Biomedical subjects

B N Bouma

Publications and source records attributed to B N Bouma.

At least 73 records · Page 4Linked to original sources

PubMed1996

Role of the A+ helix in heparin binding to protein C inhibitor.

Interactions between proteins and heparin(-like) structures involve electrostatic forces and structural features. Based on charge distributions in the linear sequence of protein C inhibitor (PCI), two positively charged regions of PCI were proposed as possible candidates for this interaction. The first region, the A+ helix, is located at the N-terminus (residues 1-11), whereas the second region, the H helix, is positioned between residues 264 and 280 of PCI. Competition experiments with synthetic peptides based on the sequence of these regions demonstrated that the H helix has the highest affinity for heparin. In contrast to previous observations we found that the A+ helix peptide competed for the interaction of PCI with heparin, but its affinity was much lower than that of the H helix peptide. Recombinant PCI was also used to investigate the role of the A+ helix in heparin binding. Full-length (wild-type) rPCI as well as an A+ helix deletion mutant of PCI (rPCI-delta 2-11) were expressed in baby hamster kidney cells and both had normal inhibition activity with activated protein C and thrombin. The interaction of the recombinant PCIs with heparin was investigated and compared to plasma PCI. The A+ helix deletion mutant showed a decreased affinity for heparin in inhibition reactions with activated protein C and thrombin, but had similar association constants compared to wild-type rPCI. The synthetic A+ helix peptide competed with rPCI-delta w-11 for binding to heparin. This indicated that the interaction between PCI and heparin is fairly non-specific and that the interaction is primarily based on electrostatic interactions. In summary, our data suggest that the H helix of PCI is the main heparin binding region of PCI, but the A+ helix increases the overall affinity for the PCI-heparin interaction by contributing a second positively charged region to the surface of PCI.

Animals↗

PubMed1996

Effect of heparin on the activation of factor XI by fibrin-bound thrombin.

Fibrin-bound thrombin is protected from inactivation by antithrombin III, while its coagulant potential is retained. In the presence of heparin, ternary complexes between thrombin, fibrin and heparin are formed. In these complexes the coagulant activity of thrombin is retained, whereas the anticoagulant activity of fibrin-bound heparin is neutralized. The limited effectiveness of heparin in the prevention of both venous thrombosis and coronary reocclusion is probably related to the protective effect of fibrin on the inactivation of thrombin by anti-thrombin III. Recently, it has been shown that factor XI can be activated by thrombin, resulting in the generation of additional thrombin via the intrinsic pathway. This additional thrombin is capable of stabilizing the clot by protecting it from fibrinolysis. We studied the effect of heparin on the activation of factor XI by fibrin-bound thrombin. First, we used fibrin monomers coupled to Sepharose to which thrombin and unfractionated heparin (UFH) were bound. Factor XI activation by thrombin was the same in the presence of fibrin-Sepharose or control-Sepharose. The addition of heparin (0.1 U/ml) resulted in a 91 and 15-fold enhancement in the presence of control-Sepharose and fibrin-Sepharose, respectively. Next, we added complexes of heparin, thrombin and fibrin monomer to factor XII and XI double-deficient plasma in the presence or absence of a reconstituting amount of factor XI. In the presence of factor XI, additional fibrin formation was observed indicating that factor XI activation by thrombin in complex with fibrin and heparin can take place in plasma. We then studied the effect of other heparin-like anticoagulants on the thrombin-mediated factor XI activation. UFH enhanced thrombin-mediated factor XI activation 68-fold, LMWH (low molecular weight heparin, Fragmin) 12-fold, danaparoid (Orgaran) 3-fold, while the pentasaccharide ORG 31540 did not result in an enhancement. Binding studies of these anticoagulants to fibrin-Sepharose showed that LMWH bound with approximately the same affinity as UFH, while danaparoid and the pentasaccharide did not bind to fibrin. We conclude that fibrin-bound thrombin is capable of factor XI activation. Furthermore, heparin bound in a complex with fibrin can act as a cofactor for this activation. This factor XI activation capacity may play a role in the limited effectiveness of heparin. Provided that thrombin-mediated factor XI activation plays an important role in vivo, danaparoid and especially the pentasaccharide may be better anticoagulants than UFH and LMWH.

