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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1992 Jul 1;89(13):6040–6044. doi: 10.1073/pnas.89.13.6040

Antithrombotic effects of synthetic peptides targeting various functional domains of thrombin.

A B Kelly 1, J M Maraganore 1, P Bourdon 1, S R Hanson 1, L A Harker 1
PMCID: PMC49433  PMID: 1385867

Abstract

To determine in vivo functional roles for thrombin's structural domains, we have compared the relative antithrombotic and antihemostatic effects of (i) catalytic-site antithrombin peptide, D-Phe-Pro-Arg; (ii) exosite antithrombin peptide, the C-terminal tyrosine-sulfated dodecapeptide of hirudin; and (iii) bifunctional antithrombin peptide, a 20-mer peptide combining catalytic-site antithrombin peptide and exosite antithrombin peptide with a polyglycyl linker. All three peptides inhibited thrombin-mediated platelet aggregation and fibrin formation in vitro. In vivo thrombus formation was measured in real time as 111In-labeled platelet deposition and 125I-labeled fibrin accumulation on thrombogenic segments incorporated into chronic exteriorized arteriovenous access shunts in baboons. Under low flow conditions, the continuous infusion of peptides reduced thrombus formation onto collagen-coated tubing by half at doses (ID50) and corresponding concentrations (IC50) of 800 nmol per kg per min and 400 nmol/ml for catalytic-site antithrombin peptide, greater than 1250 nmol per kg per min and greater than 1500 mumol/ml for exosite antithrombin peptide, and 50 nmol per kg per min and 25 nmol/ml for bifunctional antithrombin peptide. Under arterial flow conditions, systemically administered bifunctional antithrombin peptide decreased thrombus formation in a dose-dependent manner for segments of collagen-coated tubing or prosthetic vascular graft ID50 and IC50 values of 120 nmol per kg per min and 15 nmol/ml; this dose also produced intermediate inhibition of hemostatic function [bleeding time, 21 +/- 3 min vs. 4.5 +/- 0.5 min (baseline values); P less than 0.001; activated partial thromboplastin time, 285 +/- 13 sec vs. 31 +/- 3 sec (baseline), P less than 0.001]. In contrast, thrombus formation onto segments of endarterectomized aorta was potently decreased by bifunctional antithrombin peptide with an ID50 value of 2.4 nmol per kg per min and an IC50 value of 0.75 nmol/ml, a systemic dose that failed to affect hemostasis. Thus, inhibiting both thrombin's catalytic and exosite domains increases antithrombotic potency by several orders of magnitude over the inhibition of either domain alone, particularly at sites of deep arterial injury.

