Biomedical subjects

K Mertens

Publications and source records attributed to K Mertens.

At least 55 records · Page 3Linked to original sources

PubMed1996

Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding.

Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp64 by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km approximately 1, 2, and 4 x 10(3) M-1 s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent ( approximately700- and 200-fold, respectively), indicating that Asp64 replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.

Amino Acid Sequence↗

PubMed1996

Kinetics of factor VIII light-chain cleavage by thrombin and factor Xa. A regulatory role of the factor VIII heavy-chain region Lys713-Arg740.

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region Lys713-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3 +/- 0.3 x 10(6) M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713-1637)-factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713-1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713-1637)-factor VIII is clearly reduced. Furthermore, the secondorder rate constant (0.2 +/- 0.1 x 10(6) M-1 s-1) of des-(713-1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713-1637)-factor VIII light-chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des-(713-1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis.

Antibodies, Monoclonal↗

PubMed1996

Enhanced thrombin sensitivity of a factor VIII-heparin cofactor II hybrid.

Generation of thrombin at a site of vascular injury is a key event in the arrest of bleeding. In addition to the conversion of fibrinogen into the insoluble fibrin, thrombin can initiate a number of positive and negative feedback mechanisms that either sustain or down-regulate clot formation. We have modulated the thrombin sensitivity of human blood coagulation factor VIII, an essential cofactor in the intrinsic pathway of blood coagulation. We have substituted an acidic region of factor VIII corresponding to amino acid sequence Asp712-Ala736 by amino acid sequence Ile51-Leu80 of the thrombin inhibitor heparin cofactor II. Functional analysis of the resulting factor VIII-heparin cofactor II hybrid, termed des-(868-1562)-factor VIII-HCII, revealed an increase in procoagulant activity as measured in a one-stage clotting assay. Incubation of purified des-(868-1562)-factor VIII-HCII with different amounts of thrombin showed that this protein was more readily activated by thrombin when compared with des-(868-1562)-factor VIII, a control protein lacking amino acid sequence Ile51-Leu80 of heparin cofactor II. This was manifested by an increase in the second order rate constant of activation by thrombin for des-(868-1562)-factor VIII-HCII (12.0 +/- 0.48 x 10(6) M-1 s-1) compared with des-(868-1562)-factor VIII (1.77 +/- 0.21 x 10(6) M-1 s-1). Our data suggest that amino acid sequence Ile51-Leu80 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin which results in accelerated clot formation.

Amino Acid Sequence↗

PubMed1996

The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX.

In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.

Amino Acid Sequence↗

PubMed1996

Thrombin-mediated activation of endogenous factor XI in plasma in the presence of physiological glycosaminoglycans occurs only with high concentrations of thrombin.

The variable bleeding tendency associated with a genetic deficiency of factor XI (FXI) and the lack of bleeding disorders in individuals with a genetic deficiency of factor XII (FXII) suggest an alternative mechanism for FXI activation in vivo. Recently, thrombin has been shown to activate FXI. However, in plasma this activation has been shown to occur only with exogenous FXI and a non-physiological cofactor (sulphatides), and the occurrence of this reaction in a plasma environment has been questioned. Using recently developed sensitive assays for FXIa-inhibitor complexes we found thrombin-mediated and FXII-dependent activation of endogenous FXI in plasma in the presence of heparan sulphate, heparin, dermatan sulphate or dextran sulphate. Using heparan sulphate, which is present in the human vascular system, activation of about 1-2% of plasma FXI was observed, however, only after addition of very high amounts (500 nmol/l) of human alpha-thrombin to FXII-deficient plasma (at a 1 to 4 final dilution). We conclude that endogenous FXI in plasma can be activated by thrombin in the presence of various glycosaminoglycans, including the physiological compounds heparan sulphate and dermatan sulphate, but only at very high concentrations of thrombin, corresponding to 100% prothrombin activation in undiluted plasma.

Blood Coagulation↗

PubMed1995

Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa.

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Animals↗

PubMed1995

Cleavage at arginine 145 in human blood coagulation factor IX converts the zymogen into a factor VIII binding enzyme.