Blood Coagulation↗

PubMed1996

The interaction of protein S with the phospholipid surface is essential for the activated protein C-independent activity of protein S.

Protein S is a vitamin-K dependent glycoprotein involved in the regulation of the anticoagulant activity of activated protein C (APC). Recent data showed a direct anticoagulant role of protein S independent of APC, as demonstrated by the inhibition of prothrombinase and tenase activity both in plasma and in purified systems. This anticoagulant effect of protein S can be explained either by a direct interaction of protein S with one of the components of the complexes and/or by the interference with the binding of these components to phospholipid surfaces. During our investigation we noted that protein S preparations purified in different ways and derived from different sources, expressed discrepant APC cofactor and direct anticoagulant activity. In order to elucidate these differences and to study the mechanism of the APC-independent activity of protein S, we compared the protein S preparations in phospholipid-binding properties and anticoagulant activity. The dissociation constant for the binding of protein S to immobilized phospholipids ranged from 7 to 74 nM for the different protein S preparations. APC-independent inhibition of both prothrombinase and tenase activity performed on phospholipid vesicles and in plasma showed a strong correlation with the affinity for phospholipids. The APC-independent activity could be abolished by monoclonal antibodies that were either calcium-dependent and/or directed against epitopes in the Gla-region of protein S, suggesting that the protein S-phospholipid interaction is crucial for the APC-independent anticoagulant function of protein S. Protein S preparations with a low APC-independent activity expressed a high APC-cofactor activity suggesting that the affinity of protein S for phospholipids is of less importance in the expression of APC-cofactor activity of protein S. We conclude that high affinity interactions of protein S with the membrane surface are essential for the direct anticoagulant activity of protein S and we suggest that inhibition of the prothrombinase and the tenase complex by protein S is a consequence of the occupation of the phospholipid surface by protein S molecules.

Binding Sites↗

PubMed1995

Internalization of vitronectin-thrombin-antithrombin complex by endothelial cells leads to deposition of the complex into the subendothelial matrix.

Internalization of the ternary vitronectin-thrombin-antithrombin (VN-TAT) complex by human umbilical vein endothelial cells was investigated. Radiolabeled VN-TAT was bound to the cell surface at 4 degrees C, and internalization was initiated by increasing the temperature to 37 degrees C. After 30 min about half of the VN-TAT complex disappeared from the cell surface and accumulated in the subendothelial matrix. Translocation of VN-TAT complex from the luminal to the basolateral side was confirmed by electron microscopic evaluation of cross-sections of endothelial cells incubated with gold-conjugated VN-TAT complex. Furthermore, cells cultured in VN-TAT deficient serum, incubated with purified VN-TAT, and subsequently assayed for fluorescent staining using a monoclonal antibody directed against thrombin-modified antithrombin and a polyclonal antibody against vitronectin showed co-localization of both antibodies in punctates. Punctates were randomly distributed in both the xy and xz plane of endothelial cells as evidenced by confocal laser scanning microscopy. Trichloroacetic acid precipitation and SDS-polyacrylamide gel electrophoresis showed that VN-TAT was not degraded during translocation and inhibition of the microfilament system reduced release of VN-TAT to the matrix, indicating that transcytosis was responsible for translocation. These findings emphasize that VN-TAT complex is taken up by endothelial cells, not only leading to the removal of inactivated thrombin from the circulation but also to deposition of VN into the subendothelial matrix.

Antithrombin III↗

PubMed1995

Feedback activation of factor XI by thrombin in plasma results in additional formation of thrombin that protects fibrin clots from fibrinolysis.

