Substitution of Lipids with Bile Salts in the Formation of Thrombin^[*]

Summary

The generation of thrombin was studied in an activating system consisting of purified thrombin zymogens, purified autoprothrombin C, purified Ac-globulin, lipid or bile salts, and calcium chloride. With the concentration of calcium ions and pH fixed, the effect of varying the other three procoagulants was studied. Bile salts were effective substitutes for lipids in a concentration where micelles form. The approximate effectiveness from highest to lowest was: conjugated sodium salt of taurocholic acid, sodium cholate, sodium deoxycholate. Sodium dehydrocholate was ineffective. Autoprothrombin C is the enzyme for thrombin formation. For accelerating its activity best results were obtained with the simultaneous presence of optimal concentrations of calcium ions, Ac-globulin and lipids or bile salts. Reducing any one of the three to zero concentration decreased the rate and yield of thrombin generation.

The form in which the zymogen is used was found to be important. Prothrombin complex, DE AE-prothrombin and prethrombin were studied. Each substrate has its peculiar requirements for yielding thrombin. Prothrombin complex and DEAE-pro-thrombin activated far more rapidly and required 10 times less autoprothrombin C than prethrombin. The yield of thrombin from these substrates was also higher than from prethrombin. DE AE-prothrombin required the least amount of lipid. For the bile salts the required concentrations were nearly always the same from one substrate to another. To a certain extent Benadryl could also be substituted for lipids. In association with rapid thrombin generation from DE AE-prothrombin the Ac-globulin and autoprothrombin C Avere represented in approximately a 6:1 molar ratio. As compared with the weight of the enzyme large amounts of Ac-globulin and cholate were required.

DE AE-prothrombin was readily made refractory to the two-stage analytical reagents with purified platelet factor 3 and calcium ions. Combinations of sodium cholate and phosphatidyl serine were also effective, but either one alone was ineffective.

^* This investigation was supported by a research grant HE-03424-12 from the National Heart Institute, National Institutes of Health, U. S. Public Health Service.

^** North Atlantic Treaty Organization Postdoctoral Fellow from Germany. Present address: Medizinische Klinik der Medizinischen Hochschule, Hannover.

