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von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets.Briede JJ, Wielders SJ, Heemskerk JW, Baruch D, Hemker HC, Lindhout T.Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands.Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet-rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin-free planar fibrin layers and washed platelets to examine the relationship between platelet-fibrin interaction and exposure of coagulation-stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s(-1) requires fibrin-bound VWF. The cytosolic calcium concentration ([Ca(2+)]i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 +/- 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 +/- 14%) along with a transient elevation in [Ca(2+)]i from 0.05 micro mol L(-1) up to 1.1 +/- 0.2 micro mol L(-1). Platelet adhesion to fibrin at a shear rate of 50 s(-1) is mediated by thrombin but not by fibrin-bound VWF. The [Ca(2+)]i of these thrombin-activated platelets was elevated (0.2 +/- 0.1 micro mol L(-1)), but only a minority of the platelets (11 +/- 8%) exposed PS. The essential role of VWF in this thrombin-induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 +/- 23%) exposed PS and had peak values of [Ca(2+)]i of about 0.6 micro mol L(-1). Taken together, these results demonstrate that thrombin-induced exposure of PS and high calcium response on fibrin-adherent platelets depends on shear- or botrocetin-induced VWF-platelet interaction

Thrombin-induced Hyperactivity of Platelets of Young Stroke Patients.Vanschoonbeek K, Feijge MA, Keuren JF, Coenraad Hemker H, Lodder JJ, Hamulyak K, Van Pampus EC, Heemskerk JW.Dept. of Biochemistry, CARIM, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands.Activated platelets are implicated in the development of premature arterial vascular diseases, in particular ischemic stroke. Since elevated cytosolic [Ca(2+)](i) is an integrative marker of platelet activation, we determined the generation of Ca(2+) signal in stimulated platelets from 26 young patients recuperating from stroke, 20 patients with symptomatic peripheral arterial disease, and 56 healthy volunteers. Even in the presence of aspirin, the platelets from various individuals showed highly different thrombin-induced Ca(2+) responses. On average, the thrombin-induced Ca(2+) response was increased for platelets from either patient group in comparison to the controls (P

Thromb Haemost 2001 Mar;85(3):509-13 Related Articles, Books, LinkOut Contribution of platelet-derived factor Va to thrombin generation on immobilized collagen- and fibrinogen-adherent platelets.Briede JJ, Heemskerk JW, van't Veer C, Hemker HC, Lindhout T.Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, The Netherlands.Adhesion of platelets to immobilized collagen induces the expression of anionic phospholipids, e.g. phosphatidylserine (PS), in the outer leaflet of the plasma membrane of these platelets. In contrast, of the platelets that adhere to immobilized fibrinogen only a small sub-population representing 10 +/- 3% of the total population of the fibrinogen-adherent platelets has exposed PS as probed by annexin V binding. Although the presence of PS is thought to be critical for thrombin generation at the platelet surface, no information is available about the effect of this differential PS exposure on the ability of adherent platelets to support thrombin generation. Perfusion of the fibrinogen- or collagen-adherent platelets with solutions containing factor Xa and prothrombin resulted in thrombin generation that i) increased linear during the first perfusion minutes, ii) was about two-fold faster at collagen-adherent than at fibrinogen-adherent platelets and iii) was for more than 98% restricted to the surface of the adherent platelets. It appeared that the lower thrombin generating capacity of fibrinogen-adherent platelets is not due to a lower overall surface density of PS, but is caused by lower amounts of platelet-bound factor Va. Firstly, in both cases thrombin generation could be completely attenuated with antibodies against human factor Va, and secondly, in the presence of an excess of exogenous plasma-derived factor Va similar initial rates of thrombin formation were measured for collagen- and fibrinogen-adherent platelets. Our findings suggest a unique role for immobilized collagen in maintaining haemostasis.

