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A general mechanism is recognized that can cause specific enzymatic activity at interphases. It consists of 2 proteins bound in close juxtaposition at a micelle or membrane surface. One, the enzymesensu strictu, bears the active site, the other, the paraenzyme, is essential for generation or specific modification of the enzymatic activity.

Phospholipids play a role in thrombin generation because the constitute the support on which both the factor X activating enzyme (“tenase”) and the prothrombin activating enzyme (proth rcmbinase) are built.In model systems with pure synthetic phospholipids it can be shown that the binding of the coagulation factors to a phospholipid surface is a function of both phospholipid charge and membrane fluidity. A relatively high molefraction of phosphatidyl serine (PS) is a prerequisite for optimal binding of the vitamin K dependant factors. Precise measurements on the binding constant and the number of binding sites of factor X show that at molefractions of PS between 0.01 and 0.25 aproximately one PS binds per Gla-residue. Above 0.25% of PS vesicle agregation occurs.The normal intact circulating platelet shows a molefraction of PS of ̴ 0.03 in the outer leaf let of its plasma membrane and therefore hardly if at all stimulates thrombinformation.Upon activation by thrombin (2nM) together with collagen, (10μg/ml) the outer leaflet aquires increasing amounts of PS without platelet disruption taking place.This suggests a flip-flop movement of phospholipids across the membrane as part of the activation reaction of platelets. It can be shown that optimal “tenase” supporting phospholipid compositions appear before prothrombine supporting ones. The activation by thrombin and collagen is only partly inhibited by prostacyclin even at high concentrations.

An artificial factor II reagent is composed by supplementing BaSO 4 adsorbed plasma with a concentrated solution, containing factors VII and X. These factors are prepared by the addition of Echis Carinatus venom to plasma, removal of the clot which has formed within 1 hour, adsorption of the factors VII and X to DEAE Sephadex, followed by a step elution and subsequent dialysis of the factors. The reagent contains no detectable Echis Carinatus venom, factor II or thrombin. The advantages of this reagent over the conventional ones are discussed.

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. The determination of factor VII activation during incubation at 4 °C of plasma of women using oral contraceptives shows that the test described adequately measures factor VII activation. Differences in the time course of factor VII activation during this incubation in glass and plastic containers are found and implicate that rigorous standardization of blood sampling and test conditions is necessary. A possible mechanism that causes this critical dependence upon the test conditions is discussed.

Pentosan polysulphate is an heparin analogue which acts via an antithrombin III (AT III) independent pathway. We compared the effect of this drug to that of heparin and AT III infusions in AT III deficient patients. Four patients with AT III congenital deficiency received on three different occasions : (i) an infusion of human AT III concentrate (20 U/kg or 40 U/kg), (ii) an intramuscular injection of pentosan polysulphate (2 mg/kg), (iii) a subcutaneous calcium heparin injection (100 U/kg). AT III infusion inhibits the excessive thrombin generation (46 % of inhibition) observed in the plasma of AT III deficient patients during at least 12 hours, but does not modify the factor Xa formation. On the contrary, pentosan polysulphate has a marked effect on both thrombin (62 % of inhibition) and factor Xa generation (57 % of inhibition) still present 8 hours after injection. Heparin injection has the same effect, more prolonged, as pentosan polysulphate on thrombin generation but is not so effective on impairing factor Xa generation (27 % of inhibition). The marked effect of pentosan polysulphate on thrombin and factor Xa generation in these patients is due to its AT III independent mechanism of action.

Heparin is a well established drug in the treatment and prophylaxis of thrombosis. However, the in vitro methods for thecontrol of heparin therapy (aPTT, anti-Xa, anti-II) often are not sufficiently sensitive to monitor low-dose heparin therapy. We used an assay that is based upon the in vivo activation of factor VIII at the site of skin punction: the factor VIII activity in blood samples taken from a capillary wound increases with time Hurlet-Birk Jenssen et al. Path. Biol. 24, 6 (1976).Assay: 50 μl blood samples are taken 30, 60 and 90 sec after capillary puncture. The samples are diluted with 950μl, 10 mM sodium citrate, Mich, buffer and centrifugated(3 min, 4 °C). In the diluted samples the factor VIII activity is determined.In healthy volunteers the increase in factor VIII activity in the successive samples is fairly constant (1.4 - 1.8 fold) in spite of distinct interindividual differences in the factor VIII level. In patients receiving continuous intravenous heparin (20000 IE/24 h) and in a hemophiliaA patient there was no increase in factor VIII activity with the time. In individuals receiving 5000 IE heparin subcutaneously the increase in factor VIII activity was dependent upon the time interval between heparin injection and sampling. There was almost no influence of heparin on the 30 s samples but the 90 and 150 sec. samples showed distinctly prolonged clotting times at sampling points from 2 to 4 hours after heparin administration. It was not possible to detect the heparin effectby an automated anti-IIa assay or in a prothrombin time assay because the heparinlevels were below the detection limit of the tests.These results suggest that low-dose heparin therapy has a definite influence on the feedback activation of factor VIII even in concentrations that are not detectable with the usual heparin tests.

