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.

Interaction of bovine blood clotting factor Va and its subunits with phospholipid vesicles.

Thrombin-activated factor Va and factor Va subunit binding to large-volume vesicles was investigated by a technique based on the separation by centrifugation of phospholipid-bound protein from the bulk solution. This technique allows the direct measurement of free-protein concentration. It is concluded that the phospholipid binding site on factor Va is located on a basic factor Va subunit with Mr 80 000 (factor Va-LC).

Activation of human prothrombin by stoichiometric levels of staphylocoagulase.

The activation of human prothrombin by the bacterial protein staphylocoagulase proceeds via the formation of a very stable equimolar complex. Unmasking of the active center in the prothrombin moiety of the complex is not caused by limited proteolysis. The kinetics of activation of human prothrombin by staphylocoagulase has been studied.

Stimulation of the vitamin K-dependent carboxylase from bovine liver.

Vitamin K-dependent carboxylase from bovine liver is stimulated not only by reducing agents and bivalent metal ions (especially Mn2+), but also by several organic solvents (dimethyl sulphoxide, ketones and acetonitrile). The organic solvents stimulated both the carboxylation of glutamic acid residues and the formation of vitamin K epoxide. This stimulation by organic solvents was independent of the physical state of the phospholipid; it was highest at low temperatures and could only be demonstrated with vitamin K1 and not with 3-DTT-MK-O (the thioether adduct of menadione and dithiothreitol) or t-butyl hydroperoxide, which normally can substitute for vitamin K.

The adsorption of prothrombin to phosphatidylserine multilayers quantitated by ellipsometry.

We investigated by means of an automated ellipsometer the adsorption of prothrombin from a buffer solution by multilayers of 14:0/14:0- and 18:1/18:1-phosphatidylserine (PS) stacked on chromium slides. In this instrument thickness and refractive index of the adsorbed phospholipid and proteins are monitored continuously. Two equations are derived to relate the mass of stacked phospholipids and the mass of protein adsorbed to the thickness and refractive index.

Platelet membrane involvement in blood coagulation.

Intact platelets do not show procoagulant phospholipids on their exterior. These phospholipids are located at the inside leaf of the bilayer membrane. They become available by (a) disrupture of the platelets (mechanical, osmotical etc.

Evaluation of a coagulation assay determining the activity state of factor VII in plasma.

A coagulation assay is described that allows the measurement of the degree of activation of factor VII in circulating blood. The test is based on the use of both bovine and human brain thromboplastin, together with an artificial factor VII-deficient plasma. The latter can be prepared on a relatively large scale which makes it possible to measure factor VII activation in large series of patients.

Half-life time and control frequency of vitamin K-dependent Coagulation factors. Theoretical considerations on the place of factor VII in the control of oral anticoagulation therapy.

A short review about the place of coagulation factor VII in the initial phase of blood coagulation is given. A theoretical model describing the relationship between the half-life times of vitamin K-dependent coagulation factors and the control frequency of oral anti-coagulation therapy is presented. The constraints that control frequency imposes on the type of determination to be carried out are discussed.

Activation of factor IX by factor XIa--a spectrophotometric assay for factor IX in human plasma.

The activation of Factor IX by partially purified Factor XIa was followed by active site titration, gelelectrophoresis and by a spectrophotometric assay. The assay is based on the finding that the rate of Factor X activation in the presence of phospholipid and Ca2+ is linear in time and proportional to the amount of Factor IXa present and can be determined with the chromogenic substrate S2222. Conditions were found that allowed complete activation of Factor IX in human plasma by Factor XIa.

Factor Va-factor Xa interaction. Effects of phospholipid vesicles of varying composition.

The interaction between factor Xa and factor Va was investigated both in solution and in the presence of phospholipid vesicles with varying contents of phosphatidylserine. The binding parameters were inferred from the kinetics of prothrombin activation. Factor Xa and factor Va form in solution an equimolar complex with a dissociation constant of 3.

Interindividual variation in relationships between plasma heparin concentration and the results of five heparin assays.