Recently, an alternative pathway for factor XI activation has been described in which factor XI is activated by thrombin. Patients with a factor XI deficiency bleed mostly from tissues with high local fibrinolytic activity. Therefore, the role of thrombin-mediated factor XI activation in both fibrin formation and fibrinolysis was studied in a plasma system. Clotting was induced by the addition of tissue factor or thrombin to recalcified plasma in the presence or absence of tissue-type plasminogen activator, after which clot formation and lysis were measured using turbidimetry. Thrombin-mediated activation of factor XI was found to take place in plasma under physiologic conditions in the absence of a dextran sulfate-like cofactor. At high tissue factor concentrations, no effect of factor XI was seen on the rate of fibrin formation. Decreasing amounts of tissue factor resulted in a gradually increasing contribution of factor XI to the rate of fibrin formation. In addition, thrombin-mediated factor XI activation resulted in an inhibition of tissue-type plasminogen activator-induced lysis of the clot. This inhibition occurred even at tissue factor concentrations at which no effect of factor XI was observed on fibrin formation. Trace amounts of activated factor XI (1.25 pmol/L, representing 0.01% activation) were capable of completely inhibiting fibrinolysis in our system. The inhibitory effect was found to be mediated by thrombin that is additionally generated in a factor XI-dependent manner via the intrinsic pathway and is capable of protecting the clot against lysis. We also observed that formation of additional thrombin continued after the clot had been formed. We conclude that thrombin-mediated factor XI activation can take place in plasma. The presence of factor XI during coagulation results in the formation of additional thrombin within the clot capable of protecting this clot from fibrinolytic attack. The large amounts of thrombin that are formed by the intrinsic pathway via factor XI may play an important role in the procoagulant and thrombogenic state of clots and may therefore have important clinical and therapeutic implications.

Blood Coagulation↗

PubMed1995

Synergistic inhibition of the intrinsic factor X activation by protein S and C4b-binding protein.

The complement protein C4b-binding protein plays an important role in the regulation of the protein C anticoagulant pathway. C4b-binding protein can bind to protein S, thereby inhibiting the cofactor activity of protein S for activated protein C. In this report, we describe a new role for C4b-binding protein in coagulation. We observed inhibition of the intrinsic factor X activating reaction by the complex of C4b-binding protein and protein S. At the plasma concentration of protein S, the factor X activation was inhibited for 50% and addition of C4b-binding protein led to a potentiation of the inhibition to almost 90%. Because C4b-binding protein alone had no effect on the activation of factor X, we hypothesized that binding of C4b-binding protein to protein S was a prerequisite for optimal inhibition of factor X activation. C4b-binding protein lacking the beta-chain, which is unable to bind to protein S, did not potentiate the inhibitory effect of protein S. In an earlier study, we observed that C4b-binding protein increased the binding affinity of protein S for factor VIII. Therefore, a possible interaction of C4b-binding protein with factor VIII was investigated. C4b-binding protein bound to factor VIII and to thrombin activated factor VIII in a saturable and specific way. Also, factor VIII in complex with von Willebrand factor was able to bind C4b-binding protein. The beta-chain of C4b-binding protein was not required for the interaction with factor VIII because C4b-binding protein lacking the beta-chain also bound to factor VIII. Monoclonal antibodies directed against the alpha-chain of C4b-binding protein inhibited the binding to factor VIII, whereas monoclonal antibodies directed against the beta-chain had no effect on the binding to factor VIII. This finding indicates that the binding site for factor VIII on C4b-binding protein is localized on the alpha-chains of C4b-binding protein. The potentiation by C4b-binding protein of the inhibition of the factor X activation by protein S was blocked by a monoclonal antibody directed against the alpha-chain of C4b-binding protein. This finding indicates that the potentiation of the inhibitory effect of protein S was mediated via an interaction of C4b-binding protein with factor VIII. C4b-binding protein did not bind to factor V and was not able to potentiate the inhibitory effect of protein S on prothrombinase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1995

Inhibition of the intrinsic factor X activating complex by protein S: evidence for a specific binding of protein S to factor VIII.