The transition of the factor IX zymogen into the enzyme factor IXa beta was investigated. For this purpose, the activation intermediate factors IX alpha and IXa alpha were purified after cleavage of the Arg145-Ala146 and Arg180-Val181 bonds, respectively. These intermediates were compared for a number of functional properties with factor IXa beta, which is cleaved at both positions. Factor IXa alpha was equal to factor IXa beta in hydrolyzing the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide (kcat/Km approximately 120 s-1 M-1) but was less efficient in factor X activation. Factor IX alpha was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXa alpha and factor IXa beta. Factor IX alpha and factor IXa beta had similar affinity for the inhibitor benzamidine (Ki approximately 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXa beta, factor IX alpha was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXa beta and factor IX alpha by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IX alpha was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IX alpha differs from that of the fully activated factor IXa beta. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IX alpha and factor IXa beta displayed a 30-fold higher affinity for the factor VIII light chain (Kd approximately 12 nM) than the factor IX zymogen. Factor IXa alpha showed lower affinity (Kd approximately 50 nM) than factor IX alpha and factor IXa beta, which may explain the lower efficiency of factor X activation by factor IXa alpha. Collectively, our data indicate that cleavage of the Arg180-Val181 bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg145-Ala146 bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1995

The role of cleavage of the light chain at positions Arg1689 or Arg1721 in subunit interaction and activation of human blood coagulation factor VIII.

The role of Factor VIII light chain cleavage in Factor VIII activation and subunit interaction was investigated. Purified Factor VIII was dissociated into its separate subunits, and the isolated light chain was cleaved by thrombin at position Arg1689 or by Factor Xa at position Arg1721. These Factor VIII light chain derivatives then were used for reconstitution with purified Factor VIII heavy chain to obtain heterodimers that were exclusively cleaved within the light chain. Intact and cleaved light chain could effectively be reassociated with heavy chain, with concomitant regain of Factor VIII cofactor function. The association rate constant of Factor Xa-cleaved light chain was found to be 3-fold lower than that of thrombin-cleaved or intact light chain, suggesting a role of the region Ser1690-Arg1721 in subunit assembly. Dissociation rate constants, however, were independent of Factor VIII light chain cleavage. Low ionic strength was observed to promote association but to destabilize the Factor VIII heterodimer. At high ionic strength, Factor VIII dissociation was extremely slow (kappa off approximately 10(-5) s-1) for all Factor VIII light chain derivatives, indicating that Factor VIII light chain cleavage is not related to Factor VIII dissociation. Furthermore, Factor VIII light chain cleavage does not affect enzyme-cofactor assembly, since the various light chain derivatives proved equally efficient in binding to Factor IXa (Kd approximately 15 nM). Studies in a purified Factor X-activating system demonstrated that thrombin and Factor Xa activate Factor VIII to the same extent. However, Factor Xa differed from thrombin in that it cleaved at Arg1721 rather than at Arg1689. Reassociated heterodimers of Factor VIII heavy chain and intact light chain did not promote Factor X activation. In contrast, heterodimers that contained cleaved light chain exhibited substantial Factor VIIIa activity. These data demonstrate that a single cleavage at either Arg1689 or Arg1721 converts the inactive Factor VIII heterodimer into an active cofactor of Factor IXa.

Arginine↗

PubMed1994

Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V.

Abnormal coagulation factor V may underlie the thrombotic events associated with resistance to activated protein C (APC). We analysed 27 consecutive patients with documented idiopathic (recurrent) thromboembolism for the occurrence of point mutations within the APC sensitive regions of blood coagulation factor V. In 10 patients we observed a single basepair mutation resulting in a substitution of Arg506 to Gln. This mutation was significantly linked to in-vitro resistance to APC in these subjects. This mutation at Arg506 of factor V may form the molecular basis for the thrombotic events associated with APC resistance.

Adult↗

PubMed1994

Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.

The interaction between human factor IXa and factor VIII or its constituent units was investigated. Equilibrium binding studies were performed employing factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated factor VIII light chain and the factor VIII heterodimer were equally effective in factor IXa binding, demonstrating that this interaction did not require the factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with factor IXa, indicating that the high affinity for factor VIII light chain was unique for factor IXa. The factor IXa-factor VIII light chain interaction was inhibited by von Willebrand factor, but this effect was abolished by cleavage of the factor VIII light chain by thrombin. An antibody that inhibits von Willebrand factor-factor VIII complex formation did not compete for factor IXa binding. In contrast, association of factor IXa with the factor VIII light chain was inhibited by an antibody directed against the factor VIII region Gln1778-Asp1840. We propose that this sequence provides a factor IXa binding site and that its exposure requires dissociation of the factor VIII-von Willebrand factor complex.

Antibodies, Monoclonal↗

PubMed1994

Age- and gender-related changes in the hepatic metabolism of 2-methylpropene and relationship to epoxide metabolizing enzymes.