In order to characterize the enzyme system which converts abnormal prothrombin (PIVKA-II) in prothrombin we prepared liver cell-free extracts from normal healthy cows. The enzyme system devoid of either precursor or prothrombin was isolated by differential centrifugation and partly purified with the aid of column chromatography.The carboxylation of purified PIVKA-II to prothrombin was detected by means of the one-stage coagulation assay and with 14CO2 incorporation. The reaction was dependent upon microsomal supernatant, vitamin K, ATP, Mg++ ions, O2, pH and temperature. Inhibitors were: warfarin, 2.4-dinitrophenol and factor II. From our experiments we concluded that PIVKA-II can act as a precursor of prothrombin and that PIVKA-II can be converted into the normal clotting factor in an in vitro cell-free system. In contrast to the systems published by others, here the converting enzymes are added to separately purified precursor proteins, so the system provides a way to study the nature of this reaction in more detail.

The formation of 7-carboxyglutamic acid residues from glutamic acid residues is a vitamin K-dependent carboxylation reaction. The reaction has been demonstrated in various species and tissues [1,2], but the most extensive studies have been performed in the microsomal fraction of rat liver.It is generally assumed that the carboxylation reaction is coupled to the epoxidation of reduced vitamin K 13,41, but the reaction mechanism is still unclear. The purification of the carboxylating enzyme (carboxylase) from rat liver seems to be difficult and has not been reported until now. The possible invoh'ement of other cofactors, such as haem groups [5-7] is therefore still a matter of dispute. The main argument for the involvement of haem in the carboxylation reaction was the observation that cyanide inhibits rat carboxylase [5,8]. This inhibition has not been further analyzed, however, whereas other common haem ligands such as azide and carbon monoxide do not inhibit the vitamin K-dependent carboxylation I I ]. We have developed a carboxylatingenzyme system from the livers of warfarin-treated cows and obtained a 100-fold purification of the microsomal enzyme by immunospecific adsorption onto antibodies against the endogenous ubstrate [9]. This partly purified enzyme preparation is attached to Sepharose beads and is called solid-phase carboxylase (SP-carboxylase). The enzymatic activity of SP-carboxylase is strictly dependent on the presence of phospholipids and in the presence of an excess of exogenous ubstrate (Phe-ku-Glu-Glu-ku), the carboxylation rate was constant for at least 3 h at 25" C [10]. Here, we describe the results of some more detailed investiga

We determined the generation of prothrombinase activity in plasma using a mathematical analysis of the thrombin generation curve (H. C. Hemker, G. M. Willems, S. Béguin. Thromb. Haemostas. 56, 9-17, 1986).Addition of the purified factor VII, IX or X to plasma from deeply anticoagulated patients (10%) did not influence the rate and amount of prothrombinase formed. Only the amount of prothrombin in the starting plasma determined the course of thrombin generation. Adding increasing amounts of purified human clotting factor preparations to deficient plasmas showed that the treshold concentration under which factor VII, and IX start to have an effect on prothrombinase activity are 5% or lower. For factor X it is lower than 10%.From this it can be concluded that only the changes in prothrombin level must be held responsible for the anti thrombotic effect of oral anticoagulation. These conclusions are not modified if different types and concentrations of thromboplastin are used.We were able to show that at dilutions of human brain thromboplastin higher than 1:50, a factor IX and factor VIII dependent pathway plays an increasingly important role. This directly demonstrates the Josso pathway. The concentration of factor IX necessary for full activity is again We conclude that the antithrombotic effect of oral anticoagulant treatment, if it is mediated via the coagulation system, works via modification of the prothrombin level only.

