Biomedical subjects

T Lindhout

Publications and source records attributed to T Lindhout.

At least 37 records · Page 2Linked to original sources

PubMed1998

Ex vivo testing of heparin-coated extracorporeal circuits: bovine experiments.

In this study the intrinsic thrombogenicity of the extracorporeal circuits and the benefit of heparin-bonded circuits in an extracorporeal life support system without full systemic heparinization and with minimal interference of the so called material-independent factors was tested in four calves. In two circuits (group A) all blood-contacting surfaces were coated with end-point-attached heparin and the other two were non-coated (group B). Under standardized conditions the calves were perfused at a blood flow rate of 2 L/min. After only one bolus injection of heparin (250 IU/kg body weight) before cannulation, plasma heparin activity rapidly decreased in both groups: half life of about 55 minutes. This decrease of the heparin activity was accompanied by a fall of the activated clotting time (ACT) level to baseline values. The experiments using a heparin-coated circuit, had a runtime of more than 360 minutes, whereas the experiments using a non-coated circuit had to be terminated after a runtime of 255 minutes, because massive fibrin formation was noticed in the circuit. This formation was accompanied by a rapid increase in the line pressure, measured just before the inlet of the oxygenator. The macroscopic inspections after terminating the experiments and rinsing the circuit showed a clean circuit in group A. The fibrinopeptide A (FPA) level increased faster during perfusion with the non-coated circuit than in the heparin coated circuit. Lung histopathological examinations of the lungs of the animals in group A showed no fibrin deposition, whereas most of the blood vessels of the lung preparations of the animals in group B were partially or completely occluded with fibrin. These results suggest that heparin-bonding greatly reduces the thrombogenicity of the extracorporeal circuit, and therefore it can reduce the need for systemic heparinization in an extracorporeal life support system.

Animals↗

PubMed1998

Factor Xa cleavage of tissue factor pathway inhibitor is associated with loss of anticoagulant activity.

Tissue factor:factor VIIa induced activation of blood coagulation is inhibited by the complex between factor Xa and tissue factor pathway inhibitor (factor Xa:TFPI). We recently reported that phospholipid-bound factor Xa reduces the high binding affinity of factor Xa:TFPI for negatively charged phospholipids by a partial degradation of TFPI (17). The present study was undertaken to elucidate the factor Xa cleavage sites in TFPI and to delineate the consequences of this proteolysis with respect to the inhibitory activity of factor Xa:TFPI. We found that phospholipid-bound factor Xa cleaves in TFPI the peptide bonds between Lys86-Thr87 and Argl99-Ala200. Interestingly, Arg199 is the P1 residue of the third Kunitz-type protease inhibitor domain. The fast cleavage of the Arg199-Ala200 bond results in a 50-70% reduction of the anticoagulant activity of factor Xa:TFPI, as determined with a dilute tissue factor assay, but is not associated with a diminished inhibitory activity of factor Xa:TFPI towards TF:factor VIIa catalyzed activation of factor X. On the other hand, the slower cleavage of the Lys86-Thr87 peptide bond was associated with both a diminished anticoagulant and anti-TF:factor VIIa activity. Dissociation of factor Xa from the cleaved TFPI was not observed. These data provide evidence for a dual role of factor Xa since it is the essential cofactor in the TFPI-controlled regulation of TF-dependent coagulation as well as a catalyst of the inactivation of TFPI.

Anticoagulants↗

PubMed1997

Collagen but not fibrinogen surfaces induce bleb formation, exposure of phosphatidylserine, and procoagulant activity of adherent platelets: evidence for regulation by protein tyrosine kinase-dependent Ca2+ responses.

