The influence of cadmium ions on the adsorption of prothrombin onto A1(OH)3 as a means to purify prothrombin.

1. In the presence of CdSO4(1mM),Al(OH)3(1.3% w/v) completely adsorbs the coagulation factors VII, IX, and X from normal plasma, but factor II (prothrombin) is adsorbed for about 50% only.2. A purification procedure for factor II is developed, using Al(OH)3 adsorption in the presence of Cd2+ as a first step and using column chromatography only once. A 750-fold purification is obtained at a 24% yield. 3. Comparison of the prothrombin thus obtained, with prothrombin isolated by the method of Kisiel and Hanahan (Biochim. Biophys. Acta (1973) 304, 103-113) does not show significant differences in amino acid composition, N-terminal amino acid, molecular weight or immunological properties. 4. Comparison of the two prothrombin preparations in a thrombin-generating system shows that although the final yield of thrombin from a given amount of prothrombin in both preparations is the same, the initial velocity of thrombin formation from our preparation is comparable to that of native prothrombin, whereas the other preparation is converted significantly slower.

The prolongation of the thrombotest clotting time in newborns.

As judged from thrombotest dilution curves clotting inhibiting material was present in 67% of human umbilical cord plasma samples of healthy full term infants (n = 40). The correlation coefficient (r-value) between the thrombotest clotting times and the prothrombin levels was -0.46. Using sensitive enzyme immuno assays for fibrin degradation products (FbDP) and fibrinogen degradation products (FgDP), we found that no degradation products could be demonstrated in the non-inhibited group whereas small amounts of these products were present in the inhibited group. These small amounts were undetectable using conventional assays. The most striking finding was the presence of fibrin and fibrinogen degradation products. The prolongation of the thrombotest clotting time could be imitated by adding fibrinogen fragment X to umbilical cord plasma in which no clotting inhibiting material was present in the thrombotest dilution curve. We conclude that the thrombotest clotting is of limited value in the assessment of the vitamin K-dependent coagulation factors in umbilical cord plasma. As utmost care was taken to avoid proteolytic breakdown in vitro, our findings most likely reflect an enhanced fibrino(geno)lytic activity in umbilical cord plasma in vivo.

Reevaluation of some properties of fibrinogen, purified from cord blood of normal newborns.

In this study we compared some properties of fibrinogens, obtained from normal adult and cord plasma. Fibrinogen preparations were made under conditions, which minimize proteolytic breakdown in vitro. We were not able to demonstrate any significant differences between both purified fibrinogens as to the effects of pH and ionic strength on its clotting properties, the Km for thrombin, SDS polyacrylamide gelelectrophoresis behaviour or carbohydrate content. However, the phosphorus content of cord fibrinogen was 3-4 times higher than that of adult fibrinogen. The accelerating effect of calcium on the thrombin clotting time was more pronounced for newborn cord plasma and for purified cord fibrinogen preparations as compared with adult fibrinogen. This might be explained by the higher phosphorus content of the cord fibrinogen molecule. The thrombin clotting time of both purified adult and cord fibrinogen was markedly prolonged, when increasing amounts of fibrinogen degradation product fragment X were added to the fibrinogen solutions under conditions with high pH or high ionic strength. At high pH the effect of adding fragment X was more pronounced in cord fibrinogen preparations. Therefore, mixtures of purified fibrinogen and fragment X have several properties in common with fetal fibrinogen. These observations show, that some of the properties that have been attributed in the literature to a distinct fetal fibrinogen can be caused by the presence of fragment X in the cord fibrinogen preparations.

The action of Echis carinatus venom on the blood coagulation system. Demonstration of an activator of factor X.

It is shown that Echis carinatus venom activates both coagulation factor II and coagulation factor X. The activation of both proenzymes by the venom is Ca++-dependent; phospholipids are not necessary. The activation of factor II by the venom is a slow process and, in the absence of factor X, the clotting activity towards fibrinogen is generated only very slowly. Because Echis carinatus venom clots plasma readily, we postulate that under conditions where the prothrombinase complex can be formed from the factor X activated by the venom it is this complex, rather than the venom itself, that is responsible for the major part of the thrombin formation.

The activity state of factor VII in plasma: two pathways for the cold promoted activation of factor VII.

The apparent amount of factor VII as determined in a one-stage test depends on the type of thromboplastin used: bovine thromboplastin only reacts with human factor VIIa whereas human thromboplastin interacts with unactivated human factor VII as well. Therefore the ratio factor VII activity as measured with bovine thromboplastin divided by the factor VII activity as assessed with human thromboplastin reflects the state of activation of factor VII in plasma. This approach was used to study the process of cold promoted factor VII activation and the involvement of different clotting factors therein. It could be shown that cold promoted activation does not occur in the absence of factors II and XII and is reduced for about 50% in factor IX deficient plasma. The other coagulation factors have a minor influence on the process. The results indicate that the cold promoted factor VII activation is the result of activation by both activated contact products and thrombin.