Protein S is a vitamin K-dependent nonenzymatic anticoagulant protein that acts as a cofactor to activated protein C. Recently it was shown that protein S inhibits the prothrombinase reaction independent of activated protein C. In this study, we show that protein S can also inhibit the intrinsic factor X activation via a specific interaction with factor VIII. In the presence of endothelial cells, the intrinsic activation of factor X was inhibited by protein S with an IC50 value of 0.28 +/- 0.04 mumol/L corresponding to the plasma concentration of protein S. This inhibitory effect was even more pronounced when the intrinsic factor X activation was studied in the presence of activated platelets (IC50 = 0.15 +/- 0.02 mumol/L). When a nonlimiting concentration of phospholipid vesicles was used, the plasma concentration of protein S (300 nmol/L) inhibited the intrinsic factor X activation by 40%. Thrombin-cleaved protein S inhibited the endothelial cell-mediated factor X activation with an IC50 similar to that of native protein S (0.26 +/- 0.02 mumol/L). Protein S in complex with C4b-binding protein inhibited the endothelial cell-mediated factor X activation more potently than protein S alone (IC50 = 0.19 +/- 0.03 mumol/L). Using thrombin activated factor VIII, IC50 values of 0.53 +/- 0.09 mumol/L and 0.46 +/- 0.10 mumol/L were found for native protein S and thrombin-cleaved protein S, respectively. The possible interactions of protein S with factor IXa, phospholipids, and factor VIII were investigated. The enzymatic activity of factor IXa was not affected by protein S, and interaction of protein S with the phospholipid surface could not fully explain the inhibitory effect of protein S on the factor X activation. Using a solid-phase binding assay, we showed a specific, saturable, and reversible binding of protein S to factor VIII with a high affinity. The concentration of protein S where half-maximal binding was reached (B1/2max) was 0.41 +/- 0.06 mumol/L. A similar affinity was found for the interaction of thrombin-cleaved protein S with factor VIII (B1/2max = 0.40 +/- 0.04 mumol/L). The affinity of the complex protein S with C4B-binding protein appeared to be five times higher (B1/2max = 0.07 +/- 0.03 mumol/L). Because the affinities of the interaction of the different forms of protein S with factor VIII correspond to the IC50 values observed for the intrinsic factor X activating complex, the interaction of protein S with factor VIII may explain the inhibitory effect of protein S on the intrinsic factor X activating complex.(ABSTRACT TRUNCATED AT 400 WORDS)

Anticoagulants↗

PubMed1995

The affinity and stoichiometry of binding of human factor VIII to von Willebrand factor.

To study the interaction between factor VIII and von Willebrand factor (vWF), binding experiments were performed using immobilized plasma vWF. Plasma was obtained from healthy donors and from patients with severe hemophilia A. For normal and hemophilic vWF, the dissociation constants (kd) for binding of factor VIII to vWF were 0.21 +/- 0.04 and 0.22 +/- 0.05 nmol/L, respectively. At saturation, the stoichiometry was one factor VIII molecule per 50 vWF monomers. In gel-filtration experiments, vWF was saturated by 23 times more factor VIII. However, when this FVIII-vWF complex was immobilized on microtiter plates, the ratio of factor VIII/vWF decreased to the same ratio as in the solid-phase binding assay. To exclude any effect of antibody binding, colloidal gold particles with a diameter of 15 nm were coupled to purified vWF. This vWF-gold complex remained immunoreactive toward polyclonal and monoclonal antibodies, and was able to bind factor VIII, specifically, saturably, and reversibly. After incubation of vWF-gold with factor VIII, unbound and bound factor VIII were separated by centrifugation. Binding isotherms of these fluid-phase binding experiments indicated a kd of 0.32 +/- 0.09 nmol/L and a stoichiometry of approximately 0.5 factor VIII molecule per vWF monomer. We conclude that vWF-binding to a surface, with or without an antibody, may induce a conformational change causing a dissociation of bound factor VIII from vWF.