With a combined phase-contrast and fluorescence video imaging system, changes in morphology and cytosolic [Ca2+]i were investigated of fura-2-loaded platelets during adhesion to fibrinogen or collagen matrices. The Ca2+ signals were, on the level of single platelets, compared to the secretion and procoagulant responses, using fluorescent-labeled AK-6 antibody against P-selectin and labeled annexin V for detection of surface-exposed phosphatidylserine (PS), respectively. Platelets in contact with fibrinogen developed filapods and spread over the matrix, in most of the cells without detectable Ca2+ signal. Thrombin induced repetitive spiking in [Ca2+]i, followed by the expression of P-selectin but not of PS on the platelet surface. Platelet interaction with collagen resulted in spreading and transformation of the cells into blebbing, "balloon"-like structures (diameter about 5 microm). The latter morphological changes were accompanied by high and prolonged increases in [Ca2+]i, by the exposure of both P-selectin and PS, and by the ability of the platelets to convert prothrombin into thrombin. Thrombin addition accelerated the onset of the Ca2+ signals and the appearance of surface-exposed PS. Collagen-induced PS exposure was slightly reduced by treatment of the platelets with aspirin, and strongly inhibited by suppression of the Ca2+ responses with prostaglandin E1 or the Ca2+ chelator, dimethyl-BAPTA. Inhibition of protein tyrosine phosphorylation with genistein, U73343, or wortmannin resulted in spiking Ca2+ responses in many of the platelets and in almost complete reduction of bleb formation and PS exposure. In contrast, genistein did not suppress bleb formation and PS exposure of platelets stimulated with the Ca2+ ionophore A23187. We conclude that a collagen but not fibrinogen matrix acts as a potent activator of the procoagulant response through activation of tyrosine kinases and subsequent generation of sustained intracellular Ca2+ signals.

Androstadienes↗

PubMed1997

Prothrombinase is protected from inactivation by tissue factor pathway inhibitor: competition between prothrombin and inhibitor.

The inhibition of prothrombinase by tissue factor pathway inhibitor (TFPI) has been studied in the presence and absence of prothrombin. The rate constant of association of prothrombinase with full-length TFPI was 2.1x10(7) M-1.s-1 and 0.05x10(7) M-1.s-1 for the reaction with C-terminus truncated TFPI (TFPI1-161). The rate constant of dissociation was 0.65x10(-4) s-1 in both cases. The rate constant of inhibition of prothrombinase by TFPI1-161 was similar to that of solution-phase factor Xa. In contrast, phospholipids and factor Va enhanced the association rate of the reaction between factor Xa and full-length TFPI by approx. 20-fold. Although TFPI, and in particular the full-length variant of the molecule, is a potent inhibitor of prothrombinase (overall inhibition constant of 3 pM), we also found that prothrombin competed very effectively with TFPI for the active site of factor Xa in the prothrombinase complex. A 50% reduction of the rate constant of inhibition was measured in the presence of 4 nM prothrombin, i.e. 0.2% of the plasma concentration of prothrombin. The physiological significance of TFPI as an inhibitor of prothrombinase activity is thus questionable.

Animals↗

PubMed1997

Activation of blood coagulation at heparin-coated surfaces.

It is hypothesized that immobilized heparin exerts a dual role in blood coagulation. On the one hand, the heparinized surface is because of its dense negative charge, thought to initiate the intrinsic pathway of blood coagulation. On the other hand, heparin is known as a potent anticoagulant drug. However, it remains to be seen how much contact-phase activation of factor XI contributes to thrombin formation and how this process is counterbalanced by which of the anti-protease activities of immobilized heparin. In the present study we examined the generation of factors XIa, IXa, and Xa, and thrombin in recalcified normal and antithrombin-depleted plasma exposed to polyacrylamide-graft polyurethane (PU) sheets modified by multipoint attachment of two different heparin species. One of them, HAH, contained the specific antithrombin binding sequence and the other one, NAH, had a low affinity for antithrombin and had no anticoagulant activity. Our data demonstrate that in contrast to PU, PU-NAH and PU-HAH are strong mediators of factor XIa and factor IXa formation in normal and antithrombin-deficient plasma. Interestingly, compared to PU-HAH and PU-NAH, thrombin formation was only slightly diminished in antithrombin-deficient plasma exposed to PU. In contrast, thrombin formation was dramatically delayed and diminished in normal plasma exposed to PU-HAH. These findings indicate that very low amounts of factor XIa apparently suffice to induce significant amounts of thrombin. In this sense, heparinized surfaces are highly thrombogenic, but our data also indicate that this activity is effectively counterbalanced by the anti-thrombin activity of the immobilized anti-coagulant species of heparin.

