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T Lindhout

Publications and source records attributed to T Lindhout.

At least 19 recordsLinked to original sources
PubMed1992

Activation of factor X and its regulation by tissue factor pathway inhibitor in small-diameter capillaries lined with human endothelial cells.

The activation of factor X at the surface of endothelial cells was investigated under controlled flow conditions. A method is described for preparing polyethylene capillaries whose inner walls are covered with a confluent layer of human umbilical vein endothelial cells. To obtain a stable and unperturbed layer of endothelial cells it was essential to pre-perfuse the endothelialized capillaries with medium for about 18 hours. At this stage no tissue factor activity could be detected, but when the seeded cells were perfused with medium containing tumor necrosis factor (TNF) a maximum steady-state rate of factor Xa production (16 fmol factor Xa/min/cm2) was observed within 8 hours. Further experiments were performed with endothelial cells incubated for 4 hours with TNF. Factor Xa was produced at a rate of 7 fmol factor Xa/min/cm2 on perfusion of the capillaries with factor X (100 nmol/L) and factor VII (0.1 U/mL) at a shear rate of 34 s-1. The extracellular matrix preparations of these cells produced factor Xa at a 20-fold higher rate (150 fmol factor Xa/min/cm2). In both cases factor Xa formation was dependent on the presence of factor VII and was completely inhibited when the perfusate also contained 5 nmol/L recombinant tissue factor pathway inhibitor (rTFPI). Pre-perfusion with factor Xa-TFPI complex in the absence of factor VIIa caused a much lesser inhibitory effect, suggesting that TFPI-mediated neutralization of endothelial cell and matrix tissue factor activity requires the presence of factor VIIa in addition to the presence of factor Xa.

Animals↗
PubMed1992

Activation of prothrombin in the presence of human umbilical-vein endothelial cells.

Addition of Factor Xa, Factor Va and prothrombin to immobilized cultured human umbilical-vein endothelial cells resulted after a time delay in thrombin formation. The prothrombin-converting (prothrombinase) activity, however, was not associated with the cell surface. Rather, perturbation by thrombin, either formed in situ or exogenously added, induced a procoagulant phospholipid surface in the fluid phase, which, in the presence of Factor Xa and Factor Va, enabled the assembly of prothrombinase.

Annexin A5↗
PubMed1992

Ratios of anti-factor Xa to antithrombin activities of heparins as determined in recalcified human plasma.

Anti-factor Xa and anti-thrombin activities of unfractionated (UF) and low molecular weight (LMW) heparins have been measured in human plasma and with purified human antithrombin III (ATIII) in the absence and presence of 1.5 mM calcium. The anti-factor Xa and anti-thrombin activities were measured directly, by assessing the heparin-dependent pseudo-first order rate constants of inactivation of human factor Xa or thrombin. These activities were studied with the 4th International Standard for UF heparin, the 1st International Standard for LMW heparin, CY216, enoxaparin, CY222, and the synthetic pentasaccharide. In plasma, calcium equally well increased the specific anti-factor Xa catalytic activities as compared to purified ATIII. That is, 1.5 mM calcium stimulated the UF standard heparin-catalysed inactivation of factor Xa 2.1-2.4 times. In the presence of the LMW heparins the effect of calcium was smaller (1.3-1.7 times), and in plasma there was no effect of calcium on the pentasaccharide-catalysed inactivation of factor Xa. Thus, the largest effects of calcium in the inactivation reaction of factor Xa is seen with UF standard heparin. Calcium reduced the anti-thrombin activities of all the heparin preparations studied about 1.5 times when purified ATIII was used, although in plasma this effect was less clear. Consequently, in the presence of 1.5 mM calcium the ratio of the anti-factor Xa to the anti-thrombin activities of UF standard heparin approximated those of the LMW heparins. The only exception was CY222, which under all conditions retained anti-factor Xa/anti-thrombin ratios significantly higher than those of UF standard heparin.

Antithrombin III↗
PubMed1991

Low molecular weight heparin-catalyzed inactivation of factor Xa and thrombin by antithrombin III--effect of platelet factor 4.

Low molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca(2+)-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III↗
PubMed1991

Flow and the inhibition of prothrombinase by antithrombin III and heparin.

