2 and 2 were at and was at in a The to in was in the or presence of ligands, and were as a of were in and The of ligands on the activity of was by the of or by 2 thrombin in the or presence of HD22, or and were by of the were in To the that exosite ligands modulate exosite we a thrombin variant fluorescently labeled at exosite 1. is a thrombin variant with in exosite 1 with PubMed Scopus Google Scholar). The fluorescence of was as the was titrated with exosite 2 of γ′-peptide or heparin the fluorescence of in a dose-dependent and saturable The for γ′-peptide binding was a to the of when thrombin fluorescently labeled at the active site was titrated with γ′-peptide J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), heparin with a of to γ′-peptide or HD22 the fluorescence of and with a of These findings the that binding of exosite ligands to thrombin a in exosite 1. As binding of HD1 and hirudin-(54–65) to also were exosite 1 ligands with affinity that with HD1 and hirudin-(54–65) with of and To the that by fluorescence we used to the functional consequences of the long range communication between thrombin exosites. To the of we first the of exosite ligands on of a of fibrinogen that with thrombin exosite 1 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). was by and a of exosite ligands the thrombin in a the in with J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HD22 was the a of the thrombin at a of γ′-peptide at to Although it is the of HD22 on the to that of the of the ligands for exosite 2 on thrombin, with HD22 binding with the the ligands on the because on exosite As the of exosite ligands also was As HD1 and hirudin-(54–65) a the exosite ligands, the at 1 exosite ligands modulate the thrombin by allosterically the active site exosite were to the The that ligands allosterically the active site of thrombin was The of of of substrates by 2 thrombin were in the or presence of of exosite As for hirudin-(54–65) (7Naski M.C. Fenton 2nd, J.W. Maraganore J.M. Olson S.T. Shafer J.A. J. Biol. Chem. 1990; 265: 13484-13489Abstract Full Text PDF PubMed Google J. Biol. Chem. Full Text PDF PubMed Google Scholar), HD1 and HD22 the for of substrates There was to the presence of HD22, with the with and and with The two also the HD22 reduced the and with HD1 on the of of this is for exosite HD1 and hirudin-(54–65) on the with the from 2 that ligands for the exosite These also protein or ligands, modulate the active site by binding to the that the exosite ligands also the of exosite 1 and that this to activity in the used a fibrin binding to explore functional between the exosites, because the interaction of thrombin with fibrin is of the active and for thrombin of substrates in the presence of exosite Open in a new thrombin to solely exosite this interaction to whether exosite ligands an allosteric at exosite 1. To thrombin binding to γA/γA-fibrin, we the of in of fibrin with a of in the or presence of Although the fibrin was of in the of the first of the of γA/γA-fibrin with a of a with the of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the presence of HD22, binding to γA/γA-fibrin was reduced by with a at a charged the were a of thrombin binding to γA/γA-fibrin by binding was from the obtained with and γ′-peptide also were to whether the was to γ′-peptide and reduced thrombin binding to γA/γA-fibrin with of and The of γ′-peptide and to HD22 are with for thrombin with of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the exosite also was as a As a direct of thrombin binding to γA/γA-fibrin, HD1 a and inhibited binding to the with a of to a of which is to the of HD1 for of J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the exosite the exosite ligands thrombin from fibrin that are in a of exosite ligands on binding to γA/γA-fibrin clots. and were to 2 2 and HD22 γ′-peptide or at the for were by and in the was used to the are as the of thrombin to the the of and the of in the of the HD22 was the of the ligands, a of and are HD22, and thrombin binding with of and HD22 thrombin binding to it was of to on the affinity of the the presence of HD22, the of for γA/γA-fibrin was an affinity 6-fold that in the of further for the that exosite ligands interactions mediated by exosite 1. a active thrombin was used in of active thrombin to address the that the presence of an in the active site the binding of the exosites. The also was to and in the presence of exosite The of thrombin in by was used to thrombin on the binding of to HD22 also reduced the binding of to fibrin in a dose-dependent and saturable These that the presence of the chloromethyl in the active site on thrombin binding to fibrin or HD22 binding to was used to the obtained in the binding we that the binding for the interaction are to with fibrin J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), thereby as a for the the γA/γA-fibrinogen was immobilized a and was immobilized as a for γA/γA-fibrinogen with a of a to the affinity of for γA/γA-fibrin The exosite ligands were with 2 were used to thrombin and the were as the of the of exosite the presence of HD22, the of thrombin to the immobilized γA/γA-fibrinogen in a dose-dependent and saturable HD22 was to a of the with was to inhibit thrombin Similar were obtained with γ′-peptide and of and To the of the ligands on thrombin binding to γA/γA-fibrin, the immobilized γA/γA-fibrinogen was with thrombin to the fibrinogen to fibrin. to the immobilized γA/γA-fibrin with a of to the of obtained J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the obtained with exosite ligands binding to γA/γA-fibrin at the The for HD22 was which is to that obtained with immobilized and γ′-peptide, of and also with γA/γA-fibrinogen as with fibrin clots. Although that exosite ligands reduced the exosite interaction of thrombin with were exosite ligands thrombin binding to immobilized fibrinogen or with fibrin of the thrombin at the of exosite The for this is the that thrombin binding to fibrin is is is that the changes by of fibrin on the because thrombin to immobilized fibrin and fibrin with J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the thrombin binding in the presence of the exosite ligands, the of fibrin To address this we the dissociation of from preformed fibrin in the or presence of exosite ligands to whether exosite ligands thrombin to fibrin J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). in the presence of were for and in that or the in the with of the thrombin the presence of 2 of HD1 promoted of to a to that of HD22 promoted the dissociation of of from the the of HD22 was that of HD1 that of These further for the that an exosite binding mediated by exosite 1. that exosite ligands the of exosite it was of to whether the also is To explore