Thrombin exosite for fibrinogen recognition is partially accessible in ...

THEJOURNAL OF B I O ~ I CCHEMISTRY AL V d . 268, No. 23, Issue of August 15, pp. 16899-16902, 1993 0 1993 by The American Society for Bioehemistry and Molecular Biology, h e . Printed in U.S.A.

Communication

inactive precursor prothrombin. Upon stimulation, procoagulant thrombin is generated from prothrombin by a delicately controlled cascadeof zymogen activation reactions(3).The conversion of prothrombin to active thrombinhas been the subject of intense study (4-9). It has been shown that the minimum requirement for the expression of the proteolytic active site is (Received for publication, April 27, 1993, and in revised form, May 25, 1993) the cleavage of the Arg323-Ile324 peptide bond producing the activation intermediate meizothrombin(4). The full expression Feng NiSI, Qing NingS, CraigM. Jacksonn, and of the clotting activity, however, requiresa further cleavage at John W. Fenton Ill1 peptide bondArg274-Thr275 connecting the activation fragment From the $Biotechnology Research Institute, National 2 or a-thrombin (4, 1.2 with the protease domain prethrombin Research Council of Canada, Montreal, Quebec H4P 9). The protease domains as a result also bind specifically to 2R2, Canada, the Mmerican Red Cross Blood Services, fibrinogen, presumably via the macromolecular substrate (fiSouth Eastern Michigan Region, Detroit, Michigan brinogen) recognition exosite (1,2, 7, 10). Thus, it appears that 48232, a n d the IWew York State Departmentof Health, Wadsworth Center for Laboratories and Research, activation fragments 1 a n d 2 might obstruct the fibrinogen Albany, New York 12201 recognition exosite, resulting in reduced clotting activity for meizothrombin. The procoagulanta-thrombinis produced by the proThe properties of t h e fibrinogen recognition exositein thromteolytic cleavages of a minimum of two peptide bonds bin have been investigated with hirudin (10-12) and with hiruArg274-Th1.276and ArgS23-Ile324in prothrombin.The ArgS23-Ile324 cleavage is required for the expression of din tail peptides (13-151, also referred to as hirugen (16),tarspecifically for this thrombin exosite (17-19). the active site of thrombin (Morita, T., Iwanaga, S. Su- geted zuki, T. (1976) J . Biochern. (Zbkyo) 79,1089-1108; Hib- Interestingly, it has been shown that hirugen binds to meizobard, L. S., Nesheim, M. E., and Mann, K. G. (1982) Bio- thrombin, as well as prethrombin 2 and a-thrombin, but not to chemistry 21,2285-2292).It is not yet clear to what prothrombin (20). This finding led to the conclusion that t h e extent the proteolytic events are responsible for expos- hirugen or the fibrinogen binding exositeis not expressed being protein recognition exosites on thrombin. We em- fore the cleavages of either the Arg274-Thr275 or t h e ployed high resolution NMR spectroscopy to examine Ile324 peptide bond in prothrombin. This conclusion, on one interactions ofprothrombinandthrombin with syn- hand, is consistent with the results from previous studies emthetic hirudin peptides targeted toward the fibrinogen ploying the protein substrate fibrinogen (7).the Onother hand, recognition exosite of thrombin. The hirudin tail syn- all these studies utilized methods that are sensitive only to thetic analogues (acetyl-Asp66-Phe-G1u-Glu-Ile-Pro-Glu(VM) range. There tight binding interactions in the micromolar Glu-~-Leu-Glne5/Glys6-OH) exhibited similar NMFt re- is a possibility of weaker but specific binding between prolaxation enhancements (line broadening patterns and thrombin and hirudin peptides that escaped previous detection. transferred nuclear Overhauser effects) with human We have undertaken high resolution NMR studies on the inprothrombin as with human a-thrombin,indicating that both proteins bind the peptide in a similar manner. The teraction of hirudin tail peptides with both prothrombin and protein-induced relaxation enhancements are specific thrombin. Our results suggest that the fibrinogen recognition at least partially accessible to peptide to the interaction of the hirudin peptides with the fi- exosite in prothrombin is that brinogen recognition exosite of thrombin since no sig- binding witha bound conformation indistinguishable from recognized by thrombin. nificant effects were observed with either human serum

Thrombin Exosite for Fibrinogen Recognition Is Partially Accessible in Prothrombin*

albumin or with human y-thrombin, which has an impaired recognition exosite. The binding affinities were determined from N M R relaxation time measurements, which gave approximate Kd values of 500 p~ and 4 0 0 1.1~ for prothrombin and a-thrombin,respectively. Since the hirudin tail fragment binds specifically to the fibrinogen recognition exosite in a-thrombin, this exosite appears to be partially accessible in prothrombin in a proenzyme form. Thrombin isthe central regulatory enzymein hemostasis (1, 2). During normal circulation, it exists almost entirely as its

* This work was supported in part by a research grant from CibaGeigy Canada Ltd. and by National Institutes of Health Grant HL13160-22 (to J. W. F.). This is NRCC Publication 33666. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisernent" in accordance with 18 U.S.C. Section 1734 solely toindicate this fact. B To whom correspondence should be addressed: Biotechnology Research Institute, National Research Council of Canada, 6100Royalmount Ave., Montreal, Quebec H4P 2R2, Canada. Tel.: 514-496-6729; Fax: 514-496-5143.

EXPERIMENTALPROCEDURES Peptides and Proteirrs-The hirudin peptides a~etyl-Asp~~-Phe-GluGlu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln65-OH (peptide P24) and a substituted analog, acetyl-Asp55-Phe-Glu-Glu-Ile-Pro-Glu-Glu-~-Leu-~-OH (peptide P35), were synthesized as described previously (14, 19). Human serum albumin was obtained from Sigma as a dried powder and used without further purification. Human a-thrombin was prepared as described previously (21). Stock solutions of thrombin (0.64-1.25 mg/ ml) were stored in a deep freeze (