Adult↗

PubMed1995

Increased prothrombin activation in protein S-deficient plasma under flow conditions on endothelial cell matrix: an independent anticoagulant function of protein S in plasma.

Protein S is a vitamin K-dependent nonenzymatic coagulation factor involved in the regulation of activated protein C (aPC). In this study, we report an aPC-independent anticoagulant function of protein S in plasma under flow conditions. Plasma, anticoagulated with low-molecular-weight heparin allowing tissue factor-dependent prothrombin activation, was perfused at a wall shear rate of 100 s-1 over tissue factor containing matrices of stimulated endothelial cells placed in a perfusion chamber. Fractions were collected in time at the outlet and prothrombin activation was determined by measuring the activation fragment F1+2 of prothrombin. In normal plasma, a time-dependent prothrombin activation was detected by the generation of fragment1+2. Prothrombin activation had ceased after 12 minutes perfusion, independent of the amount of tissue factor present in the matrix. Depletion of protein S from plasma or inhibition of protein S in plasma by monoclonal antibodies induced a 5- to 25-fold increase of prothrombin activation on the procoagulant endothelial cell matrix. A prolonged prothrombin activation was detected in protein S-depleted plasma up to 20 minutes after onset of the thrombin generation. The increased prothrombin activation in protein S-depleted plasma could not be explained by the absence of the cofactor function of protein S for aPC because depletion of protein C from plasma did not result in increased prothrombin activation. These data provide further evidence for a strong anticoagulant function of protein S in plasma independent from activated protein C.

Antibodies, Monoclonal↗

PubMed1995

Method-dependent increase in lipoprotein(a) in insulin-dependent diabetes mellitus during pregnancy.

The current prevalent view is that plasma lipoprotein(a) [Lp(a)] concentrations are under strong genetic control. Most dietary and drug interventions seem to have little or no effect on plasma Lp(a) levels. However, evidence for a possible regulatory rol e of hormones is accumulating, for instance, fluctuations of Lp(a) levels during pregnancy have been reported. Also, in insulin-dependent diabetes mellitus (IDDM) patients, elevated Lp(a) levels have been reported. In the present longitudinal study, plasma lipid concentrations, including Lp(a), were determined in IDDM women before pregnancy, during pregnancy, and 3 months postpartum. In our study population, Lp(a) concentration was not significantly correlated with either hemoglobin A1c (HbA1c) levels of apolipoprotein(a) [apo(a)] phenotype. Changes in other lipid parameters observed during pregnancy in our IDDM population were similar to those reported during normal pregnancy. Lp(a) concentrations were quantified using two different immunochemical methods that possess different sensitivities and specificities: an immunoradiometric assay (IRMA) using two different anti-apo(a) antibodies, and an enzyme-linked immunosorbent assay (ELISA) using an anti-apo(a) and an anti-apo B antibody. Median prepregnancy Lp(a) concentrations were 118 mg/L (range, 15 to 672) as determined with the IRMA and 107 mg/L (range, 21 to 451) as determined with the ELISA. Women with IDDM showed, in general, no significant change in Lp(a) concentration during pregnancy when it was assayed with the IRMA, although a tendency to increased values was observed. When Lp(a) concentrations were determined with the ELISA, a strong and significant increase in Lp(a) from weeks 17 to 24 of pregnancy onward was found. The latter results confirm the prevalent view that during pregnancy Lp(a) levels are increased. However, the present results and those of others and Lp(a) in normal pregnancy strongly emphasize the importance of method selection when determining Lp(a) concentrations.

Adult↗

PubMed1995

Changes in haemostatic factors and activation products after exercise in healthy subjects with different ages.

