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Abstract Background Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin. Methods The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. Results An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA. Conclusion The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

The shear rate and corresponding shear stress have impacts on arterial thrombus formation. In particular, the effects of increasing concentration of platelets at the vessel wall and activation of platelets at this site increase the growth and stability of the thrombi which may result in a fatal narrowing of the arterial lumen. The efficacy of many antithrombotic agents is shear dependent as well. It is apparent that there is a need for a point-of-care device to rapidly monitor the risk for arterial thrombosis and to optimize antithrombotic therapy in vitro. The present review focuses on the essential role of shear rate on arterial thrombus formation in native human blood drawn directly from an antecubital vein., Competing Interests: Financial & competing interests disclosure One of the authors, KS Sakariassen of KellSa s.a.s., consults/advices academic and biopharma R&D within the areas of hemostasis, thrombosis and atherosclerosis. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.

Background: Conventional tests for vitamin B(12) deficiency measure total serum vitamin B12, whereas only that portion of vitamin B12 carried by transcobalamin (holotranscobalamin) is metabolically active. Measurement of holotranscobalamin (holoTC) may be more diagnostically accurate for detecting B(12) deficiency that requires therapy. We developed an automated assay for holoTC that can be used on the Abbott AxSYM immunoassay analyzer., Methods: AxSYM Active B12 is a 2-step sandwich microparticle enzyme immunoassay. In step 1, a holoTC-specific antibody immobilized onto latex microparticles captures holoTC in samples of serum or plasma. In step 2, the captured holoTC is detected with a conjugate of alkaline phosphatase and antiTC antibody., Results: Neither apoTC nor haptocorrin exhibited detectable cross-reactivity. The detection limit was < or = 0.1 pmol/L. Within-run and total imprecision (CV ranges) were 3.4%-5.1% and 6.3%-8.5%, respectively. Assay CVs were < 20% from at least 3 pmol/L to 107 pmol/L. With diluted serum samples, measured concentrations were 104%-114% of the expected values in the working range of the assay. No interference from bilirubin, hemoglobin, triglycerides, erythrocytes, rheumatoid factor, or total protein was detected at expected (abnormal) concentrations. A comparison of the AxSYM Active B12 assay with a commercial RIA for holoTC yielded the regression equation: AxSYM = 0.98RIA + 4.7 pmol/L (S(y x), 11.4 pmol/L; n = 204). Assay throughput was 45 tests/h. A 95% reference interval of 19-134 pmol/L holoTC was established with samples from 292 healthy individuals., Conclusions: The AxSYM Active B12 assay allows rapid, precise, sensitive, specific, and automated measurement of human holoTC in serum and plasma.

This review focuses on the validation of the principal initiator of human coagulation, the tissue factor (TF)/coagulation factor (F)VIIa complex, as an antithrombotic target, as well as on the discovery of a cyclic pentapeptide (PN7051), which dose-dependently inhibits TF/FVIIa-induced coagulation and thrombus formation. Target validation and studies of antithrombotic efficacy were performed with a human thrombosis model employing non-anticoagulated blood from severe homozygous FVII-deficient patients and healthy individuals at blood-flow conditions mimicking those in healthy and diseased vessels. Additional validation included an anti-TF monoclonal antibody, recombinant TF pathway inhibitor, recombinant inactivated-active site FVIIa and all-trans retinoic acid. Structural and biological characterization of PN7051 and other peptides from the same FVII domain indicate that PN7051 interferes with an essential interaction between the epidermal growth factor domain-2-like and the catalytic domains of FVIIa. A peptidomimetics approach is suggested to further improve the antithrombotic potency of PN7051.

Background: Holotranscobalamin, cobalamin-saturated transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holotranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holotranscobalamin., Methods: The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native transcobalamin in serum. The epitope of the holotranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA., Results: An antibody exhibiting >100-fold specificity for holotranscobalamin over apotranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of transcobalamin identified two structures in the N-terminal part of transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holotranscobalamin, HoloTC RIA., Conclusion: The identified antibody possesses a unique specificity for holotranscobalamin and can be used to develop a direct assay for the quantification of holotranscobalamin.