The heparin-catalyzed inactivation of activated coagulation factors by antithrombin III (AT III) has mostly been studied for isolated serine proteases. However, we decided to study the action of heparin and AT III under more physiological conditions, i.e. during the activation of factor X by factor IXa in the presence of phospholipid and calcium. Thereby we made use of a mathematical model which describes the generation of factor Xa by factor IXa, phospholipid and calcium in the presence of AT III and heparin. Fitting the experimental factor Xa generation curve to a set of equations gave the pseudo-first-order rate constants of factor Xa and factor IXa. In a first approach we examined the effect of AT III alone on factor X activation. We found that the second order rate constant of inhibition of formed factor Xa was 2 x 10 5M-1min-1 , whereas that of factor Xa in free solution was 5 x 10 5M-1min-1 , indicating that phospholipid-bound factor X competes with AT III for factor Xa. The second order rate constant of inhibiton of factor IXa, either in the presence or absence of accessory components, was 8 x 103 M-1min-1. Unfractionated heparin (UFH; 168 USP units/mg) was found to stimulate the inhibition of generated factor Xa by AT III (200 nM) with 0.1 min-1 per nM of UFH, and a synthetic pentasaccharide (PS; 4000 anti-Xa units/mg) stimulated this inhibition with only 0.03 min-1per nM. Due to the presence of phospholipid-bound factor X this stimulation was 4-fold less when compared with factor Xa in free solution. At UFH concentrations higher than 3 nM, and PS concentrations exceeding 10 nM hardly any active factor Xa generation could be measured because of the rapid inactivation of factor Xa whereas factor IXa was not inhibited. Using a factor IXa assay we found that PS, even at relatively high concentrations, had no effect on factor IXa inactivation by AT III (200 nM), both in the presence and absence of accessory components. The inactivation of factor IXa by AT III (200 nM) during factor X activation was stimulated by UFH with 1.6 x 10 -2min-1 per nM of UFH. Surprisingly, this was 4-fold more when compared with factor IXa in the absence of accessory components. We established that calcium stimulates the heparin-dependent inhibition of factor IXa.

The kinetic parameters of the conversion of prothrombin into thrombin by activated clotting factor X (factor Xa) have been determined in the absence and presence of Ca2+, phospholipid (phosphatidyl serine/phosphatidylcholine vesicles) and activated blood clotting factor V (factor Va). In free solution the Km for prothrombin is 298 μM which is well above its plasma concentration of 4μM. Under these conditions the Vmax of thrombin formation is 1.25 Moles min-1 Mole Xa -1. When phospholipid is present the km for prothrombin drops to 0.1μM while the Vmax is only slightly affected (3 Moles min-1 Mo Le Xa -1). For the complete prothrombin activating complex consisting of factor Xa, factor Va, Ca2+ and phospholipids the kinetic constants greatly favour thrombin formation. A for prothrombin of 0.26μM and a Vmax of 2130 Moles min-1 Mole xa -1 are measured under these conditions. These results help to elucidate the role of phospholipid and factor Va in prothrombin activation. The earlier observed rate enhancements caused by phospholipid and factor Va are explained as effects on the Km for prothrombin and the Vmax of thrombin formation, respectively. The changes of the kinetic parameters for prothrombinase complexes of various composition will be considered with respect to the function of the accessory components in the mechanism of prothrombin activation. Implications of these data for in vivo blood coagulation will be discussed.

The generation of factor X converting activity (“tenase”) from factor IXa, factor VIII and inosithin has been studied using > 95% pure preparations of the coagulation factors. It was found that: 1. a. The three reactants mentioned above and Ca++ ions are necessary to obtain full “tenase” activity. Factor IXa has some “tenase” activity of its own 2. b. The reactants were functional in the form in which they were added. 3. c. The time course of the development of the tenase activity and the dependence of “tenase” concentration on the concentration of the reactants are compatible with the reaction scheme: IXa + Ca++ + ph.lip. ⇌ IXa − Ca − ph.lip. VIII + ph.lip. ⇌ VIII − ph.lip. IXa − Ca − ph.lip. + VIII ⇌ tenase VIII − ph.lip. + Ca++ + IXa ⇌ tenase. 4. d. High phospholipid concentrations are inhibitory. At inhibitory phospholipid concentrations “tenase” forms more slowly than at non-inhibitory concentrations. Under these conditions there is a lag-phase in the conversion of factor X. This is suggestive of formation of tenase and of the tenase-factor X complex by lateral movement of molecules adsorbed at the phospholipid-water interphase.

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.