Inhibition of prothrombinase by antithrombin III (ATIII) and heparin was investigated in a continuous-flow system. Phospholipid-coated capillaries, containing phospholipid-bound factor Xa and factor Va, were perfused with 1.0 mumol/L prothrombin and 0.5 nmol/L factor Va. At 25 degrees C and a flow rate of 32 microL/min (shear rate 28 seconds-1) the steady-state rates of prothrombin conversion depended linearly on the surface concentration of prothrombinase up to 2 fmol/cm2. The rate of thrombin generation was 952 +/- 43 (SE) mol/min/mol prothrombinase. When ATIII was included in the perfusate for 10 minutes, the free thrombin concentration at the outlet of the capillary was markedly reduced: a 50% neutralization was obtained at 0.7 mumol/L ATIII. However, the prothrombinase activity was not inhibited, as could be established after a subsequent perfusion with prothrombin and factor Va. At an ATIII concentration typical of normal plasma (2 mumol/L) a slight neutralization of prothrombinase was observed: 10% neutralization following a 10-minute perfusion. During a perfusion with ATIII in the absence of prothrombin, or in its presence with hirudin (2 mumol/L) also included in the perfusate, a more pronounced neutralization of prothrombinase was observed: 40% residual activity was obtained after a 10-minute perfusion. From this observation the suggestion comes forward that thrombin, continuously produced at the surface, consumes ATIII in the boundary layer. In this case the true ATIII concentration in the vicinity of surface-bound prothrombinase will be but a small fraction of the initial ATIII concentration in the bulk fluid. Unfractionated heparin and an ultra-low molecular weight heparin (pentasaccharide) did enhance the ATIII-dependent neutralization of prothrombinase, but to a much lesser extent than observed with small unilaminar phospholipid vesicles as the catalytic sites for prothrombinase assembly. The findings reported here support the notion that regulation of prothrombinase by heparin under in vivo conditions occurs at the stage of its formation, ie, through inhibition of free factor Xa and/or the generation of factor Va, rather than by direct inhibition of the prothrombinase activity.

Antithrombin III↗
PubMed1991

Simulation model for thrombin generation in plasma.

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.

Computer Simulation↗
PubMed1990

Continuous flow and the prothrombinase-catalyzed activation of prothrombin.

The activation of prothrombin by prothrombinase was investigated in a continuous flow system at 25 degrees C. A glass capillary, containing a continuous phospholipid bilayer attached to the interior surface, was first perfused with factor Va. The factor Va bound to the phospholipid surface functioned as sites for the formation of prothrombinase, when subsequently a factor Xa and prothrombin containing solution was perfused. Under the conditions used, steady-state rates of prothrombin activation were attained after 4 to 15 min. The rates of prothrombinase formation increased with increasing factor Xa concentrations and flow rates, which is compatible with the assembly of prothrombinase being dependent on the flux of factor Xa to the phospholipid-bound factor Va. As long as factor Xa and prothrombin were present in the fluid phase the assembly of prothrombinase was apparently irreversible; during at least 20 min no loss of activity occurred. The steady-state rate of prothrombin activation was dependent on the surface concentration of prothrombinase, at 1.0 microM prothrombin and a shear rate of 82 s-1 the average rate was 870 mol thrombin/min per mol prothrombinase. In contrast to test tube experiments it was observed that in this flow system, the formation of alpha-thrombin is favoured above the formation of meizothrombin (des fragment 1).

Adsorption↗
PubMed1990

The anticoagulant mechanism of action of recombinant hirudin (CGP 39393) in plasma.

We studied the inhibitory action of recombinant desulphatohirudin (CGP 39393) on thrombin generation in whole plasma. Human plasma was activated either with thromboplastin or factor IXa. Hirudin delayed thrombin generation, but it was unable to prevent the explosive appearance of thrombin. The dose-dependent prolongation of the lag phase of the intrinsic and extrinsic thrombin generation curve was not the result of titration of thrombin activity by hirudin but the result of a delayed formation of the prothrombin converting complex (prothrombinase). In case of extrinsic activation, hirudin did not affect factor Xa generation, but prolonged the lag phase of the factor Va generation curve, causing its appearance when factor Xa generation was already in the decay phase. Because of its inhibitory action on the thrombin-mediated activation of factor VIII, hirudin prolonged the lag phase of the factor X converting complex that consists of factor IXa and factor VIIIa. Our observations with hirudin are in keeping with the notion that inhibition of the thrombin-mediated amplification reactions in blood coagulation is a very efficient way to delay or inhibit completely thrombin generation. However, although hirudin neutralizes stoichiometric amounts of thrombin, the interaction between in situ generated thrombin and hirudin appears not to be fast enough to prevent trace amounts of thrombin to activate factors VIII and V. Consequently, an explosive thrombin generation is observed even when free hirudin is present.

