Fibrolase and Its Evolution to Clinical Trials: A Long and Winding Road

  • Francis S. Markland
  • Stephen Swenson


Fibrolase is the fibrinolytic proteinase isolated from Agkistrodon contortrix contortrix (southern copperhead snake) venom. The enzyme was purified by a three-step HPLC procedure and was shown to be homogeneous by standard criteria. The purified enzyme is inhibited by EDTA and other chelating agents and is a zinc metalloproteinase containing one mole of zinc per molecule. The enzyme is also rapidly inhibited by alpha2-macroglobulin (α2 M). Fibrolase is composed of 203 amino acids with a blocked amino-terminus due to cyclization of the terminal Gln residue. The enzyme is a direct-acting thrombolytic agent and does not rely on plasminogen for clot dissolution. Fibrolase rapidly cleaves the A(α)-chain of fibrinogen and the B(β)-chain at a slower rate; it has no activity on the γ-chain. The enzyme exhibits the same specificity with fibrin. Fibrolase was shown to have very effective thrombolytic activity in a reoccluding carotid arterial thrombosis model in the canine. A recombinant version of the enzyme was made in yeast by Amgen, Inc. (Thousand Oaks, CA, USA) and called alfimeprase. Alfimeprase is identical to fibrolase except for a two amino acid truncation at the amino-terminus and the insertion of a new amino-terminal amino acid in the truncated protein; these changes lead to a more stable enzyme for prolonged storage. Twenty years after it was first purified alfimeprase was taken into clinical trials by Nuvelo, Inc. (San Carlos, CA), which licensed the enzyme from Amgen. Alfimeprase was successful in Phase I and II clinical trials for peripheral arterial occlusion (PAO) and central venous access device (CVAD) occlusion. However, in Phase III trials alfimeprase did not meet the expected end points in either PAO or CVAD occlusion and in a Phase II stroke trial and Nuvelo dropped further development in 2008.