Anticoagulants↗

PubMed1997

Photo-immobilization of dipyridamole (Persantin) at the surface of polyurethane biomaterials: reduction of in-vitro thrombogenicity.

Dipyridamole is a well-known vasodilator and a powerful inhibitor of activation and aggregation of blood platelets. Moreover, dipyridamole is essentially non-toxic. The drug is used extensively in clinical anti-coagulation regimes, for example pre- and post-coronary angioplasty procedures. Recently, we have found that photochemical, covalent coupling of dipyridamole to polyurethane surfaces leads to improved thromboresistance in vitro. This phenomenon is now studied in more detail. Both qualitative and more quantitative biochemical experiments were performed in order to characterize the in vitro blood compatibility of a set of polyurethane surfaces onto which dipyridamole was immobilized. First, scanning electron microscopy was used to examine the morphology of platelets which adhered during incubation with platelet-rich plasma. These experiments showed that immobilization of dipyridamole leads to a clearly decreased number of adherent platelets and to a largely diminished propensity of the surface to activate adherent platelets. Secondly, an in vitro thrombogenicity assay was run. These experiments showed that the thromboresistance increased with increasing surface density of immobilized dipyridamole. A short spacer chain separating dipyridamole from the polymer surface, was found to improve the thromboresistance further. Such a spacer chain apparently increases the efficacy of the immobilized drug. Collectively, the present results further substantiate the idea that dipyridamole retains its inhibitory activity with respect to activation and aggregation of blood platelets, when the compound is covalently attached to a polymer surface. The possible utility of these findings with respect to the development of an artificial blood vessel prosthesis is discussed briefly.

Biocompatible Materials↗

PubMed1997

Prothrombin conversion under flow conditions by prothrombinase assembled on adherent platelets.

Prothrombin activation by prothrombinase was investigated on platelets adhered onto a fibrinogen-coated coverslip mounted in a flow chamber. Once bound to the fibrinogen, platelets gradually changed their morphology: they developed pseudopods, spread over the surface and finally transformed into balloon-shaped cells. This last morphologic change required the presence of calcium and was accompanied by the exposure of procoagulant phospholipid at the outer membrane as detected by the capability of the platelets to bind fluorescein-labelled annexin V. Prothrombinase complexes were allowed to assemble on these adhered platelets by perfusion with factor Xa and varying concentrations of factor Va and prothrombin. The steady-state rate of thrombin formation during continuous flow increased with the prothrombin concentration but not with the factor Va between 0.05 and 0.5 nM. Once prothrombinase was assembled, factor Xa could be omitted from the perfusion mixture without affecting the steady state rate of thrombin production. Our study demonstrates the efficient ability of the procoagulant surface of adherent platelet to support the assembly of stable prothrombinase complexes. Thrombin production was limited by the rate of supply of prothrombin towards the catalytic surface.

Animals↗

PubMed1996

A versatile three-iodine molecular building block leading to new radiopaque polymeric biomaterials.

