An Assay of the Antithrombotic Action of Warfarin: Its Correlation with the Inhibition of Stasis Thrombosis in Rabbits

Summary

No assay of the antithrombotic action of warfarin has been available. Experiments were performed to determine whether Xa inhibitory activity - the reaction rate between activated factor X (Xa) and antithrombin III - could serve this function. 105 warfarin-treated patients demonstrated a significant 18% increase in Xa inhibitory activity compared to 51 controls, p <0.001, without any correlation between this activity and the prothrombin times in the treated patients. A similar increase in Xa inhibitory activity was obtained in rabbits treated with 2 mg of warfarin per day compared to control animals, p <0.001. Employing an assay which routinely produced venous thrombosis after clotting proteases were infused into warfarin-treated and control rabbits, three observations were made. 1. The extent of stasis thrombosis induced by injection of thrombin, Xa or activated factor IX, was significantly reduced in warfarin-treated rabbits compared to control animals, independent of alteration in the four established vitamin K-dependent zymogens. 2. In the rabbit, significant changes in prothrombin times and prothrombin and factor X activities preceded by 5 days both the increase in Xa inhibitory activity and the antithrombotic effect which became significant on the sixth day. 3. The correlation between Xa inhibitory activity of warfarin-treated rabbits and the extent of stasis thrombosis induced by Xa was significant, p<0.05. Xa inhibitory activity is one measure of the antithrombotic action of warfarin.