Snake Venom Clot Lysis Fibrinolytic Enzyme Anisoylated Plasminogen Streptokinase Activator Complex Orphan Drug Status 
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  1. Ahmed, N.K., Tennant, K.D., Markland, F.S., Lacz, J.P., 1990. Biochemical characteristics of fibrolase, a fibrinolytic protease from snake venom. Haemostasis 20, 147–154.PubMedGoogle Scholar
  2. Bajwa, S.S., Kirakossian, H., Reddy, K.N., Markland, F.S., 1982. Thrombin-like and fibrinolytic enzymes in the venoms from the Gaboon viper (Bitis gabonica), eastern cottonmouth moccasin (Agkistrodon p. piscivorus) and southern copperhead (Agkistrodon c. contortrix) snakes. Toxicon 20, 427–432.PubMedCrossRefGoogle Scholar
  3. Bolger, M.B., Swenson, S., Markland, F.S., 2001. Three-dimensional structure of fibrolase, the fibrinolytic enzyme from southern copperhead venom, modeled from the X-ray structure of adamalysin II and atrolysin C. AAPS Pharm. Sci. 3, E16.CrossRefGoogle Scholar
  4. Burch, R.M., Farmer, S.G., Steranka, L.R., 1990. Bradykinin receptor antagonists. Med. Res. Rev. 10, 237–269.PubMedCrossRefGoogle Scholar
  5. Cummings-Winfield, C., Mushani-Kanji, T., 2008. Restoring patency to central venous access devices. Clin. J. Oncol. Nurs. 12, 925–934.PubMedCrossRefGoogle Scholar
  6. Deitcher, S.R., Funk, W.D., Buchanan, J., Liu, S., Levy, M.D., Toombs, C.F., 2006. Alfimeprase: a novel recombinant direct-acting fibrinolytic. Expert Opin. Biol. Ther. 6, 1361–1369.PubMedCrossRefGoogle Scholar
  7. Deitcher, S.R., Toombs, C.F., 2005. Non-clinical and clinical characterization of a novel acting thrombolytic: alfimeprase. Pathophysiol. Haemost. Thromb. 34, 215–220.PubMedCrossRefGoogle Scholar
  8. Didisheim, P., Lewis, J.H., 1956. Fibrinolytic and coagulant activities of certain snake venoms and proteases. Proc. Soc. Exp. Biol. Med. 93, 10–13.PubMedGoogle Scholar
  9. Guan, A.L., Retzios, A.D., Henderson, G.N., Markland, F.S., 1991. Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake (Agkistrodon contortrix contortrix). Arch. Biochem. Biophys. 289, 197–207.PubMedCrossRefGoogle Scholar
  10. Han, S.M., Weaver, F.A., Comerota, A.J., Perler, B.A., Joing, M., 2010. Efficacy and safety of alfimeprase in patients with acute peripheral arterial occlusion (PAO). J. Vasc. Surg. 51, 600–609.Google Scholar
  11. Hong, T.T., Huang, J., Lucchesi, B.R., 2006. Effect of thrombolysis on myocardial injury: recombinant tissue plasminogen activator vs. alfimeprase. Am. J. Physiol. Heart Circ. Physiol. 290, H959–H967.PubMedCrossRefGoogle Scholar
  12. Kornalik, F., 1966. The influence of snake venoms on fibrinogen conversion and fibrinolysis. Mem. Inst. Butantan Simp. Int. 33, 179.Google Scholar
  13. Lee, E.K., 2002. Alteplase use for prevention of central line occlusion in a preterm infant. Ann. Pharmacother. 36, 272–274.PubMedCrossRefGoogle Scholar
  14. Loayza, S.L., Trikha, M., Markland, F.S., Riquelme, P., Kuo, J., 1994. Resolution of isoforms of natural and recombinant fibrolase, the fibrinolytic enzyme from Agkistrodon contortrix contortrix snake venom, and comparison of their EDTA sensitivities. J. Chromatogr. B. Biomed. Appl. 662, 227–243.PubMedCrossRefGoogle Scholar
  15. Lu, A., Kurosawa, Y., Luskey, K., Pyne-Geithman, G., Caudell, D., Clark, J., 2009. Hemorrhagic profile of the fibrinolytic alfimeprase after ischemia and reperfusion. Neurol. Res. 31, 209–214.PubMedCrossRefGoogle Scholar
  16. Markland, F.S., Friedrichs, G.S., Pewitt, S.R., Lucchesi, B.R., 1994. Thrombolytic effects of recombinant fibrolase or APSAC in a canine model of carotid artery thrombosis. Circulation 90, 2448–2456.PubMedCrossRefGoogle Scholar
  17. Markland, F.S., Morris, S., Deschamps, J.R., Ward, K.B., 1993. Resolution of isoforms of natural and recombinant fibrinolytic snake venom enzyme using high performance capillary electrophoresis. J. Liquid Chromatog. 16, 2189–2201.CrossRefGoogle Scholar
  18. Markland, F.S., Reddy, K.N.N., Guan, L.F., 1988. Purification and characterization of a direct-acting fibrinolytic enzyme from southern copperhead venom, in: Pirkle, H., Markland, F.S. (Eds.), Hemostasis and Animal venoms (vol. 7). Marcel Dekker, New York, pp. 173–189.Google Scholar
  19. Moise, M.A., Kashyap, V.S., 2008. Alfimeprase for the treatment of acute peripheral arterial occlusion. Expert Opin. Biol. Ther. 8, 683–689.PubMedCrossRefGoogle Scholar
  20. Moll, S., Kenyon, P., Bertoli, L., De Maio, J., Homesley, H., Deitcher, S.R., 2006. Phase II trial of alfimeprase, a novel-acting fibrin degradation agent, for occluded central venous access devices. J. Clin. Oncol. 24, 3056–3060.PubMedCrossRefGoogle Scholar
  21. No author, 2008. Alfimeprase. Drugs R D 9, 185–190.Google Scholar
  22. Ouriel, K., Cynamon, J., Weaver, F.A., Dardik, H., Akers, D., Blebea, J., Gruneiro, L., Toombs, C.F., Wang-Clow, F., Mohler, M., Pena, L., Wan, C.Y., Deitcher, S.R., 2005. A phase I trial of alfimeprase for peripheral arterial thrombolysis. J. Vasc. Interv. Radiol. 16, 1075–1083.PubMedCrossRefGoogle Scholar
  23. Ouriel, K., Veith, F.J., Sasahara, A.A., 1996. Thrombolysis or peripheral arterial surgery: phase I results. TOPAS Investigators. J. Vasc. Surg. 23, 64–73.PubMedCrossRefGoogle Scholar
  24. Randolph, A., Chamberlain, S.H., Chu, H.L., Retzios, A.D., Markland, F.S., Masiarz, F.R., 1992. Amino acid sequence of fibrolase, a direct-acting fibrinolytic enzyme from Agkistrodon contortrix contortrix venom. Protein Sci. 1, 590–600.PubMedCrossRefGoogle Scholar
  25. Rau, J.C., Beaulieu, L.M., Huntington, J.A., Church, F.C., 2007. Serpins in thrombosis, hemostasis and fibrinolysis. J. Thromb. Haemost. 5(Suppl 1), 102–115.PubMedCrossRefGoogle Scholar
  26. Reddy, G.K., 2006. Clinical utility of novel agents in the treatment of central venous catheter occlusion. Support Cancer Ther. 3, 135–139.PubMedCrossRefGoogle Scholar
  27. Retzios, A.D., Markland, F.S., 1990. HPLC-based two-step purification of fibrinolytic enzymes from the venom of Agkistrodon contortrix contortrix and Agkistrodon piscivorus conanti. Protein Expr. Purif. 1, 33–39.PubMedCrossRefGoogle Scholar
  28. Retzios, A.D., Markland, F.S., 1988. A direct-acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix: effects on various components of the human blood coagulation and fibrinolysis systems. Thromb. Res. 52, 541–52.PubMedCrossRefGoogle Scholar
  29. Swenson, S., Toombs, C.F., Pena, L., Johansson, J., Markland, F.S., 2004. Alpha-fibrinogenases. Curr. Drug. Targets Cardiovasc. Haematol. Disord. 4, 417–435.PubMedCrossRefGoogle Scholar
  30. Toombs, C.F., 2001. Alfimeprase: pharmacology of a novel fibrinolytic metalloproteinase for thrombolysis. Haemostasis 31, 141–147.PubMedGoogle Scholar
  31. Trikha, M., Schmitmeier, S., Markland, F.S., 1994. Purification and characterization of fibrolase isoforms from venom of individual southern copperhead (Agkistrodon contortrix contortrix) snakes. Toxicon 32, 1521–1531.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  1. 1.Department of Biochemistry and Molecular BiologyUniversity of Southern California, Keck School of Medicine, Cancer Research Laboratory #106Los AngelesUSA
  2. 2.USC/Norris Comprehensive Cancer CenterUniversity of Southern California, Keck School of Medicine, Cancer Research Laboratory #106Los AngelesUSA

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