Vitamin K-dependent carboxylase activity has been detected in human andbovine vessel wall. Studies comparingthe carboxylases from liver and vessel wall revealed that the enzyme systems may be regarded as isoenzymes withwidely different substrate specificities. The carboxylated product of vessel wall carboxylase has not yet been identified, but it seems plausible that it will be found amongst the Gla-containing proteins which are abundantly present in calcified atherosclerotic plaques (Gla= gammacarboxyglutamicacid, the abnormal amino acid formed by vitamin K-dependent carboxylase). Therefore we have started to characterize the protein constituents of hardened atherosclerotic plaques.The calcified areas from human aortae were solubilized in EDTA and the proteins extracted were partly purified by batch-wise adsorption onto QAE and elution with high salt. The crudeplaque-extract did not contain prothrombin, factor X or protein C. This excludes the possibility that Gla-containing coagulation factors are bound non-specifically from blood. Osteocalcin accounted for 20% of the total amount of protein-bound Gla-residues.Another Gla-containing protein waspurified from the crude plaque-extract by employing high performance liquid chromatography (HPLC). Gel filtration yielded a Gla-rich protein with anapparent Mr of 25 kD. In vitro boththe crude plaque-extract and the purified Gla-containing protein strongly inhibited the precipitation of calcium phosphate and calcium carbonate. A similar effect was not found with humanserum albumin nor with a thermallydecarboxylated plaque-extract. If also in vivo the Gla-containing proteinsproduced by vessel wall carboxylase prevent the precipitation of calcium salts remains to be investigated.

Coumarin treatment of the cow results in the appearance of a prothrombin precursor (de-carboxyprothrombin) in the blood. We have isolated from bovine liver the enzyme (prothrombin synthase) that is able to convert purified decarboxyprothrombin into prothrombin. Prothrombin synthase is localized in the microsomal fraction and extracted there from with a buffer containing 2% Triton X-100. Further purification of the enzyme was accomplished by column chromatography on Sepharose 4B, DEAE Sephacell and Sephadex G 100. The molecular weight of prothrombin synthase was found to be about 60,000 D. The enzyme activity is defined as the ability to increase the prothrombin concentration at standard conditions and it is measured as a decrease of the clotting time in the one-stage coagulation assay. In the presence of NaH14CO3, radioactive label is incorporated into decarboxyprothrombin, parallel to the generation of prothrombin.After extraction with a suitable organic solvent prothrombin synthase was separated from endogenously bound vitamin K. After extraction the water phase contained the enzyme, the activity of which was completely restored by supplementing the reaction mixtures with vitamin KH2. The organic phase contained the vitamin K as was deduced from U.V.spectra. Prothrombin synthase is dependent on Mn2+ and molecular oxygen and is inhibited by warfarin, Cl-K and Cu2+.

A reaction mixture completely devoid of factor X is described. It is shown that no pathways of blood coagulation exist that circumvent this factor. The relation between the clotting time and the inverse of the factor X concentration is shown to be rectilinear. The low KM of the rate-limiting reaction explains why trace amounts of factor X cause short clotting times.

The binding parameters (dissociation constant and the number of binding sites) for factor X binding to phospholipid bilayers in the presence of Ca-ions can be determined using the factor X-activating protein present in Russel’s Viper Venom (RVV-X). It is shown that RVV-X is only able to activate factor X molecules that are not bound to the phospholipid bilayer. Therefore, from the observed rates of factor X activation by RVV-X measured in the presence and absence of phospholipid the amount of factor X not bound to the phospholipid bilayer can be calculated. When the concentration of factor X added is known the bound factor X can be calculated and the binding parameters for factor X are obtained from a Scatchard plot. The technique presented here offers an advantage over other techniques published thus far. It is rapid and simple as compared to the Hummel andDryer technique. There is also an advantage over techniques using light scattering. Since the technique presented here is not hampered by aggregation of vesicles it is possible to study the binding of factor X to vesicles containing a high mole fraction of PS and at high CaCl2 concentrations.The binding parameters for factor X binding to vesicles of a mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) are dependent on the amount of negatively charged phospholipid present. At increasing mole fractions of PS factor X binds more tightly to the vesicles and also the number of factor X binding sites increases. However, at high amounts of PS (60%) the number of sites present decreases dramatically which is likely due to aggregation of the vesicles. Binding to vesicles composed of a mixture of phosphatidylgly- cerol (PG) and PC is considerably weaker whereas the lumber of sites present is about the same. Prothrombin and factor X compete for the same binding site as was determined by competition experiments. Our data will be discussed with respect to the mode of interaction of vitamin-K dependent clotting factors with negatively charged phospholipid interfaces.