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Selected References

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  1. Banner D. W., Hadváry P. Crystallographic analysis at 3.0-A resolution of the binding to human thrombin of four active site-directed inhibitors. J Biol Chem. 1991 Oct 25;266(30):20085–20093. [PubMed] [Google Scholar]
  2. Berliner L. J., Sugawara Y., Fenton J. W., 2nd Human alpha-thrombin binding to nonpolymerized fibrin-Sepharose: evidence for an anionic binding region. Biochemistry. 1985 Nov 19;24(24):7005–7009. doi: 10.1021/bi00345a038. [DOI] [PubMed] [Google Scholar]
  3. Bode W., Mayr I., Baumann U., Huber R., Stone S. R., Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467–3475. doi: 10.1002/j.1460-2075.1989.tb08511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cadroy Y., Horbett T. A., Hanson S. R. Discrimination between platelet-mediated and coagulation-mediated mechanisms in a model of complex thrombus formation in vivo. J Lab Clin Med. 1989 Apr;113(4):436–448. [PubMed] [Google Scholar]
  5. Cadroy Y., Maraganore J. M., Hanson S. R., Harker L. A. Selective inhibition by a synthetic hirudin peptide of fibrin-dependent thrombosis in baboons. Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1177–1181. doi: 10.1073/pnas.88.4.1177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chao B. H., Jakubowski J. A., Savage B., Chow E. P., Marzec U. M., Harker L. A., Maraganore J. M. Agkistrodon piscivorus piscivorus platelet aggregation inhibitor: a potent inhibitor of platelet activation. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8050–8054. doi: 10.1073/pnas.86.20.8050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davies M. J., Thomas A. C. Plaque fissuring--the cause of acute myocardial infarction, sudden ischaemic death, and crescendo angina. Br Heart J. 1985 Apr;53(4):363–373. doi: 10.1136/hrt.53.4.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gruentzig A. R., King S. B., 3rd, Schlumpf M., Siegenthaler W. Long-term follow-up after percutaneous transluminal coronary angioplasty. The early Zurich experience. N Engl J Med. 1987 Apr 30;316(18):1127–1132. doi: 10.1056/NEJM198704303161805. [DOI] [PubMed] [Google Scholar]
  9. Hanson S. R., Harker L. A. Interruption of acute platelet-dependent thrombosis by the synthetic antithrombin D-phenylalanyl-L-prolyl-L-arginyl chloromethyl ketone. Proc Natl Acad Sci U S A. 1988 May;85(9):3184–3188. doi: 10.1073/pnas.85.9.3184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Harker L. A., Hanson S. R. Experimental arterial thromboembolism in baboons. Mechanism, quantitation, and pharmacologic prevention. J Clin Invest. 1979 Aug;64(2):559–560. doi: 10.1172/JCI109494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Heras M., Chesebro J. H., Penny W. J., Bailey K. R., Badimon L., Fuster V. Effects of thrombin inhibition on the development of acute platelet-thrombus deposition during angioplasty in pigs. Heparin versus recombinant hirudin, a specific thrombin inhibitor. Circulation. 1989 Mar;79(3):657–665. doi: 10.1161/01.cir.79.3.657. [DOI] [PubMed] [Google Scholar]
  12. Hogg P. J., Jackson C. M. Fibrin monomer protects thrombin from inactivation by heparin-antithrombin III: implications for heparin efficacy. Proc Natl Acad Sci U S A. 1989 May;86(10):3619–3623. doi: 10.1073/pnas.86.10.3619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jakubowski J. A., Maraganore J. M. Inhibition of coagulation and thrombin-induced platelet activities by a synthetic dodecapeptide modeled on the carboxy-terminus of hirudin. Blood. 1990 Jan 15;75(2):399–406. [PubMed] [Google Scholar]
  14. Kelly A. B., Hanson S. R., Henderson L. W., Harker L. A. Prevention of heparin-resistant thrombotic occlusion of hollow-fiber hemodialyzers by synthetic antithrombin. J Lab Clin Med. 1989 Oct;114(4):411–418. [PubMed] [Google Scholar]
  15. Kelly A. B., Marzec U. M., Krupski W., Bass A., Cadroy Y., Hanson S. R., Harker L. A. Hirudin interruption of heparin-resistant arterial thrombus formation in baboons. Blood. 1991 Mar 1;77(5):1006–1012. [PubMed] [Google Scholar]
  16. Kotzé H. F., Lötter M. G., Badenhorst P. N., Heyns A. D. Kinetics of In-111-platelets in the baboon: I. Isolation and labelling of a viable and representative platelet population. Thromb Haemost. 1985 Jun 24;53(3):404–407. [PubMed] [Google Scholar]