SummaryRabbit being a common animal model to evaluate the antithrombotic effect of heparins, our purpose was to apply the heparin Standard Independent Unit (SIU) approach to rabbit plasma. To take into account the specificities of the enzymes we have measured the decay constants of factor Xa and of thrombin from autologous and heterologous origins, in presence and in absence of heparin. Different heparins or heparin fractions with a mean molecular weight from 1.7 to 10.5 kDa were used.We found that: a) the decay constants varied strongly between species and between enzymes; b) the decay constants of thrombin were always higher than those of factor Xa; c) the specific anti-factor Xa activity of heparins increased with the molecular weight and was 1.33 times higher when determined with bovine factor Xa than with rabbit factor Xa; d) the specific antithrombin activity of heparins also increased with the molecular weight but was similar when determined with rabbit and human thrombin; e) the specific anti-factor Xa activity was always lower than the specific antithrombin activity; f) the calibration of the heparins and heparin fractions against the 4th International Standard of Heparin expressed in International Units (IU) lead to a systematic overestimation of the anti-factor Xa activity and to an underestimation of the antithrombin activity.These observations indicate that it may be very important to use the SIU approach and to know the accurate activities to better understand the mechanism of the antithrombotic activity of heparins in experimental models.

SummaryWe administered a dose of unfractionated heparin (UFH) and two doses of a low molecular weight heparin (LMWH) to healthy volunteers by SC injection. The doses given were: a) UFH, 5000IU, which represents 8.7 mg of >5,400 MW active heparin (ACLM) and no Excess amounts of platelet factor 4 (PF4) were able to neutralize completely the anti-thrombin activity in normal plasma spiked with enoxaparin as well as in plasma samples obtained after SC enoxaparin injection. With a large excess of PF4 the anti-factor Xa activity could be inhibited to a maximum of 50%. This indicates that ACLM (above critical length material, MW >5400) is neutralized completely by PF4 whereas BCLM (below critical length material, MW We conclude that in PRP the activity of the BCLM fraction of injected LMWH remains after platelet activation. The possible role of this activity in thrombin inhibition and in the antithrombotic action of low molecular weight heparins is discussed.

SummaryThe 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. The amount of Factor IXa present in the plasma sample can be determined with the spectrophotometric assay and is proportional with the amount of plasma present. In plasma from patients receiving vitamin-K antagonists reduced Factor IX activity is found with the spectrophotometric assay, and the new assay method may be useful in monitoring oral anticoagulant therapy.

SummaryAmounts of human brain thromboplastin that do not stimulate thrombin generation in platelet poor plasma, were shown to advance by about 4 min an explosive formation of thrombin that occurs after recalcification in the presence of blood platelets. This synergistic effect is inhibited by the specific thrombin inhibitor hirudin and mimicked by adding low concentrations (< 5 nM) of thrombin to platelet rich plasma. It is our conclusion that, small amounts of thrombin, generated under the influence of thromboplastin induced procoagulant activity in the blood platelets. This activity is most likely mainly due to procoagulant phospholipids. Heparin inhibits this effect and retards the explosive thrombin formation. It does not, however, diminish the peak amount of thrombin eventually formed, because heparin neutralizing material released from the activated platelets quenches the heparin effect.

The individual importance of each of the four vitamin Independent clotting factors on the generation of prothrombinase activity in the plasma of orally anticoagulated patients has been investigated.Addition of purified factors VII, IX or X to plasma from deeply anticoagulated patients (International Normalized Ratio 2.8-4.8) did not influence the amount of prothrombinase activity or the amount of thrombin formed. Only the prothrombin level in the plasma determines the course of thrombin generation. Addition of increasing amounts of purified factor II, VII, IX or X to plasmas deficient in respectively factor II, VII, IX or X showed that the prothrombinase activity increases linearily with the concentration of factor II added and that the concentration below which the factors VII, IX and X start to have a measurable effect on prothrombinase activity are 5%, 20% and 30%, respectively. Half maximal prothrombinase activity was found at about 1% factor VII, 5% factor IX and 8% factor X respectively.From these observations we conclude that primarily the variation in factor II level determines thrombin generation and hence presumably the antithrombotic effect of oral anticoagulant therapy. It therefore seems likely that, for the control of oral anticoagulant therapy, tests that reflect factor II activity would be suitable.