A methacrylic monomer containing three iodine atoms, 2- [2',3',5'-triiodobenzoyl]-ethyl methacrylate (compound 1), was prepared in pure form. Compound 1 can be reacted with other methacrylates, such as methyl methacrylate (MMA), and 2-hydroxyethyl methacrylate (HEMA) with high conversion. Typically, less than 0.5% of free monomer is left after polymerization. For example, compound 1 was reacted with MMA and HEMA in the molar ratio 7:73:20, respectively. This yielded a terpolymer with Tg = 86 degrees C, Mw = 47,000 g/ mol and Mn = 22,800 g/mol. This material was characterized by various physicochemical techniques, including gel permeation chromatography, differential scanning calorimetry, thermogravimetric analysis, and nuclear magnetic resonance (NMR) spectroscopy (1H at 400 MHz, DMSO-d6 solution). In addition the material was found to exhibit low surface thrombogenicity in vitro and a low propensity to activate contacting blood platelets. Furthermore it was found that the terpolymer is markedly radiopaque: even thin objects (< 0.5 mm) could be easily visualized using X-ray fluoroscopic techniques as are routinely used in the clinic, e.g., during coronary angiography. The combined results obtained with the present terpolymer (particularly its in vitro hemocompatibility and its radiopacity) leads to the suggestion that this type of polymer could be used as cardiovascular biomaterials, for instance for the construction of a new type of endovascular stents. These would be expected to show improved biocompatibility if compared with metallic stents which are currently used, for instance in conjunction with percutaneous transluminal coronary angioplasty (PTCA). A stent prototype, constructed from the present radiopaque terpolymer, is shown and discussed briefly.

Angioplasty, Balloon, Coronary↗

PubMed1996

Tissue factor pathway inhibitor: regulation of its inhibitory activity by phospholipid surfaces.

The basic C-terminus of Tissue Factor Pathway Inhibitor (TFPI) appears to be essential for its anticoagulant activity when tested in a diluted thromboplastin prothrombin time assay. Although the data reported so far have increased our knowledge about the C-terminus as a major binding site for heparin, lipoproteins and phospholipids, it is still unclear how this region of TFPI plays a role in its anticoagulant mode of action. We earlier reported that in the presence of phospholipid the rate of association of factor Xa with full length TFPI (FL-TFPI) is about 10-fold faster than with C-terminus truncated TFPI. This in turn makes that, in vitro, full length TFPI is a more potent inhibitor of tissue factor-factor VIIa catalyzed factor X activation than truncated TFPI. Binding studies, utilizing an ellipsometer, revealed that in contrast to the complex of C-terminus truncated TFPI (TFPI1-161) and factor Xa, the FL-TFPI.factor Xa complex has a high affinity for negatively charged phospholipids. However, when examined in a tubular flow reactor containing tissue factor embedded in a phospholipid bilayer composed of 25 mol% phosphatidyl-serine/75 mol% phosphatidylcholine, no differences in the potency of FL-TFPI and TFPI1-161 to inhibit factor X activation were found. The two variants of TFPI did show an interesting difference though. We found that the quaternary complex of TF.factor VIIa.FL-TFPI.factor Xa was much more stable than the complex containing TFPI1-161. This difference could not be attributed to their different phospholipid-binding properties since the same difference in stability was found on membranes that contained only DOPC.

Binding Sites↗

PubMed1995

Prothrombin contributes to the assembly of the factor Va-factor Xa complex at phosphatidylserine-containing phospholipid membranes.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphoatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min-1) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (< 10 mol%) a linear relationship between the prothrombinase activity and the arithematical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.

Factor Va↗

PubMed1995

Inhibition of prothrombinase at macroscopic lipid membranes: competition between antithrombin and prothrombin.