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.

Platelets from a platelet factor 3-deficient patient, which was first described by Weiss et al (Am J Med 67:206, 1979), were found to be equally impaired in their ability to promote factor X and prothrombin activation. Compared to normal platelets, the patient's platelets showed upon stimulation with thrombin plus collagen a much slower generation and a considerably lower level of platelet prothrombin- and factor X-converting activities. Treatment of stimulated platelets with phospholipases revealed a decreased exposure of negatively charged phospholipid at the outer surface of the patient's platelets, relative to control's. We suggest that the combined impairment of prothrombin- and factor X-converting activities in this patient is due to a defect in the mechanism by which phosphatidylserine becomes exposed at the outer surface of stimulated 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.

Activation of factor IX by factor XIa occurs via an intermediate which has no esterase activity towards synthetic arginine esters or coagulant activity as determined with a clotting assay. Factor IXa can be active site titrated using p-nitrophenyl-p1-guanidinobenzoate (p-NPGB) as a titrant. The rate and equilibrium constants describing the active site titration will be presented. To determine whether the intermediate occurring during factor IX activation by factor XIa has its active site exposed for p-NPGB the time course of activation of factor IX by factor XIa was followed l) by active site titration of the active sites generated, 2) by gel- electrophoretic analysis in the presence of sodium dodecyl sulfate, 3) by a clotting assay for factor IXa and 4) by measurement of factor IXa using a spectrophotomefric assay. It will be shown that the intermediate occurring during activation of factor IX by factor XIa does not interact with p-NPGB indicating that the active site is not available in the intermediate.Factor IXa can be determined spectrophotometrically by measurement of the rate of factor X activation by factor IXa in the presence of phospholipid and CaCl2. The factor Xa generated is measured using the chromogenic substrate S222. Since human factor IX can be activated with bovine factor XIa and since human factor IXa can activate bovine factorX the bovine clotting factors factor XIa and factor X can be used to construct an assay for factor fx in plasma samples. Experiments will be presented in which it is shown that the spec- trophotometric assay for human factor IXa can be used to determine levels of factor IX in plasma samples of healthy individuals, in plasma samples deficient in various clotting factors and in plasma samples from patients under anti-coagulant therapy. The results are in agreement with a factor IX clotting test.

Five different ways of estimating prothrombin are applied to the plasma of persons receiving vitamin K antagonists, to know: the one-stage assay, the two-stage assay, the Echis Carinatus Venom assay, the coagulase-reacting factor assay and the immunological assay. The Protein Induced by Vitamin K Absence analogous to prothrombin (PIVKA-II) can be shown to be co-estimated in all but the one-stage assay. There are minor differences, however, between the other four tests. The most practical way to assess both prothrombin and PIVKA-II seems to be the coagulase-reacting factor assay. The difference between the one-stage assay and the others can be explained on basis of the new data on the role of vitamin K in prothrombin biosynthesis. The differences between the other tests are smaller and remain to be explained.

One Prothrombin.- Historical development of the prothrombin concept.- The primary structure of prothrombin, the role of vitamin K in blood coagulation and a thrombin-catalyzed `negative feed-back' control mechanism for limiting the activation of prothrombin.- The conversion of prothrombin into thrombin.- The conversion of prothrombin to thrombin: the function of the propiece of prothrombin.- Discrepancies between the one- and two-stage prothrombin estimations in purified prothrombin preparations.- Preparation of pure prothrombin by the Cd++ method.- Staphylocoagulase.- The interaction between prothrombin and staphylocoagulase.- Inhibition of thrombin.- Two PIVKA-II.- Postribosomal synthesis of prothrombin under the influence of vitamin K.- Structural comparison of normal and dicoumarol-induced prothrombin.- Preliminary studies on human coumarin prothrombin.- Prothrombin metabolism in healthy subjects and in two patients with congenital hypoprothrombinemia.- Metabolism of PIVKA II in man.- Factors influencing prothrombin complex clotting factor synthesis in the rat.- Three Congenital Abnormalities.- A congenitally abnormal prothrombin: prothrombin Barcelona.- Prothrombin San Juan: a complex new dysprothrombinemia.- Prothrombin Padua.- Prothrombin Brussels.- Factor X Friuli.- Four PIVKA VII, IX and X.- Synthesis of factor VII.- Factor VII in patients on oral anticoagulants.- Study of human factor IX variants with an immunoadsorption technique.- Proteins induced by vitamin K antagonists (PIVKAs).- Index of subjects.