• References

• 1 Seegers W. H. Blood clotting mechanisms: Three basic reactions. Ann. Rev. Physiol. 31: 269 1969;
• 2 Seegers W. H. Blood clotting enzymology. Academic Press; New York and London: 1967
• 3 Seegers W. H, McCoy L, Marciniak E. Blood clotting enzymology: Three basic reactions. Clin. Chem. 14: 97-115 1968;
• 4 Silver M. J, Turner D. L, Rodalewicz I, Giordano N, Holburn R, Herb S. F, Luddy F. E. Evaluation of activity of phospholipids in blood coagulation in vitro. Thrombos. Diathes. haemorrh. (Stuttg.) 10: 164-189 1963;
• 5 Daemen F. J. M, van Arkel C, Hart H. C, van der Drift C, van Deenen L. L. M. Activity of synthetic phospholipids in blood coagulation. Thrombos. Diathes. haemorrh. (Stuttg.) 13: 194-217 1965;
• 6 Garagnani A, Facchini G. Arch. Path. clin. Med. 33: 203 1956; Quoted from E. F. Curragh and D. T. Elmore.
• 7 Curragh E. F, Elmore D. T. Kinetics and mechanism of catalysis by proteolytic enzymes. Biochem. J. 93: 163-171 1964;
• 8 Stroeder J, Kuenzer W, Thoenes G, Hossbein Z. Gallensaures Natrium und Blutgerinnung. Klin. Wschr. 42: 295 1964;
• 9 Bangham A. D. Physical structure and behavior of lipids and lipid enzymes. Advanc. Lip. Res. 1: 65-104 1963;
• 10 Harmison C. R, Mammen E. F. Blood clotting enzymology. 86 Academic Press; New York and London: 1967
• 11 Wallach D. F. H, Maurice P. A, Steele B. B, Surgenor D. M. Studies on the relationship between the colloidal state and clot promoting activity of pure phosphatidylethanolamines. J. biol. Chem. 234: 2829-2833 1959;
• 12 Papahadjopoulos D, Hougie C, Hanahan D. J. Purification and properties of bovine factor X: Molecular changes during activation. J. Biochem. 3: 1931-1938 1964;
• 13 Marcus A. J. The role of lipids in blood coagulation. Advanc. Lip. Res. 4: 1-37 1966;
• 14 Kazal L. A. Interactions of phospholipids with lipoproteins, with serum and its proteins, and with proteolytic enzymes in blood clotting. Trans. N. Y. Acad. Sci. (Ser. 2) 27: 613-628 1964; /65
• 15 Edwall P, Foriteli K, Sten A. Micelle formation in bile salt solutions. Proc. Sec. Int. Cong. Surface Activity I. Gas/Liquid and Liquid/Liquid Interface. 357-373 Butterworth; London: 1957
• 16 Hofmann A. F. Detergent properties of bile salts. Correlation with physiological function. Ann Rev. Med. 18: 333-376 1967;
• 17 Baker W. J, Seegers W. H. The conversion of prethrombin to thrombin. Thrombos. Diathes. haemorrh. (Stuttg.) 17: 205-213 1967;
• 18 Seegers W. H. Prothrombin. Harvard Univ. Press; Cambridge: 1962
• 19 Seegers W. H, Landaburu R. H. Purification of prothrombin and thrombin by chromatography on cellulose. Canad. J. Biochem. 38: 1405-1410 1960;
• 20 Marciniak E, Seegers W. H. Experiments with prothrombin, prethrombin, autoprothrombin III and plasmas with prothrombin related deficiencies. New Istanb. Contr. clin. Sci. 8: 117-133 1965;
• 21 Seegers W. H, Marciniak E, Kipfer R. K, Yasunaga K. Isolation and some properties of prethrombin and autoprothrombin III. Arch. Biochem. 121: 372-383 1967;
• 22 Seegers W. H, McCoy L, Kipfer R. G, Murano G. Preparation and properties of thrombin. Arch. Biochem. 128: 194-201 1968;
• 23 Aoki N, Harmison C. R, Seegers W. H. Properties of bovine Ac-globulin concentrates and methods of preparation. Canad. J. Biochem. 41: 2409-2421 1963;
• 24 Cole E. R, Marciniak E, Seegers W. H. Procedures for the quantitative determination of autoprothrombin C. Thrombos. Diathes. haemorrh. (Stuttg.) 8: 434-441 1962;
• 25 Alkjaersig N, Abe T, Seegers W. H. Purification and quantitative determination of platelet factor 3. Amer. J. Physiol. 181: 304-308 1955;
• 26 Mertz E. T, Owen C. A. Imidazole buffer: Its use in blood clotting studies. Proc. Soc. exp. Biol. (N. Y.) 43: 204 1940;
• 27 Ware A. G, Seegers W. H. Two-stage procedure for the quantitative determination of prothrombin concentration. Amer. J. clin. Path. 19: 471-482 1949;
• 28 Seegers W. H, Smith H. P. Factors which influence the activity of purified thrombin. Amer. J. Physiol. 137: 348-354 1942;
• 29 Hofmann A. F. Clinical implications of physiochemical studies on bile salt. G-astroenteriology 45: 484-494 1965;
• 30 Murray M, Johnson S. A, Seegers W. H. Relationship of certain antihistamine drugs to the activation of purified prothrombin. Amer. J. Physiol. 178: 10-16 1954;
• 31 Seegers W. H, Cole E. R, Aoki N. Function of Ac-globulin and lipid in blood clotting. Canad. J. Biochem. 41: 2441-2460 1963;
• 32 Seegers W. H. Activation of purified prothrombin. Proc. Soc. exp. Biol. (N. Y.) 72: 677-680 1949;
• 33 Mertz E. T, Seegers W. H, Smith H. P. Prothrombin, thromboplastin, and thrombin: Quantitative interrelationship Proc. Soc. exp. Biol. (N. Y.) 42: 604-609 1939;
• 34 Seegers W. H. Prothrombin in enzymology, thrombosis and hemophilia. Charles C. Thomas; Springfield: 1967
• 35 Vahouny G. V, Weersing S, Treadwell C. R. Function of specific bile acids in cholesterol esterase activity in vitro. Biochim. biophys. Acta. (Amst.) 98: 607-616 1965;
• 36 Gobbi F, Barbieri U, Ascari E. Physical factors in the thromboplastic activity of phospholipids. Thrombos. Diathes. haemorrh. (Stuttg.) 17: 495-507 1967;
• 37 Seegers W. H, Aoki N, Marciniak E. Autoprothrombin C and the generation of thrombin from prothrombin activation mixtures. New Istanb. Contr. clin. Sci. 5: 170-176 1962;