  17. Lane D. A., Denton J., Flynn A. M., Thunberg L., Lindahl U. Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4. Biochem J. 1984 Mar 15;218(3):725–732. doi: 10.1042/bj2180725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lewis S. D., Lorand L., Fenton J. W., 2nd, Shafer J. A. Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII. Biochemistry. 1987 Dec 1;26(24):7597–7603. doi: 10.1021/bi00398a010. [DOI] [PubMed] [Google Scholar]
  19. Maraganore J. M., Bourdon P., Jablonski J., Ramachandran K. L., Fenton J. W., 2nd Design and characterization of hirulogs: a novel class of bivalent peptide inhibitors of thrombin. Biochemistry. 1990 Jul 31;29(30):7095–7101. doi: 10.1021/bi00482a021. [DOI] [PubMed] [Google Scholar]
  20. Maraganore J. M., Chao B., Joseph M. L., Jablonski J., Ramachandran K. L. Anticoagulant activity of synthetic hirudin peptides. J Biol Chem. 1989 May 25;264(15):8692–8698. [PubMed] [Google Scholar]
  21. Mirshahi M., Soria J., Soria C., Faivre R., Lu H., Courtney M., Roitsch C., Tripier D., Caen J. P. Evaluation of the inhibition by heparin and hirudin of coagulation activation during r-tPA-induced thrombolysis. Blood. 1989 Aug 15;74(3):1025–1030. [PubMed] [Google Scholar]
  22. Naski M. C., Fenton J. W., 2nd, Maraganore J. M., Olson S. T., Shafer J. A. The COOH-terminal domain of hirudin. An exosite-directed competitive inhibitor of the action of alpha-thrombin on fibrinogen. J Biol Chem. 1990 Aug 15;265(23):13484–13489. [PubMed] [Google Scholar]
  23. Pomerantz M. W., Owen W. G. A catalytic role for heparin. Evidence for a ternary complex of heparin cofactor thrombin and heparin. Biochim Biophys Acta. 1978 Jul 21;535(1):66–77. doi: 10.1016/0005-2795(78)90033-8. [DOI] [PubMed] [Google Scholar]
  24. Rydel T. J., Ravichandran K. G., Tulinsky A., Bode W., Huber R., Roitsch C., Fenton J. W., 2nd The structure of a complex of recombinant hirudin and human alpha-thrombin. Science. 1990 Jul 20;249(4966):277–280. doi: 10.1126/science.2374926. [DOI] [PubMed] [Google Scholar]
  25. Savage B., McFadden P. R., Hanson S. R., Harker L. A. The relation of platelet density to platelet age: survival of low- and high-density 111indium-labeled platelets in baboons. Blood. 1986 Aug;68(2):386–393. [PubMed] [Google Scholar]
  26. Schatz R. A. A view of vascular stents. Circulation. 1989 Feb;79(2):445–457. doi: 10.1161/01.cir.79.2.445. [DOI] [PubMed] [Google Scholar]
  27. Schneider P. A., Hanson S. R., Price T. M., Harker L. A. Confluent durable endothelialization of endarterectomized baboon aorta by early attachment of cultured endothelial cells. J Vasc Surg. 1990 Mar;11(3):365–372. [PubMed] [Google Scholar]
  28. Skrzypczak-Jankun E., Carperos V. E., Ravichandran K. G., Tulinsky A., Westbrook M., Maraganore J. M. Structure of the hirugen and hirulog 1 complexes of alpha-thrombin. J Mol Biol. 1991 Oct 20;221(4):1379–1393. [PubMed] [Google Scholar]
  29. Stone S. R., Hofsteenge J. Effect of heparin on the interaction between thrombin and hirudin. Eur J Biochem. 1987 Dec 1;169(2):373–376. doi: 10.1111/j.1432-1033.1987.tb13622.x. [DOI] [PubMed] [Google Scholar]
  30. Vu T. K., Hung D. T., Wheaton V. I., Coughlin S. R. Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation. Cell. 1991 Mar 22;64(6):1057–1068. doi: 10.1016/0092-8674(91)90261-v. [DOI] [PubMed] [Google Scholar]
  31. Vu T. K., Wheaton V. I., Hung D. T., Charo I., Coughlin S. R. Domains specifying thrombin-receptor interaction. Nature. 1991 Oct 17;353(6345):674–677. doi: 10.1038/353674a0. [DOI] [PubMed] [Google Scholar]
  32. Weitz J. I., Hudoba M., Massel D., Maraganore J., Hirsh J. Clot-bound thrombin is protected from inhibition by heparin-antithrombin III but is susceptible to inactivation by antithrombin III-independent inhibitors. J Clin Invest. 1990 Aug;86(2):385–391. doi: 10.1172/JCI114723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wilcox J. N., Smith K. M., Schwartz S. M., Gordon D. Localization of tissue factor in the normal vessel wall and in the atherosclerotic plaque. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2839–2843. doi: 10.1073/pnas.86.8.2839. [DOI] [PMC free article] [PubMed] [Google Scholar]

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