SummaryWe studied the mode of action of the low molecular weight heparin PK10169 and two of its constituent fractions: EMT 966 High Molecular Weight Fraction and EMT 967 Low Molecular Weight Fraction.EMT 966 like standard heparin, acts primarily on thrombin formed and not on prothrombinase (S type heparin). In contrast EMT 967 has no direct effect on thrombin. At high concentrations, it inhibits the prothrombinase complex (P type heparin). PK10169, that contains the two EMTs shows both activities: anti thrombin and antiprothrombinase (mixed type heparin).The addition of increasing amounts of EMT 967 to a constant amount of EMT 966 does not influence the breakdown constant of endogenous thrombin which is determined by the concentration of EMT 966 only. This demonstrates the absence of competition for AT III between the two components of PK10169.In platelet rich plasma, EMT 966 inhibits and postpones thrombin generation more efficiently than unfractionated heparin, probably because it is less sensitive to neutralization by platelet components (platelet factor 4). Amounts of EMT 967 that hardly inhibit thrombin generation in platelet rich plasma enhance the effect of EMT 966 probably by neutralizing platelet factor 4.

SummaryThe enzyme kinetics of competitive inhibition under conditions prevailing in clotting tests are developed and a method is given to measure relative amounts of a competitive inhibitor by means of the t — D plot.

SummaryIt is demonstrated that clotting time in a thrombotest experiment bears a linear relationship to the inverse of factor X concentration under conditions in which the ratio of factor X to the factors II and VII is equal to or smaller than the ratio in normal plasma.A similar relationship occurs with other thromboplastins as long as factor V and fibrinogen are present in excess, i. e. when the dilutions are made with BaSO4 absorbed plasma.Contact activation causes deviation from the observed straight-line relationship.

SummaryPIVKA, the circulating anticoagulant protein found in vitamin K deficiency can, on kinetical grounds, be recognized as an analogue of factor X. The existence of analogues of other vitamin K-dependent clotting factors cannot be ruled out, but need not be assumed to explain the experimental results.

SummaryObservations of the clotting time in one-stage tests in the extrinsic system show, after suitable correction, that there is a rectilinear relationship between the clotting time and the inverse of the concentration of the rate-limiting factor. This cannot be explained by the “cascade” or “waterfall” mechanism proposed as a scheme for the interaction of the blood-clotting factors concerned. This relationship can, however, be explained by a slightly different mechanism.

SummaryWhen normal citrated plasma is stored at 37° C and pH 7.8 the factor VIII activity drops to about 50% of its initial value during the first 8-12 h. In the following 4 days practically no further drop in activity is found. If the logarithm of the factor VIII activity is plotted against time a curve is obtained which can be described as biphasic. To explore the underlying mechanism of this phenomenon the influence of temperature, pH, Ca++ concentration and some other clotting factors was investigated. Between temperatures of 21° C and 45° C the inactivation of factor VIII was biphasic, the decrease of factor VIII being faster in both phases at higher temperatures. The inactivation at these temperatures showed a Q10 of about 2. At 52° C nearly all factor VIII activity disappeared within 8 h. Possibly the precipitation of fibrinogen at this temperature is of influence. Between pH 6.4 and 8.5 the decrease in factor VIII in the first phase was obviously slower at lower pH and the level of the second phase maintained at a higher factor VIII activity. No alteration of the normal inactivation pattern was seen in plasma from patients with congenital deficiencies of factors XII, IX or V or in normal plasma adsorbed with BaS04 which has factors II, VII, IX and X markedly decreased, nor was there any difference between platelet rich and platelet poor plasma. Low calcium concentrations (Resinplasma) markedly increased the rate of inactivation in the first phase, but did not influence the second phase.Four hypotheses are given to explain the biphasic inactivation of factor VIII: a) The presence of an inactivating substance in the first phase or a stabilizing factor in the second phase of factor VIII inactivation. b) The existence of two independent substances with factor VIII activity with different inactivation rates. c) Reversible denaturation of factor VIII in one or more steps. d) Factor VIII exists in plasma in two interdependent molecular forms. It is discussed that in view of the results of the experiments hypotheses a and b are not very likely. At present we cannot differentiate experimentally between c and d.