Amino Acid Sequence↗
PubMed1990

Heparin-stimulated inhibition of factor IXa generation and factor IXa neutralization in plasma.

Generation and inhibition of activated factor IXa was studied in factor XIa-activated plasma containing 4 mmol/L free calcium ions and 20 mumol/L phospholipid (25 mol% phosphatidylserine/75 mol% phosphatidylcholine). Interference of other (activated) clotting factors with the factor IXa activity measurements could be avoided by using a highly specific and sensitive bioassay. Factor IXa generation curves were analyzed according to a model that assumed Michaelis-Menten kinetics of factor XIa-catalyzed factor IXa formation and pseudo first order kinetics of inhibition of factor XIa and factor IXa. In the absence of heparin, factor IXa activity in plasma reached final levels that were found to increase with increasing amounts of factor XIa used to activate the plasma. When the model was fitted to this set of factor IXa generation curves, the analysis yielded a rate constant of inhibition of factor XIa of 0.7 +/- 0.1 min-1 and a kcat/Km ratio of 0.29 +/- 0.01 (nmol/L)-1 min-1. No neutralization of factor IXa activity was observed (the estimated rate constant of inhibition of factor IXa was 0). Thus, in the absence of heparin, the final level of factor IXa in plasma is only dependent on the initial factor XIa concentration. While neutralization of in situ generated factor IXa in normal plasma was negligible, unfractionated heparin dramatically enhanced the rate of inactivation of factor IXa (apparent second order rate constant of inhibition of 5.2 min-1/per microgram heparin/mL). The synthetic pentasaccharide heparin, the smallest heparin chain capable of binding antithrombin III, stimulated the inhibition of in situ generated factor IXa, but sevenfold less than unfractionated heparin (k = 0.76 min-1 per microgram pentasaccharide/mL). We found that free calcium ions were absolutely required to observe an unfractionated heparin and pentasaccharide-stimulated neutralization of factor IXa activity. Factor XIa inhibition (psuedo first order rate constant of 0.7 min-1) was not affected by unfractionated heparin or pentasaccharide in the range of heparin concentrations studied.

Activation Analysis↗
PubMed1990

The effect of sulfation on the anticoagulant and antithrombin III-binding properties of a heparin fraction with low affinity for antithrombin III.

Heparin with low affinity for antithrombin III (ATIII) and devoid of anticoagulant activity was chemically oversulfated and fractionated by affinity for ATIII. The oversulfated material showed ATIII binding properties, as monitored by intrinsic fluorescence enhancement of ATIII. The fluorescence increase was comparable to that of the AT III high affinity fraction of native heparin. The estimated dissociation constants however, showed a 10-fold weaker binding of the oversulfated material to ATIII, Kd = 6.4 x 10(-8) M, as compared to native heparin, Kd = 0.63 x 10(-8) M. Concomitant with the binding-induced allosteric change in ATIII, the oversulfated material stimulated the ATIII-thrombin and ATIII-factor Xa reactions. The high affinity fractions of native heparin and the sulfated material were almost equally effective in enhancing the rate of thrombin neutralization by ATIII. However, a 3-fold faster rate of factor Xa inactivation was found with the native high affinity material.

Antithrombin III↗
PubMed1989

In situ-generated thrombin is the only enzyme that effectively activates factor VIII and factor V in thromboplastin-activated plasma.

We investigated the activation of the nonenzymatic protein cofactors factor VIII and factor V in plasma when coagulation was initiated by thromboplastin. With sensitive bioassays, we were able to measure specifically the generation of activated factor VIII and activated factor V in plasma. Our results showed that when plasma was triggered with a relatively high concentration of thromboplastin, factor VIII and factor V were completely activated at the clotting time of plasma. However, when the generation of thrombin, but not that of factor Xa, was delayed by addition of hirudin to the plasma, factor Va was generated only at the time thrombin generation overcame the hirudin inhibition. In addition, generation of factor VIIIa correlated with thrombin generation and not with factor Xa generation. Furthermore, addition of large amounts of factor Xa to hirudinized plasma did not show detectable factor VIII or factor V activation. We concluded that in plasma activated with thromboplastin the enzyme responsible for activation of factor V and factor VIII is thrombin, not factor Xa.

Blood Coagulation↗
PubMed1989

Antithrombin III-dependent anti-prothrombinase activity of heparin and heparin fragments.