• References

• 1 Abildgaard U, Fagerhol MK, Egeberg O. 1970; Comparison of progressive antithrombin activity and the concentration of 3 thrombin inhibitors in human plasma. Scandinavian Journal of Clinical and Laboratory Investigation 26: 349
• 2 Alexander B, Colman R. 1960; Evidence for a new serum thromboplastic factor. Thrombosis et Diathesis Haemorrhagica (Suppl.) 4: 66
• 3 Bachmann F, Duckert F, Roller F. 1958; The Stuart-Prower factor assay and its clinical significance. Thrombosis et Diathesis Haemorrhagica 2: 24
• 4 Biggs R, Denson KW E, Akman R, Borrett R, Hadden M. 1970; Antithrombin III, antifactor Xa and heparin. British Journal of Haemotology 19: 283
• 5 Boxer GJ, Bang NU, Ferroni JW, Benson MD. 1977; Plasma amyloid P-component inhibits heparin activity. Blood Suppl (Suppl. 01) 50: 260
• 6 Breckenridge A. 1978; Oral anticoagulant drugs: Pharmacokinetic aspects. Seminars in Hematology 15: 19
• 7 Carlisle TL, Shah DV, Schlegel R, Suttie JW. 1975; Plasma abnormal prothrombin and microsomal prothrombin in various species. Proceedings of the Society of Experimental Biology and Medicine 148: 140
• 8 Coon WW, Willis III PW. 1972; Thromboembolic complications during anticoagulant therapy. Archives of Surgery 105: 209
• 9 Davis BJ. 1964; Disc electrophoresis-II: Method and application to human serum proteins. Annals of New York Academy of Science 121: 404
• 10 Discipio RG, Hermodson MA, Yates SG, Davie EW. 1977; A comparison of human prothrombin Factor IX (Christmas factor), Factor X (Stuart factor), and protein S. Biochemistry 16: 698
• 11 Domenet JG, Evans DW, Stephenson H. 1966; Anticoagulants in congestive heart failure. British Medical Journal 4: 866
• 12 Gitel SN, Stephenson RC, Wessler S. 1977; a In vitro and in vivo correlation of clotting protease activity: Effect of heparin Proceedings of the. National Academy of Sciences (USA) 74: 3028
• 13 Gitel SN, Stephenson RC, Wessler S. 1978; The activated factor X-antithrombin III reaction rate: A measure of the increased thrombotic tendency induced by estrogen-containing oral contraceptives in rabbits. Haemostasis 7: 10
• 14 Gitel SN, Wessler S. 1975; Plasma antithrombin III: A quantitative assay of biological activity. Thrombosis Research 7: 5
• 15 Gitel SN, Wessler S, Medina VM. 1977; b The rabbit as an animal model for the activated factor X-antithrombin III-heparin reaction. Circulation Research 41: 187
• 16 Henstell HH, Kugerman M. 1967; The nature of heparin antithrombin action. Thrombosis et Diathesis Haemorrhagica 18: 167
• 17 Hjort P, Rappaport SL, Owren PA. 1955; A simple, specific one-stage prothrombin assay using Russell’s viper venom in cephalin suspension. Journal of Laboratory and Clinical Medicine 46: 89
• 18 Hurn M, Barker NW, Mann FD. 1947; Variations in prothrombin and antithrombin following the administration of Dicumarol. American Journal of Clinical Pathology 17: 111
• 19 Kisiel W, Ericsson LH, Davie EW. 1976; Proteolytic activation of protein C from bovine plasma. Biochemistry 15: 4893
• 20 Roller F. 1959. What is the present status of Factor X. In: Brinkhous KM. (ed) Hemophilia and Other Hemorrhagic. States University of North Carolina Press; Chapel Hill: p 231
• 21 Krenkel JK. 1969; Introduction: symposium on the choice of animal models for the study of disease processes in man. Federation Proceedings 28: 160
• 22 Laemmli UK. 1970; Cleavage of the structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680
• 23 Magnusson S, Sottrup-Jensen L, Peterson TE. 1974; Primary structure of the vitamin K-dependent part of prothrombin. FEBS Letters 44: 189
• 24 Nelsestuen GL, Zytkovicz TH, Howard JB. 1974; The mode of action of vitamin K: Identification of y-carboxyglutamic acid as a component of prothrombin. Journal of Biological Chemistry 249: 6347
• 25 Ødegard OR, Teien AN. 1976; Antithrombin III, heparin cofactor and antifactor Xa in a clinical material. Thrombosis Research 8: 173
• 26 Quick AJ. 1937; The coagulation defect in the sweet clover disease and the hemorrhagic chick disease of dietary origin. A consideration of the source of prothrombin American Journal of Physiology 118: 260
• 27 Rosenberg RD. 1977; Chemistry of the hemostatic mechanism and its relationship to the action of heparin. Federation Proceedings 36: 10
• 28 Sagar S, Stamatahs JD, Higgins AF, Nairn D, Maffei FH, Thomas DP, Kakkar VV. 1976; a Efficacy of low-dose heparin in prevention of extensive deep-vein thrombosis in patients undergoing total-hip replacement. Lancet 1: 1151
• 29 Sagar S, Stamatakis JD, Thomas DP, Kakkar VV. 1976; b Oral contraceptives, antithrombin III activity, and postoperative deep vein thrombosis. Lancet 2: 509
• 30 Seegers WH. 1968; Antithrombin as proteinase inhibitor. Annals of the New York Academy of Science 146: 593
• 31 Spaet TH. 1959. The present status of Factor X. In: Brinkhous KM. (ed) Hemophilia and Other Hemorrhagic. States University of North Carolina Press; Chapel Hill: p 240
• 32 Stenflo J. 1976; A new vitamin K-dependent protein: purification from bovine plasma and preliminary characterization. Journal of Biological Chemistry 251: 355
• 33 Stenflo J, Fernlund P, Egan W, Roefstroff P. 1974; Vitamin K-dependent modifications of glutamic acid residues in prothrombin. Proceedings of the National Academy of Sciences (USA) 71: 2730
• 34 Stenflo J, Suthe JW. 1977. Vitamin K-dependent formation of γ-carboxyglutamic acid. In: Snell EE, Boyer PD, Meister A, Richardson CC. (eds) Annual Reviews of Biochemistry. Annual Reviews Inc; Palo Alto, CA: p 157
• 35 Sutoe JW, Jackson CM. 1977; Prothrombin structure, activation, and biosynthesis. Physiological Reviews 57: 1
• 36 Wessler S, Ballon JD, Katz JH. 1957; Studies in intravascular coagulation: V. A distinction between the anticoagulant and antithrombotic effects of Dicumarol. New England Journal of Medicine 256: 1223
• 37 Wessler S, Gitel SN. 1975. Biochemical rationale for low-dose heparin in deep venous thrombosis. In: Fratantoni J, Wessler S. (eds) Prophylactic Therapy for Deep Venous Thrombosis and Pulmonary Embolism. U S Government Printing Office; Washington, D C: p 177
• 38 Wessler S, Gitel SN, Wan LS, Pasternack BS. 1976; Estrogen-containing oral contraceptive agents: A basis for their thrombogenicity. Journal of the American Medical Association 236: 2179
• 39 Wessler S, Reimer SM, Sheps MC. 1959; Biologic assay of thrombosis-inducing activity in human serum. Journal of Applied Physiology 14: 943
• 40 Wilcoxon F. 1947; Probability tables for individual comparisons in ranking methods. Biometrics 3: 119
• 41 Witte S, Dirnberger P. 1955; Die Antithromboischen Gerinnungsfaktoren bei Thrombosekrankheiten. Deutsches Archiv fur Klinische Medizin 202: 184
• 42 Yin ET, Eisenkramer L, Butler JV. 1975. Heparin interaction with activated Factor X and its inhibitor. In: Bradshaw RA, Wessler S. (eds) Heparin: Structure, Function and Clinical Implications. Plenum Press; New York: p 239
• 43 Yin ET, Wessler S. 1968; Bovine thrombin and activated Factor X: Separation and purification. Journal of Biological Chemistry 243: 112
• 44 Yin ET, Wessler S, Stoll PJ. 1971; a Identity of plasma activated Factor X inhibitor with antithrombin III and heparin cofactor. Journal of Biological Chemistry 246: 3712
• 45 Yin ET, Wessler S, Stoll PJ. 1971; b Biological properties of the naturally occurring plasma inhibitor to activated Factor X. Journal of Biological Chemistry 246: 3703
• 46 Zytkovicz TH, Nelsestuen GL. 1975; ³H Diborane reduction of vitamin K-dependent calcium-binding proteins: Identification of a unique amino acid. Journal of Biological Chemistry 250: 2968