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A simulation model for the production of thrombin in plasma is presented. Values of the reaction rate constants as determined in purified systems are used and the model is tested by comparison of simulations of factor Xa, factor Va and thrombin generation curves with experimental data obtained in thromboplastin-activated plasma. Simulations of the effect of hirudin indicate that factor V is predominantly activated by thrombin and not by factor Xa. The model predicts a threshold value for the factor Xa production which, if exceeded, results in explosive and complete activation of prothrombinase. The dependence of this threshold value on different negative feedback reactions, e.g. the inactivation of thrombin and factor Xa by antithrombin III (+ heparin), is investigated. The threshold value, for control plasma in the range of 1–10 pM total factor Xa production, can be raised two orders of magnitude by accelerated inactivation of factor Xa and prothrombinase but is hardly affected by a tenfold increase in the rate of thrombin inactivation or by increased production of activated protein C. This latter effect, however, results in a more gradual input-response relation between factor Xa input and the extent of prothrombinase activation.

It is one of the main charms of blood coagulation that advanced biochemistry is continuous to everyday medical practice and vice versa. Common clinical facts hide the most enchanting enzymology. The prothrombin time (PT, thromboplastin time, Quick time) and the activated partial thromboplastin time (APTT) are perfect examples. Both are commonly used in the clinical laboratory as global tests for the function of the coagulation system and for the control of anticoagulant therapy. They are at the basis of the classical one-stage clotting factor determinations. Practice has taught us that the PT is perfectly suited for the control of anticoagulant therapy but not for measuring the effect of heparin, whereas the APTT can serve both purposes. Only recently was it found out why: the length of the APTT is primarily determinated by the time necessary for the activation of factor VIII by thrombin [1]. Now thrombin, of course, is the final enzyme of the coagulation cascade, whereas factor VIII is one of the factors necessary for its formation. The product actiyates one of the proteins instrumental in its own formation: a clearcut case of feedback activation. Obviously, if thrombin is quickly O l99l S. KargerAG, Basel 030 | -0 | 47 I 9 | | 02 | 4-Ot 89s2.7 5 / O

The aim of this study was the development of a simple chromogenic factor VIII assay for practical clinical use. The criteria that the assay fulfils are: (1) The method is so sensitive that even 1% factor VIII in human plasma is easily detected. (2) The method is linear in the amount of factor VIII from 0 to 200% in plasma. (3) The pipetting scheme is very simple; two reagents are prepared, reagent 1 (factor IXa, thrombin, Ca2+ and phospholipids) and reagent 2 (factor X). Then we pipet at t = 0 s, 100 microliters diluted plasma + 100 microliters reagent 1 in a reaction tube; at t = 30 s, 100 microliters reagent 2 in the same tube and at t = 90 s, 200 microliters of the reaction mixture in a cuvette with 700 microliters EDTA buffer (stop buffer) and the formed factor Xa is measured with a chromogenic substrate. (4) The reaction components are stable during at least a whole working day. Factor VIII was measured in an assay using bovine clotting factors, so one avoids the risk of viral infections, which one might catch by working with clotting factors isolated from human plasma.

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.

Prothrombin is determined with the aid of a recently developed assay, based on the amidolysis of a chromogenic substrate. The assay proved to be reliable when it was compared with more conventional coagulation assays in the control of oral anticoagulant therapy, both in the therapeutic range and in a case of overdosage. As is the case in coagulation tests, heparin therapy remains a disturbing circumstance. The prothrombin concentration was measured (a) in the plasma of 50 long-term anticoagulated patients, and the results were compared with those obtained with a one-stage coagulation assay and with those obtained with Thrombotest® determinations, and (b) during vitamin K administration in the plasma of a patient with a severe intoxication of a vitamin K antagonist.

A small piece of vascular tissue punched from a rat aorta is able to clot plasma. This coagulation process is promoted by blood platelets, especially after their activation. Thrombin, generated by this clotting process, plays a key role in vessel-wall induced platelet activation. Vascular prostacyclin inhibits vessel-wall-induced clotting of platelet-rich plasma, possibly by inhibiting platelet activation. Type and amount of dietary fats were shown to influence vessel-wall-induced clotting via at least four different mechanisms, namely: by modifying vascular prostacyclin formation; by affecting the clotting potency of the vascular tissue per sec; by an effect on some platelet property, probably connected with platelet activation; by influencing a plasma factor. Each of these mechanisms, as well as the nature of vessel-wall-induced coagulation, requires further investigation.

