Factor V and Thrombin in the Intrinsic Clotting System^[*]

Summary

Allegedly factor V 1-stage bioassays and an established mode of 2-stage thrombingeneration tests are performed with three alternative prime activators : (A) thrombokinase; (B) Russell’s viper venom (Stypven); (C) tissue thromboplastin (Simplastin), each with adequate cofactors. Study of effects of thrombin and factor V mixtures or preincubates shows a marked apparent potentiation in the 1-stage tests, but no real evidence of such in the 2-stage methods, although these are equally sensitive to changes in factor V activity. Cited (13) new observations in the 1-stage experiments record: (a) that no preincubation of thrombin and factor V is necessary; (b) that thrombin-treated factor V (= Vt) potentiates anew with a second thrombin addition ; and (c) that Vt + thrombin again shows a shift in the Sephadex G-200 elution pattern similar to the difference between the original V and Vt. These facts all constitute a convincing refutation of the theory that factor V is activated by thrombin to a postulated ‘Va’. What is obviously needed is a complete reinterpretation of the uniquely 1-stage results. A novel approach is based on testings of selective combinations of the 5 basic components (prothrombin, thrombokinase, Ca⁺⁺, phospholipid, and factor V) required for thrombin generation by <1 ìg/ml thrombokinase. The results permit the conclusion that the true explanation is to be found in activities of the prime activator, especially thrombokinase, as controlled by availabilities of the 3 cofactors, namely, factor V, Ca⁺⁺ions, and phospholipid. Factor V was previously shown to be a determinant of thrombokinase activity during thrombin generation. In no way should the evidence be misinterpreted as any V → Va activation. A new enzymic and colloidal clotting mechanism (12) is reaffirmed, in which factor V has a dual role.

^* Supported by USPHS Research Grant No. HE-01510.

• References

• 1 Borckgrevink C. F, Pool J. G, Stomorken H. A new assay for factor V (proaccelerin-accelerin) using Russell’s viper venom. J. Lab. clin. Med 55: 625 1960;
• 2 Breckenridge R. T, Ratnoff O. D. The activation of proaccelerin by bovine thrombokinase (with notes on the antithrombic activity of phenylmethylsulfanylfluoride). Blood 27: 527 1966;
• 3 Davie E. W, Ratnoff O. D. Waterfall sequence for intrinsic blood clotting. Science 145: 1310 1964;
• 4 Esnouf M. P, Macfarlane R. G. Enzymology and the blood clotting mechanism. Advanc. Enzymol 30: 255 1968;
• 5 Ferguson J. H. Lipoids and Blood Platelets with Reference to Blood Coagulation and the Hemorrhagic Diseases. Univ. N. Carolina Press; Chapel Hill: 1960
• 6 Ferguson J. H. Methods of studying new clotting factors. Thrombos. Diathes. haemorrh. (Stuttg.) Suppl. ad 04: 226 1960;
• 7 Ferguson J. H. Thromboplastic enzymes. Fed. Proc 23: 762 1964;
• 8 Ferguson J. H. Thromboplastic enzymes, thrombokinase, thromboplastin, and blood clotting: a critical review. Hemat. Rev 01: 69 1968;
• 9 Ferguson J. H. Biosasays of blood clotting mechanisms: basic biochemical and clinical significance. Hemat. Rev. (In press.)
• 10 Ferguson J. H, Ennis E. GW. Enzymes and blood clotting. II, Autprothrombin C, Russell’s viper venom (Stypven), and trypsin. Thrombos. Diathes. haemorrh. (Stuttg) 09: 62 1963;
• 11 Ferguson J. H, Ennis E. GW. One-stage ‘factor V’ bioassays: specific and other determinants in blood-clotting test systems. Proc. Soc. exp. Biol. (N. Y) 130: 486 1969;
• 12 Ferguson J. H, Ennis E. GW, Iatridis P. G, White N. B. Basic aspects of blood clotting: thrombokinase and cofactors in the conversion of prothrombin to thrombin. Thrombos. Diathes. haemorrh. (Stuttg) 18: 647 1967;
• 13 Ferguson J. H, Hitsumoto S, Ennis E. GW, White N. B. Factor V and thrombin in the extrinsic clotting system. Proc. Soc. exp. Biol. (N. Y) 130: 440 1969;
• 14 Ferguson J. H, Marcus A. J, Robinson A. J. Purified phospholipids and blood coagulation. Blood 22: 19 1963;
• 15 Ferguson J. H, Wilson E. G, Iatridis S. G, Rierson H. A, Johnston B. R. Enzymes and blood clotting. I. Trypsin as an accessory factor. J. clin. Invest 39: 1942 1960;
• 16 Kerwin D. M, Milstone J. H. Removal of thrombokinase from commercial thrombin. Thrombos. Diathes. haemorrh. (Stuttg) 17: 247 1967;
• 17 Lewis J. H, Ferguson J. H. Thrombin formation. I. The role of calcium, Ac-globulin, and tissue thromboplastin. J. clin. Invest 27: 778 1948;
• 18 Lewis J. H, Ferguson J. H, Fresh J. W, Zucker M. B. Primary hemorrhagic diseases. J. Lab. clin. Med 49: 211 1957;
• 19 Lewis M. L, Ware A. G. A simmple procedure for separation of prothrombin and accelerator globulin from citrated human plasma. Proc. Soc. Exp. Biol. (N. Y) 84: 636 1953;
• 20 Lewis M. L, Ware A. G. A one-stage determination of accelerator globulin. Proc. Soc. exp. Biol. (N. Y) 84: 640 1953;
• 21 Macfarlane R. G. An enzyme cascade in the blood clotting mechanism, and its function as a biological amplifier. Nature (Lond) 202: 498 1964;
• 22 Milstone J. H. Thrombokinase as prime activator of prothrombin: historical perspectives and present status. Fed. Proc 23: 742 1964;
• 23 Milstone J. H, Oulianoff N, Milstone V. K. Outstanding characteristics of thrombokinase from bovine plasma. J. gen. Physiol 47: 315 1963;
• 24 Prentice C. R. M, Ratnoff O. D, Breckenridge R. T. Experiments on the nature of the prothrombin-converting principle: alteration of proaccelerin by thrombin. Brit. J. Haemat 13: 898 1967;
• 25 Quick A. J, Stefanini M. The concentration of labile factor of the prothrombin complex, in human, dog, and rabbit blood: its significance in the determination of prothrombin activity. J. Lab. clin. Med 33: 819 1948;
• 26 Rapaport S. I, Hjort P. F, Patch M. J. Rabbit factor V: different effects of thrombin and venom, a source of error in bioassay. Amer. J. Physiol 211: 1477 1966;
• 27 Surgenor D. M, Wilson N. A, Henry A. S. Factor V from human plasma. Thrombos. Diathes. haemorrh. (Stuttg) 05: 1 1960;
• 28 Ware A. G, Seegers W. H. Plasma accelerator globulin: partial purification, quantitative determination, and properties. J. biol. Chem 172: 699 1948;
• 29 White N. B, Ferguson J. H, Hitsumoto S. Preparation and activity of factor Y from small quantities of bovine plasma. Amer. J. med. Sci 255: 143 1968;
• 30 Wright I. S. Nomenclature of blood clotting factors. Thrombos. Diathes. haemorrh. (Stuttg) 03: 435 1959;
• 31 Yin E. T, Wessler S. Bovine thrombin and activated factor X. J. biol. Chem 243: 112 1948;