The kinetics of factor X activation by activated clotting factor IXa was measured either in the presence or absence of phospholipid, Ca-ions and factor VIIIa. This study was carried out with purified bovine clotting factors. The rate of factor Xa formation was measured using the chromogenic substrate S 2222. Both the Km for factor X and the Vmax of factor Xa formation were determined for different compositions of the factor X activating complex in order to get more insight in the role of phospholipid, Ca2+ and factor VIII in factor X activation. With factor IXa alone, the activation of factor X is a very inefficient process. The presence of phospholipids predominantly lowers the Km for factor X while in contrast factor VIIIa mainly increases the Vmax of factor Xa formation. The implications of these findings for the mechanism of, factor X activation in the intrinsic pathway will be discussed.

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.

In order to study the function of γ-carboxyglutamic acid residues (gla) in factor X(a) we compared the properties of factor X(a) and abnormal factor X(a) from anticoagulated cowl (PIVKA-X(a)), We studied Ca++ and phospholipid binding, spectral changes upon Ca++-binding and the activation by a specific fraction from+ Russells Viper venom (RW). In factor X but+ not in PIVKA-X a cooperative binding of about 5 Caions as well as a cooperative effect of Caions in spectral shift was observed.PIVKA-X can be converted into an active esterase by RVV. This reaction is Ca++ dependent. The Hill coefficient of this dependence is 2.8 for factor X and 1.0 for PIVKA-X. Factor Xa binds to micelles of an equimolar mixture of (dioleoyl) phosphatidyl-serine and dioleoyl phosphatidylcholine in the presence of Ca++ ions. Consistent with the inability of PIVKA-X to bind Ca++ ions cooperatively, PIVKA-X does not bind to phospholipid vesicles. Kinetic experiments on the activity of factor Xand PIVKA-X , in the absence of Ca+ions and phospholipids towards amides shows that both enzymes have identical Km values for each of the substrates but different kcat values. The results indicate a positive effect of gla residues on the rate of hydrolysis of amides. These studies suggest that the gla residues not only function to anchor factor X(a), on phospholipid via Ca++ ions, but that they also assist in obtaining conformational changes in the protein molecule as distant as to influence the active site. Indeed even the “anchor function” may depend upon such changes.

Bovine descarboxyprothrombin and descarboxyfragment-1 can be used as substrates for rat and bovine vitamin K-dependent carboxylase. In both enzyme systems, however, these substrates have a high K m (0.3–0.4 mM). A better substrate ( K m = 0.001–0.003 mM) was prepared from bovine descarboxyprothrombin by limited proteolysis with subtilisin Carlsberg. This substrate is called Fragment-Su and is composed of the amino acids 13–29 of descarboxyprothrombin.

The theory of the kinetics of enzyme cascades is developed. Two types of cascades are recognized, one in which the products are stable ( open cascades ) and another in which the products are broken down ( damped cascades ). It is shown that it is a characteristic of a cascade that the final product appears after a certain lag phase. After this lag phase, the velocity of product formation can be very rapid. It is shown that whereas open cascades will always show a complicated time–product relation, damped cascades can under certain circumstances resemble a simple enzymic reaction. Because the relation between the over-all reaction velocity in the extrinsic coagulation cascade and the concentration of any of the proenzymes in this cascade is a hyperbolic one, it is concluded that this cascade is of the damped type rather than the open type.

After measurement of the conversion of prothrombin to thrombin in a purified system, a model has been constructed for the kinetics of the formation of prothrombin converting activity. In the model phospholipid provides a surface on to which coagulant factors bind.

SummaryIt is shown that factors VIII and IX react with phospholipid in a way fully analogous to the reaction of factors V and X. From this it is concluded that a complex of factors VIII and IX adsorbed onto a phospholipid micelle may be the entity that activates factor X in the intrinsic pathway. The implications of this finding for the concepts of the reaction mechanism of blood coagulation are discussed. It is shown that instead of the original 7-step cascade of Macfarlane, a 4-step cascade consisting partly of complex enzymes is more probable.

SummaryA method is described to prepare artificial reagents to test the factors VII and X individually.A procedure is given to obtain reference tables suitable for the use of these reagents as well as for any other type of specific one-stage in the extrinsic system by means of a computer. The programme is obtainable in Algol, Fortran or Focal.