The effect of age and gender on the in vitro biotransformation of 2-methylpropene, an alkene metabolized to 2-methyl-1,2-epoxypropane, was studied. The epoxide concentration and the epoxide metabolizing enzymatic activities were investigated in male and female Brown Norway rats of different ages. Liver tissue of senescent rats was exposed to smaller 2-methyl-1,2-epoxypropane concentrations than that of young animals, although changes during ageing were rather modest. With advancing age a feminization of male glutathione S-transferase and cytosolic epoxide hydrolase activities was found, as well as a significant decline of the female microsomal epoxide hydrolase activity and an increase of the cytochrome P-450 content in the oldest female rats.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1994

The activation of human blood coagulation factor X on the surface of endothelial cells: a comparison with various vascular cells, platelets and monocytes.

Rates of factor X activation on endothelial cells were compared with activation rates on other vascular cells, platelets, monocytes and negatively charged phospholipid vesicles. Factor VIIa-mediated factor X activation was observed on smooth muscle cells and fibroblasts in the absence of cell-perturbing agents, whereas endothelial cells required activation in order to allow extrinsic activation of factor X. On the other hand, unperturbed endothelial cells did promote intrinsic, factor VIII/IXa-dependent activation of factor X. The rate of factor X activation on these cells was about one-sixth of that on ionophore A23187-stimulated platelets. Also, smooth muscle cells and fibroblasts were able to activate factor X through the intrinsic pathway, although to a lesser extent than endothelial cells. Monocytes were ineffective in this respect. Prothrombin fragment 1, the prothrombin fragment containing the gamma-carboxyglutamic acid domain known to mediate binding of vitamin K-dependent coagulation factors to phospholipid surfaces, inhibited factor VIII/IXa-dependent factor X activation on endothelial cells (IC50 3.2 microM) to a lesser extent than on phospholipid vesicles (IC50 0.2 microM). Therefore, besides negatively charged phospholipids, other membrane constituents seem to be involved in endothelial cell mediated, intrinsic activation of factor X. Perturbation of endothelial cells with phorbol myristate acetate (PMA) or lipopolysaccharide (LPS) was without effect on intrinsic activation of factor X. This observation indicates that membrane constituents of endothelial cells involved in factor VIII/IXa-dependent activation of factor X are constitutively expressed.

Blood Coagulation↗

PubMed1994

Prevention of side effects by hemoglobin solutions; the selection of optimal test models, especially concerning thrombogenicity.

Modification of hemoglobin (Hb) by crosslinking and polymerization results in an improved oxygen release capacity and a prolonged vascular retention time. Modification improves the efficacy and prevents certain side effects. It eliminates leakage of Hb through the kidneys and accumulation in the tubuli. Another important issue is the degree of purification of Hb solutions. Traces of membrane fragments may cause immunogenic and thrombogenic side effects. To determine the contamination with erythrocyte membrane fragments, we developed assays for glycophorin-alpha and phospholipids. Special models were evaluated for testing the maximum allowable level of membrane contamination. As an in vitro model for thrombogenicity we used confluent monolayers of human umbilical vein endothelial cells. These cells were incubated with Hb solutions and subsequently tested on tissue factor (TF) procoagulant activity. TF was tested by the factor VII-catalyzed activation of factor X. The lower detection limit of this assay for endotoxin was 0.5 ng/ml. Hb did not cause any tissue factor expression even after prolonged incubation. No cooperation was found within endotoxin. As an in vivo test on thrombogenicity we developed a guinea pig model in which we can follow the generation of fibrinopeptide A (FPA). This is one of the most sensitive markers for thrombin activation in vivo. When slightly contaminated Hb solutions (phospholipid content 2 nmol/ml) were infused in the presence of factor Xa at a dose (9 micrograms/kg) which in itself did not induce FPA generation, we observed an increase in FPA levels in the plasma from 1.2 +/- 0.4 ng/ml to 5.2 +/- 0.7 ng/ml. Factor Xa is used to mimic a stressed clinical condition with activated coagulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Isolation and characterization of single-chain protein S.

Protein S is a vitamin K-dependent cofactor of activated protein C in the proteolytic cleavage and concomitant inactivation of the coagulation factors Va and VIIIa. Protein S is sensitive to proteolysis by thrombin which reduces its functional activity. Uncontrolled proteolytic breakdown, leading to the generation of a two-chain molecule, is commonly encountered during the purification of both human and bovine protein S. In this study we demonstrate that human, single-chain, intact protein S can be isolated from plasma in a single step by affinity chromatography using a monoclonal antibody, CLB PS 52, directed to an epitope located within the thrombin-sensitive region of protein S. The product of purification was readily cleaved by thrombin after Arg49, resulting in a two-chain molecule which demonstrated a lower reactivity towards CLB-PS 52 than the parent single-chain protein. This study for the first time shows that intact protein S can be isolated directly from plasma using a monoclonal antibody selected for its ability to recognize uncleaved protein S.