We report a study on the importance of factor IX activation in thromboplastin-dependent coagulation in plasma. Diluted, CaCl2-containing thromboplastin solutions at constant phospholipid concentration were used to trigger the coagulation in plasma from patients with congenital factor IX and factor VIII deñciency in the presence and absence of added factors IX and VIII, and the generation of thrombin activity was monitored. When coagulation is triggered with the high thromboplastin concentrations normally used in clinical routine tests, the generation of thrombin activity in plasma of patients with congenital factor IX deficiency before and after reconstitution with purified factor IX appears identical. When, however, coagulation is triggered with low thromboplastin concentrations, a clear dependency of the generation of thrombin activity on the concentration of factor IX becomes evident at factor IX concentrations lower than 30 nM (about 40% clotting factor activity). Factor VIII is a compulsory cofactor for this factor IX activity because the prothrombinase activity at optimal factor IX concentration is still critically dependent upon the amount of factor VIII present. The lower the amount of thromboplastin, the higher the importance of factor IX and factor VIII activation in thromboplastin-dependent coagulation. This suggests a role of this pathway in pathophysiological thrombin formation.

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. Platelets stimulated by ionophore A23187 or by the combined action of collagen plus thrombin show a much shorter clotting time than can be accounted for by minor platelet lysis or release and activation of factor V from the platelets. The results with this clotting assay essentially confirm previous observations [Bevers et al.: Eur. J. Biochem. 122:429–436, 1982] using a spectrophotometric method with highly purified coagulation factors and a chromogenic substrate to measure the rate of thrombin formation with activated platelets.

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, 21.4, 11.0 and 15.0%. After correction for the assay variations the remaining biological variations are respectively 11.7, 21.2, 9.7 and 14.8%. Plasma from 98 patients receiving long-term anticoagulant therapy was assayed with ‘Thrombotest’, a clotting test in whole blood introduced by Owren and in these plasmas the activity of each of the vitamin K-dependent factors was assayed with spectrophotometric methods. For the clotting factors IX and VII, novel spectrophotometric methods were applied and the plasma activities thus measured were compared to results obtained with factor IX and VII clotting assays. Chromogenic activities of the different factors were correlated among each other and with 1/Thrombotest values. When the therapeutic range for Thrombotest values is set between 5 and 12.5% the corresponding therapeutic ranges for the activity of the factors II, VII, IX and X are respectively 12.6–36.1, 27.0–52.3, 23.1–49.3 and 18.9–36.2% (expressed as a percentage of the activity in normal pool plasma). The chromogenic assays for the factors II, VII, IX and X provide the same information on the therapeutic state of the patients in respectively 86.7, 78.6, 81.6 and 89.8% of the cases. Finally we discuss the suitability of the different assays to monitor oral anticoagulant therapy.

A short review on the discovery of the relation between vitamin K and the synthesis of clotting factors and on the development of anticoagulant therapy is given. The pro-coagulant activity induced by vitamin K resides in the postribosomal modification of N-terminal glutamic acids in γ-COOH glutamic acids. The mode of action of oral anticoagulants is explained. The concentration-effect relations and the choice of an oral anticoagulant is discussed.

The bovine coagulation factors II, VII, IX and X were separated from other plasma proteins, and each factor was obtained in a largely pure state by various chromatographic procedures in the presence of diisopropylfluorophosphate (DFP). Factor X was separated from factors II, VII, and IX by DEAE Sephadex chromatography, followed by G-100 filtration and hydroxylapatite chromatography. Separation of factors II and IX from factor VII was achieved by chromatography on DEAE cellulose, followed by G-100 gel filtration and hydroxylapatite chromatography. Factors II and IX were separated by preparative polyacrylamide electrophoresis. After DEAE cellulose chromatography, factor VII was purified by gradient elution from hydroxylapatite.

Thrombotest dilution curves and specific inhibitor experiments were carried out in the abnormal factor X (factor X Friuli) plasma and in coumarin plasma. Thrombotest dilution curves obtained with Friuli plasma suggested the presence of a PIVKA-like inhibitor which was not confirmed by the specific experiments. The inhibitory capacity found in Friuli plasma was-0.09 arbitrary units, whereas that found in coumarin-treated patients with a factor X level ranging from 1 to 14% varied between 0.34 and 0.21 arbitrary units.

A preparation of coagulation factors II, VII, IX, and X was obtained by adsorption onto Al (OH)3 from normal plasma. The subsequent eluate was subjected to gel filtration experiments. It was noticed that the purification obtained on Sephadex G-100 columns was hardly dependent upon the characteristics of the buffer. Factor VII is partly separated from the other factors. Factors II and IX appear to be of equal size, whereas factor X is slightly larger. Molecular weights of about 72,000–74,000 were calculated for factors II, IX, and X, and 57,000–59,000 for factor VII. The coagulation factors are bound to dextran blue, depending upon the ionic strength. Factors II and IX are much more tightly bound than factors VII and X. At an ionic strength of 0.15 and pH 7.0, both, factor II and factor IX interact strongly with dextran blue, whereas the other factors remain free. On the basis of this effect, nearly complete separation can be achieved. The interaction between the factors and dextran blue decreases with increasing pH.