The kinetics of inhibition of prothrombinase during prothrombin conversion by antithrombin and antithrombin-heparin complexes was studied in a tubular flow reactor. Prothrombinase was assembled at a macroscopic phospholipid membrane, composed of 25 mol % phosphatidylserine and 75 mol % phosphatidylcholine, deposited on the inner wall of a glass capillary, by perfusion with a factor Xa-factor Va mixture. Measurement of thrombin production allowed estimation of the amount of prothrombinase present at the capillary wall. Perfusion with a mixture of prothrombin and antithrombin or antithrombin-heparin complexes caused a progressive decline of the prothrombinase activity. The rate of inactivation steeply decreased with increasing prothrombin concentrations, indicating competitive inhibition. Analysis of competitive inhibition data requires estimation of the time-dependent substrate concentration, Co, near the prothrombin converting surface using earlier developed transport theory [Billy, D., et al. (1995) J. Biol. Chem. 270, 1029-1034]. It appears that the inhibition rate is proportional to the fraction of enzyme, Km/(Km+Co), not occupied by substrate. The value of Km of prothrombinase estimated from the dependence of the inhibition rate on the prothrombin concentration (Km = 2-3 nM) is in excellent agreement with the value estimated from the substrate conversion rate (Km = 3 nM). Therefore inhibition of prothrombinase by antithrombin and antithrombin-heparin complexes is fully competitive with the substrate: prothrombin. Our results show that prothrombinase assembled on macroscopic lipid surfaces by virtue of its low Km value is protected for inhibition due to highly effective competition of prothrombin with antithrombin for the active site of factor Xa.

Animals↗

PubMed1995

Prothrombin activation by prothrombinase in a tubular flow reactor.

Thrombin production by the phospholipid-bound complex of blood clotting factors Xa and Va (prothrombinase) was studied in a tubular flow reactor. The inner wall of a glass capillary was coated with a phospholipid bilayer of 25% phosphatidylserine and 75% phosphatidylcholine. Prothrombinase was assembled on this bilayer by perfusion with a mixture containing an excess of factor Va (2 nM) and a limiting amount of factor Xa (1-100 pM), either in the absence or presence of prothrombin. The rate of assembly of prothrombinase in the presence of prothrombin appeared to be limited by the transfer rate of factor Xa to the phospholipid surface. A good agreement was found between the predicted mass transfer coefficient for factor Xa and the observed pre-steady state rate of thrombin production. The eventually obtained steady state rates of thrombin production were proportional to the prothrombin concentration and independent of the surface density of prothrombinase. The observed rate of thrombin production was in excellent agreement with the predicted mass transfer rate for prothrombin. Transport-limited prothrombin conversion was observed for prothrombinase densities exceeding 1 fmol/cm2, which corresponds to 0.05% occupation of available binding sites. The kinetic parameters of the reaction were determined at low prothrombinase densities (0.02-0.04 fmol/cm2). Even in this situation the Michaelis-Menten equation had to be corrected for substrate depletion near the catalytic surface. We hereto employed an accurate approximation of the mass transfer coefficient. The kinetic parameter kcat was 60 s-1 and the intrinsic Km had a surprisingly low value of 3 nM. Both parameters were not influenced by the wall shear rate.

Biotechnology↗

PubMed1995

Antithrombin activity of surface-bound heparin studied under flow conditions.

Polyacrylamide-grafted polyetherurethane sheets were modified by end-point and multipoint attachment of heparin. The surface-bound heparin was firmly attached. No release of heparin activity could be detected when the surface was rinsed at a wall shear rate of 2000 s-1. Uptake of antithrombin and thrombin inactivation were investigated under well-defined flow conditions by the use of a spinning device with an attached disk-shaped heparinized surface. It is demonstrated that the rate of thrombin inactivation at the antithrombin-heparin surface equals the maximal rate of transport of thrombin toward the surface when the surface coverage of antithrombin exceeds 10 pmol/cm2. This result indicates that a higher intrinsic catalytic efficiency of a surface does not necessarily result in a higher antithrombin activity. We varied the heparin content of the surfaces between 0 and 35 micrograms/cm2 by increasing the number of functional groups to which heparin could be covalently attached. The uptake of antithrombin increased with the heparin content of the surface, but the stoichiometry decreased from 2 to 0.5 pmol antithrombin/micrograms heparin. Apparently, antithrombin could not bind to heparins buried in the poly(acrylamide) layer. The rate of thrombin inactivation at surfaces with low heparin content (2 micrograms/cm2) fells below the transport limit of thrombin and became proportional with the heparin content of the surface. Although the contribution of surface-bound heparin to the neutralization of fluid-phase thrombin was found to be negligible compared with the effect of fluid-phase antithrombin at physiologic relevant concentrations, these heparinized surfaces markedly delayed the onset of thrombin generation in platelet-rich plasma.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombins↗

PubMed1995

Inhibition of prothrombinase by antithrombin-heparin at a macroscopic surface.