We studied exercise-induced changes in coagulation and fibrinolytic factors and activation products in different age categories. Thirty-eight sedentary males, divided in three age categories (cats I-III; 20-30, 35-45 and 50-60 y) were subjected to a standardized exercise test. Pre-exercise levels (cats I-III resp) of FVII:c (105 +/- 5, 121 +/- 6 and 123 +/- 7% NP), fibrinogen (2.35 +/- 0.12, 2.55 +/- 0.10 and 2.66 +/- 0.09 mg/ml), prothrombin activation fragment F1 + 2 (0.80 +/- 0.10, 0.80 +/- 0.11 and 1.22 +/- 0.16 nM), t-PA (5.2 +/- 0.6, 9.2 +/- 1.0, 8.6 +/- 1.2 ng/ml) and PAI-I (42.8 +/- 7.5, 67.6 +/- 7.6, 62.2 +/- 10.9 ng/ml) showed differences that seemed related to age. Regression analysis revealed associations with anthropometry (FVII:c, fibrinogen, F1+2, t-PA, PAI-1) rather than with age. Exercise-induced changes in coagulation (increase in von Willebrand factor and FVIII:c and a shortening of APTT) and fibrinolytic potential (increase in t-PA and u-PA) were of comparable magnitude for the three age categories. Hardly any change in F1 + 2 (6%) was observed, while thrombin-antithrombin complexes (93%), plasmin-antiplasmin complexes (79%) and D-dimer (77%) almost doubled during maximal exercise. We conclude that anthropometric differences play a more significant role than age on constitutive levels of haemostatic factors in participants up to 60 years of age. The magnitude of exercise-induced changes is comparable in the age categories under study, and simply super-imposed on constitutive (pre-exercise) levels. Clear evidence for prothrombin activation is lacking, but plasmin formation is enhanced during exercise.

Adult↗

PubMed1994

Analysis of protein S C4b-binding protein interactions by homology modeling and inhibitory antibodies.

A monoclonal antibody (mAb 6F6) directed against the beta-chain of C4b-binding protein (C4BP) was previously shown to inhibit the binding of protein S to C4BP. To localize the epitope of this antibody, 23 overlapping synthetic peptides (15-mers) covering the entire sequence (1-235) of the beta-chain of C4BP were used. When the immobilized peptides were screened for their ability to bind mAb 6F6, only peptide beta(51-65) showed high-affinity binding. The apparent affinity of mAb 6F6 for immobilized peptide beta(51-65) was somewhat similar to that for native C4BP with Kd approximately 1 nM for C4BP and approximately 9 nM for peptide beta(51-65). Peptide beta(51-65) inhibited the binding of the mAb 6F6 to immobilized C4BP with half-maximal inhibition at 30 microM peptide. Clotting assays of protein S anticoagulant cofactor activity using a factor Xa-1-stage assay with activated protein C allow measurement of free protein S in solution since only free protein S is active. Studies using such clotting assays showed that preincubation of C4BP with either mAb 6F6 or polyclonal anti-beta(31-45) antibodies inhibited the formation of the complex between C4BP and protein S. Previous studies showed that, although peptide beta(51-65) itself does not inhibit complex formation, peptide beta(31-45) does bind directly to protein S and does inhibit protein S binding to C4BP. The three-dimensional structure of the first SCR (residues 2-60) of the C4BP beta-chain was made on the basis of homology modeling.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1994

Human protein S inhibits prothrombinase complex activity on endothelial cells and platelets via direct interactions with factors Va and Xa.