Recombinant human transcobalamin (TC) was probed with 17 monoclonal antibodies (mAbs), using surface plasmon resonance measurements. These experiments identified five distinct epitope clusters on the surface of holo-TC. Western blot analysis of the CNBr cleavage fragments of TC allowed us to distribute the epitopes between two regions, which spanned either the second quarter of the TC sequence GQLA...TAAM(103-198) or the C-terminal peptide LEPA...LVSW(316-427). Proteolytic fragments of TC and the synthetic peptides were used to further specify the epitope map and define the functional domains of TC. Only one antibody showed some interference with cobalamin (Cbl) binding to TC, and the corresponding epitope was situated at the C-terminal stretch TQAS...QLLR(372-399). We explored the receptor-blocking effect of several mAbs and heparin to identify TC domains essential for the interaction between holo-TC and the receptor. The receptor-related epitopes were located within the TC sequence GQLA...HHSV(103-159). The putative heparin-binding site corresponded to a positively charged segment KRSN...RTVR(207-227), which also seemed to be necessary for receptor binding. We conclude that conformational changes in TC upon Cbl binding are accompanied by the convergence of multiple domains, and only the assembled conformation of the protein (i.e. holo-TC) has high affinity for the receptor.

Transcobalamin (TC), a vitamin B12 (cobalamin, Cbl) binding protein in plasma, promotes the cellular uptake of the vitamin by receptor-mediated endocytosis. Inherited TC deficiency is an autosomal recessive disorder characterized by megaloblastic anaemia caused by cellular vitamin B12 depletion. It may be accompanied by neurological complications, including a delay in psychomotor and mental development. This report describes three sisters with inherited TC deficiency resulting from a splicing defect in the TC gene. A point mutation was identified in intron 3 splice site of the TC gene that activates a cryptic splice site in exon 3. The transcript generated has an in-frame deletion of 81 nucleotides and the resulting truncated protein is unstable and not secreted by the cells. Until now, genetic studies have been reported in only five patients with TC deficiency and the molecular defect was different in each of them, which gives evidence for a genetic heterogeneity of the disease.

We have previously reported the finding of a cyclic dodecapeptide representing loop I of the second EGF-like domain of FVII, which inhibited TF-dependent FX activation (Orning et al. 1997). The biological activity was localized to the tripeptide motif, Glu-Gln-Tyr. We have now synthesized a cyclic analog of this motif, Cys-Glu-Gln-Tyr-Cys (PN7051), evaluated its anticoagulant and antithrombotic properties and performed a detailed structural characterization of the peptide. PN7051 is a dose-dependent inhibitor of TF-dependent FX activation and coagulation of plasma with IC50 values of 10+/-2 microM and 1.3+/-0.2 mM, respectively. It shows inhibitory efficacy on acute thrombus formation in an ex vivo model of human thrombosis using native blood. Fibrin deposition, platelet-fibrin adhesion, platelet-thrombus formation, and thrombin-antithrombin complex formation were all inhibited by PN7051 at IC50 values between 0.3 and 0.7 mM. The cyclic peptide is a non-competitive inhibitor of FX activation with no significant active-site effects on FXa or FVIIa, indicating it affects FVII/TF/FX complex formation and function. Studies on the structure activity relationship revealed that Gln3-Tyr4, but not Glu2 were of importance for inhibition. In line with biological results, NMR measurements of PN7051 suggested that the Gln and Tyr residues configure a structural feature that contributes to the anticoagulant activity. Modeling of the Glu99Gln100Tyr101 motif in FVII and comparison with the solution structure of PN705 I suggest that the cyclic pentapeptide exerts its antithrombotic effect by interfering with the docking of Tyr101 into a hydrophobic pocket in the catalytic domain thereby disrupting an essential interaction between the second EGF-like and the catalytic domains of FVII.

Background: In India, most people adhere to a vegetarian diet, which may lead to cobalamin deficiency., Objective: The objective was to examine indicators of cobalamin status in Asian Indians., Design: The study population included 204 men and women aged 27-55 y from Pune, Maharashtra, India, categorized into 4 groups: patients with cardiovascular disease (CVD) and diabetes, patients with CVD but no diabetes, patients with diabetes but no CVD, and healthy subjects. Data on medical history, lifestyle, and diet were obtained by interviews and questionnaires. Blood samples were collected for measurement of serum or plasma total cobalamin, holotranscobalamin (holoTC), methylmalonic acid (MMA), and total homocysteine (tHcy) and hemetologic indexes., Results: MMA, tHcy, total cobalamin, and holoTC did not differ significantly among the 4 groups; therefore, the data were pooled. Total cobalamin showed a strong inverse correlation with tHcy (r = -0.59) and MMA (r = -0.54). Forty-seven percent of the subjects had cobalamin deficiency (total cobalamin <150 pmol/L), 73% had low holoTC (<35 pmol/L), 77% had hyperhomocysteinemia (tHcy >15 micromol/L), and 73% had elevated serum MMA (>0.26 micromol/L). These indicators of impaired cobalamin status were observed in both vegetarians and nonvegetarians. Folate deficiency was rare and only 2.5% of the subjects were homozygous for the MTHFR 677C-->T polymorphism., Conclusions: About 75% of the subjects had metabolic signs of cobalamin deficiency, which was only partly explained by the vegetarian diet. If impaired cobalamin status is confirmed in other parts of India, it may have important health implications.