Although the science of biochemistry has its roots in attempts to apply the knowledge of chemistry to benefit the sick, its evolution has long since brought it to independence, even to the extent that there are biochemists to be found who cannot help regarding with a certain dismay their unfortunate colleagues who have to make a living by working in the esoteric surroundings of a hospital. On the other hand, many a physician is of the opinion that one would have to be a scientist of Boerhaave’s stature to master both the practice of medicine and the basic science of biochemistry, and so they devote themselves to clinical medicine, willfully resigning themselves to forgetting every bit of biochemistry that does not directly help to cure patients. Recent progress in the field of biochemistry unquestionably means that a choice must be made, but although it is true that the examples given above illustrate extremes, they are also the choices most commonly made. Yet, in a volume meant to open a series called after the man who was a master in the basic sciences as well as of clinical medicine, we think it might be appropriate to remind ourselves of the benefit still to be derived from close collaboration between clinical medicine and biochemistry.

The determination of plasma enzyme levels after a myocardial infarction proves to be a useful tool in estimating the size of an infarcted area. A quick but rough estimation can be obtained by measuring lactate dehydrogenase (LDH) (or alpha-hydroxybutyrate dehydrogenase (α-HBDH)) until the maximal value is reached about 48 hours after the infarction occurred (for patients treated with urokinase, about 30 hours). Six to eight samples taken at intervals of about six hours are sufficient for this purpose.

SummaryIn vitamin K deficiency (either absolute or induced by oral anticoagulants) two types of prothrombin occur. One is not distinguishable from normal prothrombin. It generates thrombin quickly in a medium in which the factors V, VII and X, thromboplastin and Ca++ are present in sufficient amounts. The other is converted into thrombin much more slowly under the same conditions. In the onestage prothrombin assay only the first form is measured, in a two-stage prothrombin assay both forms are estimated. This accounts for the well-known discrepancy between these two tests in vitamin K deficiency. The abnormal prothrombin can be considered one of the Proteins Induced by Vitamin K Absence. The occurrence of this kind of proteins fits in the concept of the action of vitamin K as a co-factor in a system that converts polypeptide-precursors into coagulation factors.

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.

SummaryApplication of the methods of enzyme-kinetic analysis to the results of clotting tests is feasible and can yield useful results. However, the standard methods of enzyme kinetics are not applicable without modifications imposed by the peculiarities of the blood-clotting enzyme system. The influence of the following complicating circumstances is calculated :1. Substrate is not present in excess.2. Only relative measures exist for concentrations of substrate or enzymes.3. Enzymes and substrates are often added together.4. Reagents are not pure.5. Clotting-time is our only measure for clotting-velocity.Formulas are deduced, which makes it possible to recognize the effect of these complications.

SummaryApplication of enzyme kinetics to the results of thrombotest estimations in correlation with specific clotting factor estimations has led to the recognition of a protein moiety that occurs in plasma in vitamin K deficiency and acts as a competitive inhibitor of thrombin formation. A hypothesis is given by which the occurrence of this inhibitor is explained in terms of a biphasic synthesis of the vitamin K-dependent clotting factors.

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.

THE activities of the clotting factors II, VII, IX, and X are mutually lowered to a similar degree in chronic hepatic damage as well as during prolonged real or relative (coumar in-induced) vitamin K-deficiency1,2. In the experiments described here, Owren's thrombotest (slightly modified, Fig. 1) is used for the assessment of the extent of depression. Each plasma sample is tested in a series of dilutions (D) and the coagulation time (t, mean of 4 readings) measured in a coagulometer3,4. All plasmas and plasma dilutions are kept in carefully siliconized glassware or plastic material. When t is plotted against D, a straight line is obtained (Fig. 1).

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.

1. 1. Accurate determinations of the shape of the curve relating the ATPase activity of rat-liver mitochondria with the dinitrophenol concentration have shown that a double optimum is obtained at about 0.1 mM and 0.2 mM 2,4-dinitrophenol, respectively, at pH 7. 2. 2. Low concentrations of Amytal have a greater effect on the latter peak, whereas low concentrations of antimycin have a greater effect on the former. 3. 3. The curves relating dinitrophenol-induced ATPase activity to concentration of antimycin or of Amytal show a clearly defined plateau indicating that only part of the ATPase is susceptible to each inhibitor. The effects of Amytal and antimycin are largely additive, and the ATPase is almost completely inhibited by Amytal and antimycin, together. 4. 4. In the absence of dinitrophenol, both inhibitors induce an ATPase, the curves representing the ATPase activity in the presence and absence of dinitrophenol, respectively, running together at concentrations of the inhibitor equal to and higher than the amount giving the maximum ATPase activity in the absence of dinitrophenol. 5. 5. A detailed study of the relationship between ATPase activity, measured in the presence and absence of dinitrophenol, and the concentration of the two inhibitors suggests that the dinitrophenol-induced ATPase at pH 7.0 is made up of two enzyme systems: 62% by an Amytal-sensitive system inducible by dinitrophenol (optimal concn., 0.2 mM) or antimycin; 33% by an antimycin-sensitive system inducible by dinitrophenol (optimal concn., 0.1 mM) or Amytal. 6. 6. It is proposed that the former system involves enzymes concerned in the first phosphorylation step and the second enzymes of the second phosphorylation step. 7. 7. Various theories of oxidative phosphorylation are discussed in the light of these results.