SummaryThe interaction between stearates and coagulation factors consists of adsorption of the factors and partial activation resulting in thrombin formation. Factors V and XI are most readily adsorbed, closely followed by factor X.Activation is seen most clearly with low concentrations of the adsorbant. It can be inhibited by omission of Ca++ ions and addition of hirudin, thus it involves most probably the generation of thrombin. No thrombin is found in the reaction mixture due to the strong adsorption of thrombin on stearates. The effects of activation are small compared to the effect of adsorption time, pH, temperature and hirudin.Activity of factor V can be demonstrated in the adsorbed state but the factor cannot be eluted. Factor X shows no activity when adsorbed, but can be eluted from the powder. This suggests that the biological activity of factor V resides in a hydro-phylic part of the molecule, whereas the active centre of factor X probably is hidden but not destroyed by hydrophobic surfaces. The implications of these findings for the concept of prothrombinase formation on phospholipids are discussed.

SummaryIt is confirmed that a protein from Russell’s viper venom has a direct activating action on human as well as bovine factor V.Unactivated human factor V has a molecular weight as estimated by gel filtration of ∼410,000, after activation the molecular weight is ∼110,000, thus suggesting the dissociation of a tetramer.The approximative molecular weight figures for unactivated and activated bovine factor V are 400,000 and 195,000, resp. This suggests the dissociation into a dimer accompanying the process of activation.

SummaryCommercial thrombin was shown to contain appreciable amounts of thrombokinase. Passage through a DEAE-cellulose column substantially reduced the thrombokinase titer, although significant contamination could still be detected. Similarly, repeated barium sulfate adsorptions failed to remove the thrombokinase completely.However, barium sulfate adsorptions followed by passage through a column of DEAE-cellulose resulted in an essentially complete removal of the contaminant. Thrombin, so obtained, did not accelerate activation of prothrombin in the presence of calcium, phosphatide and barium carbonate adsorbed serum.Thrombokinase, separated by chromatography, was able to activate prothrombin very slowly in the presence of oxalate. Comparably dilute thrombin fractions and the unfractionated commercial preparation either did not possess this capability, or else this capability was obscured by a concomitant loss of thrombin.

SummaryThe therapeutic range in oral anticoagulation, which is defined as the range of hypocoagulability resulting in optimal thrombosis prophylaxis and an acceptable bleeding tendency, is monitored by assay procedures of which the majority are modifications of Quick’s original prothrombin time test. With respect to individual patients on stabilized anticoagulation, the different modifications commonly used give the same information, the reliability depending more on the accuracy than on the sensitivity of the tests. The results do not appear to be comparable in terms of conventional reference curves or prolongation ratios of the coagulation times found, but on the basis of painstakingly defined normal and patient plasma, the mean of the factor II and factor X activity present in patients’ plasma proved to be a useful common denominator. In terms of this common denominator, the therapeutic range to be aimed at lies between (8 to) 10 and 20(to 25)percent.

The interaction between stearates and coagulation factors consists of adsorption of the factors and partial activation resulting in thrombin formation. Factors V and XI are most readily adsorbed, closely followed by factor X. Activation is seen most clearly with low concentrations of the adsorbant. It can be inhibited by omission of Ca++ ions and addition of hirudin, thus it involves most probably the generation of thrombin. No thrombin is found in the reaction mixture due to the strong adsorption of thrombin on stearates. The effects of activation are small compared to the effect of adsorption time, pH, temperature and hirudin. Activity of factor V can be demonstrated in the adsorbed state but the factor cannot be eluted. Factor X shows no activity when adsorbed, but can be eluted from the powder. This suggests that the biological activity of factor V resides in a hydro-phylic part of the molecule, whereas the active centre of factor X probably is hidden but not destroyed by hydrophobic surfaces. The implications of these findings for the concept of prothrombinase formation on phospholipids are discussed.

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.

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+.

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.