Protein S is a vitamin K-dependent non-enzymatic coagulation factor involved in the regulation of activated protein C (APC). In this paper we report an APC-independent anticoagulant function of protein S. We observed an inhibition of prothrombinase activity on endothelial cells and platelets which was half-maximal at physiological concentrations of free protein S in plasma. On endothelial cells, thrombin-cleaved protein S (PSt) as well as protein S in complex with C4b-binding protein (C4BP) inhibited prothrombinase activity to the same extent as protein S did. In solid-phase binding assays, direct binding of protein S and PSt to factor V and factor Va were observed. Protein S-C4BP complex did not bind to factor V or factor Va, implicating that the factor V(a) binding site on protein S is lost by the interaction with C4BP. A direct inhibition of factor Xa activity by protein S was also observed. Incubation of factor Xa with protein S revealed a noncompetitive inhibition of factor Xa by protein S with a Ki of (4.9 +/- 0.8) x 10(-7) M. Both protein S and protein S-C4BP complex were able to inhibit factor Xa to the same extent, whereas PSt had lost its inhibitory activity. This suggests that the conformational change induced by cleavage of the amino-terminal thrombin-sensitive loop results in a loss of a factor Xa binding site on protein S. The inhibitory effect of protein S involves interactions with both factor Va and factor Xa. The interaction of protein S with factor Xa is influenced by cleavage of the thrombin-sensitive loop, whereas C4BP blocks the interaction of protein S with factor Va. Inhibition of the prothrombinase complex by either forms of protein S might be an important mechanism in regulating thrombin generation in blood coagulation. The importance of the APC-independent anticoagulant action of protein S was emphasized by experiments in which the addition of protein S to normal plasma induced a prolongation of clotting time in a dilute activated partial thromboplastin time (dAPTT) assay. Furthermore, inhibition of endogenous protein S by the addition of monoclonal antibodies against protein S to normal plasma, induced a shortening of the clotting time in a dAPTT assay.

Blood Platelets↗

PubMed1994

Activated factor X and thrombin formation triggered by tissue factor on endothelial cell matrix in a flow model: effect of the tissue factor pathway inhibitor.

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma-TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.

Animals↗

PubMed1994

Sulfhydryl compounds influence immunoreactivity, structure and functional aspects of lipoprotein(a).

Human plasma Lp(a) is susceptible to various sulfhydryl compounds. In this study we present evidence indicating that after treatment of Lp(a) with sulfhydryl compounds, immunoreactivity is changed, structural changes occur and functional characteristics regarding the numerous kringle structures in apo(a) disappear. Purified Lp(a) was subjected to variable concentrations (0.01-10 mM) of various sulfhydryl compounds: DTT, 2-mercapto-ethanol (BME), N-acetylcysteine (NAC) and homocysteine (HCys). Free SH groups were blocked by iodoacetamide. Reduced and alkylated Lp(a) was tested in two ELISAs, one detecting apo(a) alone and one detecting apo(a)-apoB complexes. In both ELISAs polyclonal antibodies were used. For comparison a commercial apo(a) IRMA utilizing two monoclonal antibodies was used. The results indicate that a similar decrease in response of both ELISAs is observed, whereas the IRMA response is less affected. Western blotting of "DTT treated" Lp(a) after SDS-PAGE under nonreducing conditions showed that separate apo(a) and apoB-100 bands became detectable at 1 mM DTT. Native PAGE (2.5-16%) indicated structural changes of Lp(a) beginning to occur at 0.03 mM DTT. Epsilon-aminocaproic acid-inhibitable binding of "DTT-treated" Lp(a) to Desafib-X decreased with increasing DTT concentrations in concert with a loss of the capacity of Lp(a) to inhibit plasminogen activation upon treatment with DTT. The observed immunological and functional changes of Lp(a) indicate that apo(a) kringle function is severely affected by sulfhydryl compounds.

Dithiothreitol↗

PubMed1994

Dexamethasone affects platelet aggregation and fibrinolytic activity in rats at different doses which is reflected by their effect on arterial thrombosis.