SummaryThe biological disappearance rate of coagulation factors II, VIT, IX and X from plasma is decreased in myxoedema and increased in thyreotoxicosis. In patients with elevated body temperatures, an increase of up to three times the normal rate was observed. The clinical implications of these findings are briefly discussed.

Thrombin generation in platelet-rich plasma (PRP) involves complex interactions between platelets and coagulation proteins. We previously reported that the addition of fibrin to PRP enhances tissue-factor initiated thrombin generation by approximate to 40%, and the current studies were designed to assess the mechanism(s) underlying thrombin generation in the absence and presence of fibrin. Blocking platelet GPIIb/IIIa + alpha v beta 3 receptors with a monoclonal antibody (MoAb) inhibited basal thrombin generation, but did not affect the enhancement produced by fibrin. In contrast, blocking GPIb with any of three different MoAbs had no effect on basal thrombin generation, but essentially eliminated fibrin enhancement of thrombin generation. When thrombin generation was tested in PRP deficient in von Willebrand factor (VWF), both basal and fibrin-enhanced thrombin generation were markedly reduced, and the addition of factor VIII did not normalize thrombin generation. Botrocetin, which induces the binding of vWF to GPIb, enhanced thrombin generation. In all studies, the ability of PRP to support thrombin generation correlated with the production of platelet-derived microparticles and serum platelet-derived procoagulant activity. Thus, two separate mechanisms, both of which depend on vWF, appear to contribute to platelet-derived procoagulant activity: one is independent of fibrin and relies primarily on GPIIb/IIIa, but with a minor contribution from alpha v beta 3; and the other is fibrin-dependent and relies on GPIb. These data may have implications for understanding the mechanisms of the abnormalities in serum prothrombin times reported in Bernard-Soulier syndrome, hemorrhage in von Willebrand disease (vWD), and the increased risk of thrombosis associated with elevated VWF levels. (C) 1999 by The American Society of Hematology.

SummaryThe relation between clotting time and the inverse of prothrombin concentration is shown to be rectilinear.

Heparin and heparin fragments in the molecular mass range 1,700-20,000 Da were examined for their ability to accelerate the antithrombin III (AT III)-dependent inhibition of human factor Xa and the prothrombin converting complex (prothrombinase) during human prothrombin activation. The prothrombinase reaction was modeled by a 3-parameter 2-exponential equation to determine the initial rate of prothrombin activation and the pseudo-first order rate constants of inhibition of prothrombinase and in situ generated thrombin activity. The catalytic specific activities of the heparins increased with increasing molecular size for both the inhibition of prothrombinase and factor Xa. A 10-fold increase over the entire Mr range was found. In contrast to results obtained by others (Ellis, V., Scully, M. F., and Kakkar, V. V. (1986) Biochem. J. 233, 161-165; Barrowcliffe, T. W., Havercroft, S. J., Kemball-Cook, G., and Lindahl, U. (1987) Biochem. J. 243, 31-37), all the heparins showed a 5-fold higher rate of inhibition of factor Xa when compared with the inhibition of prothrombinase, indicating that the factor Va-mediated protection of factor Xa from inhibition by AT III/heparin is independent of the molecular size of the heparin. Our original approach has also revealed a hitherto unrecognized phenomenon, namely, in addition to the accelerating effect of the heparins on the rate of formation of the inactive AT III-factor Xa complex, heparins with Mr greater than 4,500 reduce the initial rate of thrombin generation in the presence of AT III in a concentration-dependent way. We hypothesize that the formation of the dissociable ternary AT III-heparin-factor Xa complex results in a (partial) loss of factor Xa activity towards its natural substrate prothrombin.