The antithrombin-dependent inhibition of prothrombinase, assembled at a macroscopic surface, was studied under flow conditions utilizing a tubular flow reactor that consists of a phospholipid-coated glass capillary. Prothrombinase activity was determined from steady-state rates of thrombin production upon perfusion with prothrombin and from factor Va-associated factor Xa activity present in the flow reactor. The prothrombinase density was maintained at a low level (0.03 fmol/cm2) to assure that the rate of thrombin production reflected the amount of prothrombinase present in the capillary. Perfusion of the flow reactor with antithrombin resulted in an exponential decrease of prothrombinase activity in time. The second order rate constant (8.5 x 10(4) M-1min-1) is comparable with the rate of inactivation of free factor Xa. Inhibition was much faster when antithrombin was complexed with heparin. The second order rate constants of inhibition decreased with decreasing heparin chain length: 9.6 x 10(7), 4.5 x 10(7) and 0.39 x 10(7) M-1min-1 for unfractionated heparin, low molecular weight heparin and synthetic pentasaccharide heparin, respectively. In the presence of prothrombin (0.2 microM), however, the heparin-dependent rate of inhibition of prothrombinase was about 50-fold lower. The heparin-independent inhibition of prothrombinase by antithrombin (4 microM) in the presence of prothrombin (0.2 microM) was virtually negligible. At a 70-fold higher surface density of prothrombinase (2 fmol/cm2) prothrombinase activity was much faster inactivated. The rate of thrombin production, however, was not affected. In conclusion, at low prothrombinase densities, prothrombin efficiently protects prothrombinase from inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Antithrombin III↗

PubMed1995

Kinetics of the inhibition of tissue factor-factor VIIa by tissue factor pathway inhibitor.

Tissue factor-factor VIIa catalysed activation of factor IX is inhibited by the complex of tissue factor pathway inhibitor (TFPI) and factor Xa. At present, no information is available as to what extent the kinetics of complex formation between TFPI and factor Xa during factor X activation contribute to the overall rate of inactivation of the factor X converting complex. We have determined the kinetic parameters of the individual reactions, i.e. factor X activation, formation of the TFPI-factor Xa complex, and inactivation of tissue factor-factor VIIa by the TFPI-factor Xa complex. We modelled the overall reaction by assuming a two-step reaction: factor Xa generated by tissue factor-factor VIIa forms a reversible complex with TFPI and in the second step this complex forms a reversible quaternary complex with tissue factor-factor VIIa. The validity of the model was demonstrated by analysis of factor Xa generation curves in the presence of TFPI. Independently determined constants for factor X activation (kcat = 12 s-1, Km = 70 nM) and inhibition of tissue factor-factor VIIa by TFPI-factor Xa complex (rate constant of inhibition of 1.1 x 10(8) M-1S-1) were used. The association rate constant of the formation of the TFPI-factor Xa complex was estimated by fitting the model to the data.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1995

Inhibition of factor X activation at extracellular matrix of fibroblasts during flow conditions: a comparison between tissue factor pathway inhibitor and inactive factor VIIa.