The effect of oral administration of dexamethasone to rats on the haemostatic system was investigated. Dexamethasone was given once daily for 5 consecutive days. Plasma PAI-1 antigen levels were increased dose dependently (up to 210 +/- 29% of control values at a dose of 3 mg/kg) whereas no significant effects on plasma t-PA antigen levels were observed (131 +/- 6% compared with control values). In addition, treatment with 1 mg/kg dexamethasone decreased t-PA activity in tissue extracts of the aorta, heart and liver (65%, 28% and 58%, respectively) whereas tissue u-PA activity was not influenced. In vivo fibrinolytic activity was significantly decreased after dexamethasone treatment at a dose of 3 mg/kg but not at a dose of 1 mg/kg. The effect of dexamethasone on in vivo platelet aggregation was studied in an arterial thrombosis model. Dexamethasone treatment resulted in a two-fold decrease in arterial thrombosis at a dose of 0.1 mg/kg. At a dose of 1 mg/kg a less pronounced but significant decrease was observed. We conclude that in haemostasis the primary effect of dexamethasone treatment is an inhibition of arterial thrombosis by inhibition of platelet aggregation which is neutralized at higher doses by a decreased fibrinolytic activity.

Animals↗

PubMed1994

Fibrinolytic activity in blood is distributed over a cellular and the plasma fraction which can be modulated separately.

Blood fibrinolytic activity is mediated by plasma and cellular components. We have studied blood fibrinolytic activity in different species and investigated the distribution pattern in rats after modulation with PAF, dexamethasone, or retinoic acid. Whole blood and plasma activity were measured in an assay system using human or endogenous fibrin as substrate. When human fibrin was used as substrate marked species differences in distribution of fibrinolytic activity were observed. In rat and murine blood most fibrinolytic activity was associated with the plasma fraction (70% and 50% respectively) while in human and canine blood the plasma fraction contained only 30% of the blood fibrinolytic activity. When endogenous fibrin was used as substrate the distribution pattern of fibrinolytic activity in rat blood changed dramatically. Less than 25% of the blood fibrinolytic activity was now present in the plasma fraction. The fbrinolytic system was further investigated in rats using specific inhibitors of proteolytic activity. Blood fibrinolytic activity could be inhibited for 33% by antibodies raised against t-PA and 60% inhibition was obtained in the presence of amiloride. No significant effect of elastinal (an inhibitor of elastase) could be detected. Plasma fibrinolytic activity was not affected by these inhibitors. The fibrinolytic activity in plasma could be enhanced about 100-fold after i.v. PAF administration (10 micrograms/kg). This extra fibrinolytic activity could be fully blocked by antibodies raised against t-PA. Oral administration of dexamethasone or retinoic acid affected blood fibrinolytic activity by modulating selectively the activity mediated by the cellular fraction. Dexamethasone treatment (1 mg/kg) resulted in a 59% decrease of this fibrinolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Surface independent factor XI activation by thrombin in the presence of high molecular weight kininogen.

A deficiency of one of the proteins of the contact system of blood coagulation does not result in a bleeding disorder. For this reason activation of blood coagulation via this system is believed to be an in vitro artefact. However, patients deficient in factor XI do suffer from variable bleeding abnormalities. Recently, an alternative pathway for factor XI activation has been described. Factor XI was found to be activated by thrombin in the presence of dextran sulfate as a surface. However, high molecular weight kininogen (HK), to which factor XI is bound in plasma, and fibrinogen were shown to block this activation suggesting it to be an in vitro phenomenon. We investigated the thrombin-mediated factor XI activation using an amplified detection system consisting of factors IX, VIII and X, which was shown to be very sensitive for factor XIa activity. This assay is approximately 4 to 5 orders of magnitude more sensitive than the normal factor XIa activity assay using a chromogenic substrate. With this assay we found that factor XI activation by thrombin could take place in the absence of dextran sulfate. The initial activation rate was approximately 0.3 pM/min (using 25 nM factor XI and 10 nM thrombin). The presence of dextran sulfate enhanced this rate about 8500-fold. A very rapid and complete factor X activation was observed in the presence of dextran sulfate. Although only minute amounts of factor XIa were formed in the absence of dextran sulfate, significant activation of factor X was detected in the amplification assay within a few minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical Phenomena↗