The cyclic dodecapeptide, disulfide-cyclo-[H-Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys(Acm)-Glu-Gln-Tyr-Cys-OH], which corresponds to the 91-102 sequence of the second epidermal growth factor domain of human blood coagulation factor VII, was synthesized using solid-phase procedures. It was shown to be an inhibitor at the key step in the induction of coagulation by the extrinsic pathway, i.e. the factor VII/tissue factor-catalyzed activation of coagulation factor X. The solution structure of this peptide was investigated by NMR spectroscopy and was computer-modeled via molecular mechanics. Structures were calculated based on 112 distance and nine dihedral angle constraints. The resulting backbone structures were classified into two structural subsets: one which exhibited a twisted '8'-shaped folding and another describing an open, circular 'O' outline. The local backbone structures of segments Asn3-Glu4-Asn5, Gly7-Cys8 and Gln10-Tyr11 were well preserved among the two subsets. Apart from the unrestrained N- and C-termini, Gly6 and Glu9 sites exhibited marked local disorder between the two subsets, suggesting localized flexible hinges likely to govern tertiary structure interconversion between the two subsets. Two transient hydrogen bonds were identified from pH chemical shift titrations by matching the pKa values of NH and carboxylate groups, which supported the occurrence of the '8' structure, and agreed with temperature coefficients of peptidyl NH resonances. Structure-function relationships of the peptide were discussed in terms of the likely physiological function of the disulfide-bonded loop in factor VII which the peptide represents.

The presence of a cysteine residue at or near the active site of leukotriene A(4) hydrolase (EC 3.3.2.6) was suggested by inactivation of the enzyme with sulfhydryl-blocking reagents and by protection against inactivation afforded by substrates and competitive inhibitors. The aminopeptidase activity was more susceptible to inactivation than the epoxide hydrolase activity. The sulfhydryl-modifying reagent methyl-methane thiosulfonate reacted with one thiol as judged by kinetic data and titration with 5, 5'-dithiobis-2-nitrobenzoate. Inactivation was a time- and dose-dependent process of apparent pseudo-first-order and maximal at 80-85%. The inactivation rate was nonsaturable and strongly influenced by ion strength. The second-order rate constant increased from 0.9 to 4.3 M(-1) s(-1) in the presence of 0.2 M NaCl. Albumin, a stimulator of the aminopeptidase activity, increased apparent inactivation rates by shifting pK(a) for the modification from 8.2 to 7.8. The inactivated enzyme partially regained activity upon treatment with beta-mercaptoethanol. Peptide substrates and competitive inhibitors protected against inactivation. Bestatin, a competitive inhibitor, afforded complete protection with a K(D) = 0.15 microM, similar to K(i) = 0.17 microM for inhibition of peptidase activity. Treated enzyme had an unchanged K(m) but a reduced V(max). The epoxide hydrolase activity was only weakly affected by methyl-methane thiosulfonate with a maximal inactivation of 15-20% after prolonged treatment. Pretreatment of leukotriene A(4) hydrolase with the reagent did not protect against mechanism-based inactivation by its lipid substrate, leukotriene A(4). On the other hand, leukotriene B(4) was a competitive inhibitor of aminopeptidase activity and protected against modification by methyl-methane thiosulfonate. Our results suggest the presence of a cysteine at or close to subsite S'(1) of the active site of leukotriene A(4) hydrolase and that modification of this residue interferes with the function of the aminopeptidase activity, but not the epoxide hydrolase activity. This is the first report to distinguish the two catalytic activities of leukotriene A(4) hydrolase by chemical means., (Copyright 1999 Academic Press.)

