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.

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