Tissue factor pathway inhibitor (TFPI) is a naturally occurring factor Xa-dependent inhibitor of factor VIIa/tissue factor activity. In the present study, we examined the importance of the TFPI C-terminus and 3rd Kunitz-like domain for the inhibitory capacity of TFPI towards factor VIIa/tissue factor-catalyzed factor X activation and compared the inhibition with that of inactivated factor VIIa (factor VIIai). The extra-cellular matrix of fibroblasts, mounted in a parallel-plate flow chamber, were perfused with reaction mixtures that contained factors X, VIIa, and varying amounts of TFPI or factor VIIai. Inhibition was evaluated from the time course of factor Xa production at the outlet of the flow chamber. The factor VIIa/tissue factor-catalyzed factor Xa production was inhibited by factor VIIai and compatible with a direct competition between factor VIIai for tissue factor. In contrast, TFPI showed a progressive inhibition of factor Xa production; the initial rate of factor X activation, however, was not inhibited by TFPI. Inhibition of factor Xa generation already in progress was seen for TFPI but not factor VIIai. In both cases we found that the truncated TFPI variants were as potent as full length TFPI. As to the stability of the enzyme-inhibitor complexes, TFPI-/Xa/VIIa/tissue factor and factor VIIai/tissue factor, marked differences were observed. About 60% of the factor VIIa/tissue factor activity was recovered from the truncated TFPI/Xa/VIIa/tissue factor complex after 150 min of perfusion with reaction mixtures that contained factors X and VIIa. In contrast, full length TFPI did not dissociate from the complex, nor could factor VIIai be displaced by a large excess of factor VIIa.

Drug Stability↗

PubMed1994

Kinetics of the inhibition of human factor Xa by full-length and truncated recombinant tissue factor pathway inhibitor.

The inhibition equilibrium and kinetics of association and dissociation of the binding of three types of recombinant tissue factor pathway inhibitor (TFPI), namely full-length TFPI, C-terminal-truncated TFPI, and TFPI without the third Kunitz domain (TFPI1-161), to factor Xa have been measured. Formation and dissociation of the complexes were monitored by continuous measurement of the changes in the rate of hydrolysis of a peptidyl-p-nitroanilide substrate. Progress curves of product formation were fitted to a set of equations describing a one-step bimolecular inhibitory reaction in the presence of a competing substrate. For full-length TFPI the rate constants of association (kon) and dissociation (koff) were (5.1 +/- 0.7) x 10(6) M-1.s-1 and (2.6 +/- 0.9) x 10(-4)s-1 respectively. Thus, although the inhibition constant (50 pM) is far below the plasma concentration (2.5 nM) of TFPI, the half-time for transition to equilibrium in plasma is rather long (66s). The truncated forms of TFPI differ in that they have a 4-fold lower kon value but a similar dissociation rate constant. Therefore the inhibition constant, Ki, is 4-fold higher (0.2 nM) and the half-time to achieve equilibrium is prolonged to 250 s. The kon values of full-length and C-terminal-truncated TFPI, but not that of TFPI1-161, were found to decrease with increasing ionic strength.

Binding Sites↗

PubMed1994

Studies on two new radiopaque polymeric biomaterials.

Two new polymeric materials (polymers A and B) containing covalently bound iodine were prepared. These polymers were evaluated with respect to their possible use as radiopaque implant biomaterials--that is, materials that are visible in a noninvasive manner using routine X-ray absorption imaging techniques. Polymer A is a copolymer of methyl methacrylate (MMA) and 1 (80 and 20 mol%, respectively). Polymer B was prepared from MMA, 1, and 2-hydroxyethyl methacrylate (HEMA) (mol ratio 65:20:15, respectively). Compound 1 was synthesized from 4-iodophenol and methacryloyl chloride. The resulting polymers were characterized with GPC, DSC, NMR, and by measuring both the advancing and receding contact angles. Thrombogenicity of the polymers was determined by an in vitro thrombin generation test procedure. The maximum concentration of free thrombin was 76 +/- 1 nM for polymer A, and 64 +/- 3 nM for polymer B. The lag times (i.e., time onset of thrombin generation) were 392 seconds for polymer A and 553 seconds for polymer B. For PVC-T, which is known as a passive material, a lag time of 583 seconds was found. This indicates that polymer B is comparable to PVC-T, and more passive than polymer A. Polymer A exhibited minor activation of platelets. Polymer B did not induce platelet activation at all. The polymers exhibited, even as fibers with a diameter of ca. 0.3 mm, good radiopacity with routine imaging X-ray techniques in the clinic.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorptiometry, Photon↗