In order to determine their value for estimating the heparin concentration in plasma, we established the relationship between test result and heparin concentration in plasma from various individuals, for five assays used with heparin treatment. Only assays which can be carried out routinely in clinical laboratories were considered. The thrombin time and the whole blood recalcification time give pointless and ambiguous information respectively, concerning the heparin level.

Studies on the mechanism of the vitamin K-dependent carboxylation reaction. Carboxylation without the concurrent formation of vitamin K 2,3-epoxide.

In addition to the three known forms of vitamin K (vitamin K quinone, vitamin K hydroquinone, and vitamin K epoxide), a fourth metabolite, hydroxyvitamin K, was found in reaction mixtures containing a vitamin K-dependent carboxylating enzyme system. When sulfite was added to such reaction mixtures, the formation of hydroxyvitamin K was substantially enhanced, whereas no epoxide was formed anymore. The vitamin K-dependent carboxylation was stimulated at these sulfite concentrations.

A comparison between vitamin K-dependent carboxylase from normal and warfarin-treated cows.

Detergent-solubilized microsomal preparations that catalyse the vitamin K-dependent gamma-carboxylation of glutamic acid residues in peptide and protein substrates, have been obtained from the livers of normal and warfarin-treated cows. The preparations from warfarin-treated animals contained more endogenous substrate than those from normal cows, but otherwise the two preparations were indistinguishable. The enzymes vitamin K reductase and gamma-glutamyl carboxylase, may function independently of each other in this system.

On the clot-promoting activity of human platelets in a one-stage prothrombinase assay.

The procoagulant activity of activated platelets in a one-stage prothrombinase assay is reevaluated. It is shown that the apparent procoagulant activity of platelets activated by ADP or collagen can be explained by minor cell lysis accompanying platelet activation. The reduction in clotting time observed with thrombin activated platelets can be explained by a combined effect of minor cell lysis and release and activation of factor V from the platelets.

Use of chromogenic peptide substrates in the determination of clotting factors II, VII, IX and X in normal plasma and in plasma of patients treated with oral anticoagulants.

Spectrophotometric methods were used to assay the clotting factors II, VII, IX and X in plasma of 33 subjectively healthy human donors and in plasma of 98 patients receiving long-term oral anticoagulant therapy. In 33 normal subjects the interindividual variations in the plasma activities of the clotting factors II, VII, IX and X are respectively 12.2, 21.

Identification of phospholipid as an essential part of bovine vitamin K-dependent carboxylase.

Vitamin K-dependent carboxylase from bovine liver contains phospholipid (primarily phosphatidylcholine), which is essential for its in vitro activity. Sepharose-bound carboxylase can be depleted of phospholipids, either by washing the enzyme with detergents or by phospholipase treatment. The enzyme can be reconstituted by adding mixed micelles of phosphatidylcholine and cholate to the Sepharose-bound proteins.

The role of phospholipid and factor VIIIa in the activation of bovine factor X.

The kinetic parameters of bovine factor X activation by bovine factor IXa have been determined in the absence and presence of Ca2+, thrombin-activated bovine factor VIII (VIIIa), and phospholipid (dioleoylphosphatidylcholine/dioleoylphosphatidylserine, 75/25; mol/mol). Factor IXa in the absence of Ca2+, factor VIIIa, and phospholipid is able to catalyze factor X activation. The Km for factor X is 299 microM which is well above its concentration in bovine plasma, about 0.

Inhibition of activated factors II, VII, IX, and X by synthetic organic compounds directed against the active-site seryl residue.

A series of 53 organic chemicals belonging to the groups of organic phosphates, sulfonyl derivatives and carbamates were screened for their activity against the coagulation factors IIa (thrombin), VIIa, IXa and Xa. Relatively specific inhibitors for the factors IIa, VIIa and Xa were found.

Contribution of the platelet factor V content to platelet factor 3 activity.

The procoagulant activity obtained from bovine thrombocytes has been compared to that of lipids isolated from platelets, with and without the addition of purified bovine factor V. A one-stage assay, which consisted of delipidated bovine plasma containing RVV-activated factor X, was used to assess the activity. At low lipid concentrations no difference in coagulant activity was found between sonicated vesicles of extracted platelet lipid and lysed platelets.