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.

Antithrombin III↗
PubMed1989

A further insight into the binding of blood clotting factors to membranes.

The active site of factor Xa, labelled with dansylglutamylglycylarginine (DnsEGR) is sensitive to association with Ca2+, factor Va and phospholipids. When bound to factor Va, DnsEGR-factor-Xa does not change the composition of the binding site of factor Va, as shown by fluorescence energy-transfer experiments between the Trp residues of factor Va and pyrene-labelled phospholipids. Prothrombin was cleaved by alpha-chymotrypsin into two parts: N-terminal residues 1-41 (peptide 1-41) containing the gamma-carboxyglutamic acid residues (Gla), and des-(1-41)-prothrombin; their membrane association was investigated. Peptide 1-41 contains the aromatic residues Tyr and Trp in positions 24 and 41, respectively, and is suitable for fluorescence spectroscopy. The absence of fluorescence energy transfer between these residues suggests that they are more than 2.8 nm apart. Binding of Ca2+ and of phospholipids involves essentially the Tyr residue, while the C-terminal characteristics of the Trp residue remain unchanged. The conformational change which takes place on binding does not shorten the distance between Tyr and Trp beyond 2.8 nm. Our conclusion is that peptide 1-41 has an extended conformation. This result is compatible with the disordered character of the Gla region found in the crystalline structure of fragment 1 of prothrombin. Ca2+ induces a greater fluorescence energy transfer between prothrombin and membranes labelled with pyrene but has no influence on the binding of des-(1-41)-prothrombin. Moreover, the binding curves of des(1-41)-prothrombin are similar to those of prothrombin in the absence of Ca2+. It is concluded that the Ca2+-independent association of prothrombin with membranes involves essentially that part of the prothrombin molecule deleted in the Gla region.

Affinity Labels↗
PubMed1989

The mode of action of low molecular weight heparin preparation (PK10169) and two of its major components on thrombin generation in plasma.

We 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: antithrombin 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.

Heparin, Low-Molecular-Weight↗
PubMed1989

The effect of trace amounts of tissue factor on thrombin generation in platelet rich plasma, its inhibition by heparin.

Amounts 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 (less than 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.

Blood Platelets↗
PubMed1989

Inhibition of phospholipid and platelet-dependent prothrombinase activity in the plasma of patients with lupus anticoagulants.

Prothrombinase activity was analysed in the plasma of a series of patients with lupus anticoagulants (LAC). In the presence of purified PS-PC (20-80%) vesicles the prothrombinase activity triggered by kaolin was retarded by 2-3 min with respect with normal plasma. The maximal values of prothrombinase activity increased by increasing the amount of phospholipid vesicles. However, in the plasma of the patients they were always lower than those of normal plasma at each phospholipid concentration. Platelet-dependent prothrombinase activity was subsequently investigated. Again, both a delay in appearance and reduced peak values of prothrombinase activity were observed in the plasma of the patients. This inhibition was partially overcome by the addition of an excess of purified phospholipids. Finally, the effect of LAC IgG on platelet rich plasma-dependent prothrombinase activity was investigated. The main effect observed was a delay of the peak time of prothrombinase activity, while the maximal peaks were affected only by one IgG preparation. We conclude that LAC antibodies can react with both purified negatively-charged phospholipids and platelet procoagulant phospholipids and inhibit prothrombinase activity in a similar way in both cases.

Adult↗
PubMed1988

The mode of action of heparin in plasma.

The influence of heparin on prothrombin conversion in plasma was estimated by measuring prothrombin disappearance with the aid of staphylocoagulase or by calculation from the thrombin generation curve, compensating for simultaneous thrombin inactivation by plasma protease inhibitors. In thromboplastin-activated plasma the effect of heparin on prothrombinase (factor Xa-factor Va-phospholipid) is negligible compared to that on thrombin probably because both the extrinsic factor X activating complex and phospholipid bound factor Xa are insensitive to AT III-heparin action. In contact-activated plasma prothrombinase generation is inhibited by heparin, because heparin lowers the ambient concentrations of thrombin so that the feedback activation of factor VIII by thrombin is diminished. Consequently, the delayed factor Xa generation causes a lag phase in prothrombinase generation. We conclude that heparin in plasma acts by its thrombin scavenging properties. No direct action on prothrombinase or on the factor X activating enzyme needs to be assumed if one takes into account the effect of thrombin depletion on thrombin-mediated feedback reactions.

Heparin↗