Some Effects of Calcium and Strontium Ions on Three Basic Reactions of Blood Coagulation

SummaryCalcium ions accelerated all three basic reactions of blood coagulation at an optimum concentration of 0.01 M. Strontium ions were practically ineffective as substitutes for calcium ions in the formation of autoprothrombin C (factor Xa), but functioned just as effectively as calcium ions in the formation of thrombin. Inhibition was the primary effect of strontium ions in fibrin formation. It is suggested that calcium ions function differently in each reaction. Echis carinatus venom was found to form thrombin directly from its precursor and, likewise, formed autoprothrombin C from its precursor.

Antithrombin

SummaryAn evaluation of the mode of action of antithrombin in the temporary inhibition of purified 3.7 S bovine thrombin was made according to traditional enzyme inhibition theory. Using enzyme clotting activity and concentration of active sites synonymously, it was observed that the binding of antithrombin to thrombin followed a second order reaction that could be distinguished into an essentially irreversible phase and a slower, potentially reversible phase. The level of steady state enzyme inhibition depended upon the ratio of the initial concentrations of enzyme to antithrombin. In addition, there occurred a rapid enhancement of enzyme esterolytic activity during the concomitant loss in clotting activity. This enhancement was also observed for acetylated thrombin which had no proteolytic activity. Under high concentrations of antithrombin, this enhancement subsequently deteriorated. A working hypothesis, including equations, for a mutual depletion system is presented to account for the known parameters of enzyme inhibition by antithrombin. We compared the neutralization of thrombin C with autoprothrombin C and found that each enzyme responds to antithrombin according to similar kinetic relationships.

Blood Coagulation

SummaryPreparations of platelet factor 3 accelerated the conversion of purified autoprothrombin III (F-X) to autoprothrombin C (F-X?). The reaction was slow and the yields were low. Purified Hageman factor or purified autoprothrombin II (F-IX?) functioned as platelet cofactors. Each increased the yield of autoprothrombin C from purified autoprothrombin III above that obtained with platelet factor 3 alone. Hageman protein thus has a role in the direct acceleration of the first basic reaction in blood coagulation. Purified prothrombin was made refractory to the two-stage analytical reagents with Hageman factor.

Structural Changes in Prothrombin during Activation: A Theory

SummaryProperties of the thrombin zymogen are quite different when in the form of prothrombin complex, DEAE-prothrombin (prothrombin) or prethrombin. When removed from the prothrombin complex, prothrombin spontaneously became refractory to the two-stage analytical reagents. No new N-terminal amino acids formed in association with this activation. A first step in prothrombin activation might be related to conformation. Repeatedly one mole of alanine was found as N-terminal amino acid for prothrombin and it is thus a single chain protein. Prethrombin did not have this alanine, but lysine and threonine, were found. Prethrombin, like thrombin, thus had two chains. After the conversion of prethrombin to thrombin with purified autoprothrombin C, a new N-terminal alanine amino acid was found attached to a peptide. Additionally peptides with N-terminal serine, lysine, and glycine were found. Threonine and isoleucine were again found as the N-terminal amino acids for 3.7 S thrombin and 3.2 S thrombin. As a working hypothesis, a perspective on the possible structure of prothrombin is outlined on the basis that it contains two moles of thrombin. Prethrombin probably forms when Ala1-peptide splits from a polypeptide chain which forms a loop held together by a disulfide bridge. Proteolysis probably also occurs in this loop. The postulated disulfide bridge would be the one which holds the A and B chains of thrombin together. In the conversion of prethrombin to thrombin, the first mole of thrombin would be set free. Then the Ala2-peptide released might correspond to the Ala1-peptide removed when prethrombin originally formed. Ala2-peptide thus would be related to the second mole of thrombin which could arise by further proteolysis including a split in the loop held together by a disulfide bridge. It is postulated that acidic peptides are attached to the main prothrombin polypeptide chain, to prethrombin, and to 3.7 S thrombin as satellite material.

Activation of Factor X in Bovine Prothrombin Preparations

SummaryChromatographic fractionation of factor X (autoprothrombin III) and factor Xa (autoprothrombin C), obtained from bovine prothrombin preparations, on amberlite IRC-50 demonstrates activation of factor X under the conditions of prothrombin activation. The increase in factor Xa protein during prothrombin activation is due to incorporation of a non-active protein into the fraction. Prothrombin Activating Derivative B (PAD-B) is activated by concentrated salt solutions and not by stypven.

Note on the Inactivation of Autoprothrombin C with Diisopropylfluorophosphate or Phenylmethanesulfonyl Fluoride

SummaryIn relatively high concentrations DFP was found to inactivate autoprothrombin C. PMSF also inactivated the enzyme. A useful modification in our assay procedure for autoprothrombin C is described.

Substitution of Lipids with Bile Salts in the Formation of Thrombin

SummaryThe 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.