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We developed a method which enables as to compute the course of prothrombinase activity in clotting plasma (H.C. Hemker, G.M. Willems, S. Béguin: Thromb. Haemostas. 56, 9-17, 1986) and used this for a study of the effect of pentosan polysulphate (PPS) on thrombin generation.When added to normal plasma in the concentration range of 0-8 μg/ml PPS induces a linear increase of the pseudo first order decay constant of endogenous thrombin like heparin does, 1 ug of PPS being equivalent to 0.045 Aig of heparin. Contrary to heparin this action is partly (∼ 65%) dependent upon AT III and partly (∼ 35%) upon heparin cofactor II.In normal plasma PPS causes an inhibition of both extrinsic and intrinsic prothrombinase formation. Only in the intrinsic system an increase of the lag time of prothrombinase appearance is observed. Unlike heparin, PPS does not inhibit factor IXa induced thrombin formation neither does it inhibit prothrombinase formation in the presence of preactivated factor VIII. The prolongation of the lag times must therefore be ascribed to inhibition by PPS of the activation of factor VIII.The inhibition of extrinsic prothrombinase formation by PPS increases with progressive dilution of thromboplastin and is not seen in haemophilia A or B plasma. This demonstrates the existance of a factor VIII and IX dependent process in extrinsic coagulation that gains in importance when the potency of factgr VII-tissue factor complex decreases, i.e. the Josso pathway.PPS, but also heparin causes an unexplained increase of prothrombinase action in haemophIIic plasma. The same phenomenon may be expected to exist in normal plasma, be it obscured by a concomitant inhibition. This, together with the incomplete inhibition of factor VIII activation by PPS makes that we cannot use this inhibitor as a means to quantitate the Josso pathway. The best estimate that we can obtain is that, in the presence of 2% thromboplastin, the factor IX dependent activation of factor X contributes more then 20% to prothrombinase generation.

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.

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.

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.