Potentiation of the Anti-factor VIII Effect of Thrombin in Plasma by the Plasminolytic Derivatives of Fibrinogen and Fibrin

SummaryThe activity of factor VIII in mixtures of native plasma or blood with fibrinogen derivatives at concentrations high enough to inhibit prothrombin consumption, is greatly reduced. On the other hand the activity of factor VIII was found normal in similar mixtures of adsorbed native plasma with fibrinogen derivatives, where no generation of thrombin could have taken place. The fibrinogen derivatives increased significantly the inactivating effect of low concentrations of thrombin on the activity of factor VIII of mixtures of adsorbed oxalated plasma with the derivatives. The same inactivating effect was exerted by fibrinogen derivatives heated at 60° C for 15 minutes and by plasmin lysed fibrin. In contradistinction addition of intact fibrinogen or fibrinogen derivatives to heat-defibrinated plasma protected factor VIII from inactivation by thrombin. When both, intact fibrinogen and its derivatives, were added simultaneously at the same concentrations at which they were added singly, the protective effect was intermediate between that exerted by intact fibrinogen and fibrinogen derivatives alone. Under these conditions the fibrinogen derivatives appeared as if they potentiated the inactivating effect of thrombin. Probably by inhibiting the formation of structural fibrin they increased the amount of thrombin, which remained unadsorbed and free to inactivate factor VIII.

Human Coumarin Prothrombin

SummaryThe abnormal prothrombin produced in patients under coumarin treatment has been isolated from oxalated plasma. After removal of normal prothrombin by adsorption on to small amounts of barium sulfate, coumarin prothrombin proved to be adsorbable on to larger amounts of barium sulfate. After elution into citrate the coumarin prothrombin was precipitated with ethanol and chromatographed on DEAE cellulose. Using a similar linear gradient than for normal prothrombin, the coumarin prothrombin was eluted ahead of normal prothrombin. Immunological studies performed on chromatographed coumarin prothrombin showed that it shared with normal prothrombin the main antigenic determinants and a complete line of identity was obtained when tested against antihuman normal prothrombin antiserum. On crossed Immunoelectrophoresis the electrophoretic mobility of coumarin prothrombin, unlike normal prothrombin, was not modified by the addition of calcium ions. Coagulation studies revealed that coumarin prothrombin could not be activated by physiological activators whereas conversion to thrombin was possible by non physiological activators such as staphylocoagulase and Echis carinatus venom. The amounts of thrombin generated under staphylocoagulase activation per 100 antigen units were similar to those generated by normal prothrombin. The amounts of thrombin obtained under the action of the venom were less than from normal prothrombin. In addition, the activation of coumarin prothrombin proved to be slower than for normal prothrombin.

Observations on Factor VIII (Antihaemophilic A Factor) Stability in Rabbit and Rat Plasma Estimation of Factor VIII in Rat Blood by a New Method

Summary1. Factor VIII activity of rat and rabbit plasma can be measured with a modification of the thromboplastin generation test.2. Using this method it can be shown that factor VIII activity is unstable on storage and on repeated freezings and thawings in oxalated plasma from the two animal species. The factor VIII activity is especially unstable in oxalated rat plasma, from which it disappears almost completely by four or more repeated freezings and thawings.3. A rat oxalated plasma, which had been frozen and thawed ten times, could be used as a factor VIII-free reagent in a simple one-stage test system for measurements of this factor in rat or rabbit plasma.4. The method developed may be used with rat tail capillary blood as test material, making repeated observations on rats possible. The use of the method is discussed.

The Use of a Stabilized Partial Thromboplastin Reagent in the Partial Thromboplastin Test

A stable sensitive partial thromboplastin reagent is prepared from an ether extract of acetone-dried bovine brain. The normal oxalated plasma partialthromboplastin time range with this reagent is 66-86 sec. Lyophilized plasmas deficient in coagulation factors give prolonged partial thromboplastin test times. Some of the properties of this reagent are discussed.

Oxalate Induced Correction of the Plasma Antithromboplastin Deficiency in the Aging Arteriosclerotic Subject

SummaryIntravenous heparin will correct the enhanced thromboplastin generation of oxalated plasma obtained from the human arteriosclerotic patient. It will not,however, correct the deficiency of antithromboplastin activity in non-oxalated plasma obtained from the pathological subject. This deficiency of antithromboplastin disappears if oxalate ion is introduced into the heparinized plasma.

Influence of Serum Incubation Conditions on Asolectin Thromboplastin Generation Results

SummaryThromboplastin generation in a system employing Asolectin, a soybean phosphatide, suspension as a platelet substitute is greatly influenced by the time and temperature of serum incubation.Reproducible results and good separation of populations of normal and PTC deficient sera were obtained in the asolectin TGT system when test sera were incubated for 1 hour at 20° C prior to the thromboplastin generation test. Longer time or higher temperatures of serum incubation resulted in progressively less reliable, separation of pathologic and normal populations in asolectin TGT systems than in fresh platelet TGT systems. Cephalin suspension was shown in a smaller number of tests to behave similarly to asolectin suspension when used as a platelet substitute in TGT systems.The 0.14 M sodium citrate eluate of citrated normal or Hemophilia B sera (incubated 1 hour at 37° C) had the ability to shorten the prolonged asolectin TGT minimal time of “over-incubated” normal sera to values obtained with the same sera in fresh platelet TGT systems. This correction, however, was probably non-specific since Hemophilia B sera results were sometimes similarly normalized. Citrated (but not oxalated) plasma incubated at 37° C for 24 hours, then recalcified to manufacture serum, and incubated an additional hour at 20° C showed no loss of the fresh 1 hour 20° C serum activity in asolectin thromboplastin generation, further suggesting that the thermolabile component of normal serum influencing asolectin TGT is a reaction product evolved during clotting.