this HD1 and hirudin-(54–65) were for on exosite binding of thrombin to γ′-peptide. the of thrombin binding to the immobilized γ′-peptide was an affinity to the of obtained in direct binding J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). As a was the in the presence of exosite HD22 and γ′-peptide inhibited binding to the immobilized γ′-peptide with of and The of HD22 to γ′-peptide is with affinity for thrombin with of and 1 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). When the was in the presence of exosite 1 ligands, a of thrombin binding also was HD1 inhibited binding to immobilized γ′-peptide, hirudin-(54–65) inhibited binding by The for HD1 and hirudin-(54–65) were and was that HD1 was HD22, thrombin with J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These are with obtained for γA/γA-fibrinogen an exosite the capacity of the exosite to To that communication between the exosites, the binding was it an exosite we that to γ′-peptide in an interaction that by exosite 1 to γ′-peptide with a of a to the affinity of for γ′-peptide. on the binding of to immobilized γ′-peptide with the that it J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). HD1 reduced binding to γ′-peptide, a that with the presence of a of exosite 1 on which was used as a reduced or binding to immobilized γ′-peptide by As HD22 reduced and binding to γ′-peptide to a in with the exosite interaction of with γ′-peptide. These provide further of the allosteric between the exosites. of thrombin's central in hemostasis, of activity is The exosites the active site of thrombin to the of thrombin by substrates to the active site or by thrombin of substrates or and the exosites also to allosterically modulate thrombin's When exosite an surface is from the active site Di Cera E. Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google Scholar). ligands that to exosite to modulate thrombin activity with and substrates L.W. Esmon C.T. J. Biol. Chem. Full Text PDF PubMed Google Olson S.T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google H. J. PubMed Scopus Google Scholar). fluorescently labeled we that ligands the exosites in a inter-exosite communication J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that binding of to exosite 2 on thrombin of a from exosite 1. this Olson S.T. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). the a thrombin variant labeled in exosite 1 was used to the of binding to exosite HD22, γ′-peptide, and heparin changes at exosite and binding were to obtained by other These findings provide evidence of an allosteric linkage between the exosites. To explore the functional consequences of this inter-exosite were used to the fluorescence ligands and were used in that the interaction of the that thrombin the of exosite 1 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the of exosite ligands on thrombin binding to fibrin was exosite ligands of affinity and were to the of thrombin to fibrin by HD22 reduced the affinity of thrombin for fibrin by These that there is a between the exosites. Furthermore, that the also is exosite ligands thrombin binding to γ′-peptide, which is an exosite 2-mediated interaction J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that HD22 thrombin to preformed fibrin a that the that the presence of the ligands the thrombin binding to fibrin. provide further evidence that the exosites of thrombin are The by which the exosites interactions are to to the exosites. Although to fibrinogen is to the of exosite 2 the the interaction with fibrin exosite 1 J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Esmon C.T. J. Biol. Chem. Full Text PDF PubMed Google Scholar). is also by evidence from the interaction of thrombin with fibrin fragment which binding mediated exosite 1 L.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The of thrombin in with HD22 to of thrombin in with an exosite or heparin sites of to exosite 2 Long PubMed Scopus Google E. Huntington J.A. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google T.P. PubMed Scopus Google Scholar). also of exosite 2 by the ligands used in J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. PubMed Scopus Google T.P. PubMed Scopus Google Scholar). The findings of and functional it that the that we are the of ligands to exosite hindering the the of the interactions from long range There is and that of exosite 1 changes in the active site. also is that there is an allosteric between exosite 1 and the site Carrell Bush L.A. Di Cera E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Cera E. PubMed Scopus Google Scholar). is an allosteric effector that modulates the and anticoagulant of allosteric a of in the of the thrombin that from exosite 1 to the site. the it is that the site between the active site and exosite the of thrombin in with the γ′-peptide, the to exosite 2 and the of the site Mosesson M.W. Di Cera E. Chem. PubMed Scopus Google Scholar). because a on thrombin it is that the allosteric the site to exosite 2 Cera E. PubMed Scopus Google Scholar). there to for the in this other thrombin the of of the of by to the that the allosteric changes are this a of of the active site by chloromethyl Cera E. PubMed Scopus Google Scholar). of an allosteric between the exosites, provide evidence that binding of γ′-peptide to exosite 2 alters exosite 1 M.C. 45: PubMed Scopus Google Scholar). that ligands for exosite the active site, the active site. is with the that thrombin is a allosteric Although this evidence for inter-exosite there are of ligands that exosites These such as with or Although at with the it is to that are or such that the ligands for exosites are to The is of the binding of a that exosites Proc. Natl. Acad. Sci. U.S.A. PubMed Scopus Google J. D. J. PubMed Scopus Google Scholar). binding a on the of thrombin to from is unclear whether the allosteric between the exosites that the allosteric that in thrombin to domains Cera E. PubMed Scopus Google Scholar). of exosites on the active site J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), it is that of exosites a from that by of exosites. of factors V or VIII (4Esmon C.T. Lollar P. J. Biol. Chem. 1996; 271: 13882-13887Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), which exosites on thrombin for optimal activity, of the findings the of mechanisms by which thrombin The that an exosite 2 thrombin's interaction with fibrin also a which thrombin's activity it to utilize exosite ligands to thrombin, thereby it to by Exosite ligands also the to by thrombin binding to and the of thrombin, such as of which is mediated by exosite These exosite ligands with a to that of such as which the active site of thrombin, or hirudin and which exosite 1 and the active site 2002; PubMed Scopus Google Scholar).