Amino Acid Sequence↗

PubMed1993

Specific glycosaminoglycans support the inhibition of thrombin by plasminogen activator inhibitor 1.

In the absence of accessory components, plasminogen activator inhibitor 1 (PAI-1) rapidly forms equimolar, inactive complexes both with tissue-type (t-PA) and with urokinase-type (u-PA) plamsinogen activator. In the presence of either the glycoprotein vitronectin or the glycosaminoglycan heparin, PAI-1 is endowed with additional, efficient thrombin-inhibitory properties (Ehrlich et al., 1990, 1991a). Here, we have investigated the interaction between PAI-1, thrombin, and glycosaminoglycans in more detail. Inhibition of thrombin by PAI-1 was quantitatively analyzed in the presence of a wide range of concentrations of heparin, heparan sulfate, dermatan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, keratan sulfate, and hyaluronic acid by measuring residual amidolytic activity. In addition, a qualitative analysis was performed by determining the formation of SDS-stable, equimolar complexes between thrombin and PAI-1 in the presence of various glycosaminoglycans. Heparin, at concentrations between 0.1 and 1 microgram/mL, significantly promoted thrombin inhibition by PAI-1 as well as SDS-stable complex formation. Suboptimal inhibition was observed with dermatan sulfate, chondroitin 4-sulfate, and heparan sulfate at concentrations that are at least 1 order of magnitude higher than that required for optimal inhibition in the presence of heparin. Virtually no inhibition of thrombin and SDS-stable complex formation was detected with any of the other glycosaminoglycans at concentrations between 0.1 and 1 microgram/mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Glutathione dependent detoxication in adult rat hepatocytes under various culture conditions.

In order to obtain more information concerning the effects of culture and medium conditions on the glutathione dependent detoxication system in hepatocyte cultures, glutathione reductase (GR) and glutathione peroxidase (GPx) activities were studied in both pure cultures of adult rat hepatocytes and their co-cultures with rat epithelial cells. Cells were isolated either with an oxygen saturated Krebs Henseleit buffer (KHB) or with a non-gassed Hepes buffer. As medium conditions, additions of 10% fetal calf serum (FCS), 25 mM nicotinamide, 0.1 microM selenium and 2% dimethylsulphoxide, respectively, to the culture medium were examined. It was found that co-cultures of rat hepatocytes can cope better with oxidative stress than pure cultures do. This conclusion was reached from the following observations. When oxygenated KHB was used as isolating buffer, GR and GPx activities increased during the first days of pure culture and then slowly decreased. This was observed for all the medium conditions studied and no significant differences between the different media could be observed. For co-cultures, however, after some initial variations GR and GPx activities reached stabilized levels which were not only significantly lower than those observed for pure cultures, but were also maintained throughout the whole culture period. Supplementation of the medium had no effect on these findings with the exception of high GPx activities when Se was added to the co-culture medium. When Hepes buffer with a low oxygen content was used in cell isolation, pure cultures showed significantly lower GR and GPx activities than those first mentioned.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.

Recombinant factor VIII variants with overlapping deletions spanning the region Lys713-Ile1668 have been expressed in mammalian cells, and analysed for biological activity both in vitro and in vivo. Two distinct assay systems were used to measure the activity in vitro. The one-stage coagulation assay served to assess factor VIII procoagulant activity while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa-dependent factor X activation. Deletion of the entire B-domain (Ser741-Arg1648) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This functional inconsistency was further addressed by comparing purified factor VIII-del(713-1637) with factor VIII-del(868-1562), a mutant with normal in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the cofactor activity from factor VIII-del(713-1637) than needed for factor VIII-del(868-1562) or plasma factor VIII. The physiological significance of this finding was assessed in dogs with haemophilia A. Both deletion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half-life and recovery. Employing the cuticle bleeding time model, factor VIII-del(868-1562) was found to be indistinguishable from plasma factor VIII, whereas factor VIII-del(713-1637) was less effective. The increased thrombin-resistance of factor VIII-del(713-1637) thus limits both procoagulant activity and haemostatic efficacy in cuticle bleeding. These observations suggest that the heavy-chain sequence Lys713-Arg740, although dispensable for factor VIII cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.

Amino Acid Sequence↗

PubMed1993

Evaluation of Maudgal lens six months after implantation.

We studied the implantation and follow-up of Maudgal compressible disc lens in 102 eyes with a follow-up of at least six months. The degree and incidence of decentration of this lens in the bag is remarkably low. A postoperative visual acuity of 5/10 or more was achieved in 96% patients and 7/10 or better in 86.2% cases without having an associated pathology at the time of surgery.

Cataract Extraction↗