Coagulation factor VII bound to its cofactor tissue factor is the physiological initiator of blood coagulation. The interaction between factor VII and tissue factor involves all four of the structural modules found in factor VII, with the most significant contribution coming from the first EGF-like domain. In this study, the synthesis and biological activity of several analogues derived from the first EGF-like domain of FVII comprising the sequence 45-83 are reported on. The six cysteine residues found in the native protein were replaced by Abu. The peptides were isolated from a multicomponent mixture following standard Fmoc solid phase synthesis. Purification and characterisation of the heterogeneous product showed that aspartimide formation was a major side-reaction, occurring predominantly at the Asp46-Gly47 and Asn57-Gly58 dipeptides. Although relatively common in peptide synthesis, the extent to which this side-reaction had taken place was considered surprising. Reported herein are the analytical methods used to isolate and characterise several of the modified products. Also, the inhibitory effect of these peptides on the formation and enzymatic activity of the factor VIIa/tissue factor complex have been compared. Surprisingly, the peptide containing an iso-Asp residue at position 57 possessed 66-fold higher inhibitory activity compared with the original target peptide. A possible explanation for this increase in observed activity is presented.

Synthetic peptides based on the putative factor X recognition site of human (Thr-Leu-Tyr-Tyr-Trp-Lys-Ser-Ser-Ser-Ser), rabbit (Thr-Leu-Tyr-Tyr-Trp-Arg-Ala-Ser-Ser-Thr), and murine tissue factor (Ile-Ile-Thr-Tyr-Arg-Lys-Gly-Ser-Ser-Thr) were dose-dependent inhibitors of human tissue factor/factor VIIa catalyzed factor X activation with IC50 values of 220, 17, and 33 microM, respectively. The mouse results were highly surprising given the low homology between the human and mouse sequence (40%) and that mouse tissue factor, in contrast with rabbit tissue factor, does not support the procoagulant activity of human factor VIIa on factor X. The inhibitory mechanism of the murine peptide was noncompetitive with respect to factor X but competitive with respect to tissue factor, indicating the peptide competes with tissue factor (or the tissue factor/factor VIIa complex) for binding to factor X. The peptide could be N-terminally truncated by two Ile without loss of inhibitory activity or changed inhibitory mechanism. Substitution of two Gly for the two Ile, which increased solubility, decreased IC50 to 17 microM whereas scrambling the peptide made it inactive.

A missense mutation at codon 100 in the second epidermal growth factor-like domain, resulting in Gln100-->Arg, was detected in 19 out of 21 available severely factor VII (FVII) deficient patients in Norway. Seventeen patients were homozygous, and the two remaining were compound heterozygotes. In the homozygous patients, FVII antigen was measured to 10-28%, and activity to 0.6-6.5% of that in normal pooled plasma. Recombinant FVII containing the mutation was expressed transiently in CHO cells to a mean antigen level of 57% of the wild type FVII protein, and with a specific activity of 6% of wild type. The mutant protein had a 14-fold reduction in affinity for tissue factor (TF), whereas binding of FX seemed unaffected. In line with the experimental data, molecular modelling of the mutation based on the coordinates of the tissue factor/FVIIa complex showed that substituting arginine for glutamine disrupts the interface between the catalytic and second epidermal growth factor-like domains.

Factor VIIa (FVIIa) is the enzymatically active constituent of the FVIIa/tissue factor (TF) complex, the initiator of the extrinsic pathway of blood coagulation. The zymogen FVII and FVIIa are composed of discrete domains, two of which are homologous to the epidermal growth factor (EGF). This investigation examined the significance of the FVII EGF-2 domain in the processes leading to activation of factor X (FX). Peptides 47 residues in length and corresponding to the amino acid sequence of the EGF-2 domain of human FVII were prepared by solid-phase synthesis methods. Peptide variants with all six Cys residues replaced by L-2-aminobutyryl residues (1), or containing one (2a-c), two (3a,b) or three (4) disulfide bonds, were obtained by application of various S-protecting groups and oxidation methods. Peptide 4, containing the cystine bridge arrangement corresponding to that found in the native protein, was prepared by a two-step regioselective disulfide bond formation method. An evaluation of the anti-coagulant properties of peptides 1-4 revealed that all peptides, with the exception of the two-cystine isomer containing non-native disulfide pairings (3b), were potent inhibitors of TF/FVIIa-mediated activation of FX. The fully constrained peptide 4 was found to be twice as active as its completely non-constrained counterpart 1, the two peptides showing IC50 values of 1.6 +/- 0.5 microM (1) and 0.8 +/- 0.2 microM (4) with respect to TF/FVIIa-dependent FX activation. The results of this study demonstrate the functional importance of the EGF-2 domain of FVII in the induction of coagulation by the extrinsic pathway.

Several studies have indicated a profound role for factor VII(a) [FVII(a)] in venous and arterial thrombogenesis. In the present study, we quantified the inhibitory efficacy of dansyl-glutamyl-glycyl-arginyl-recombinant FVIIa (DEGR-rFVIIa) on acute thrombus formation. Thrombus formation was elicited by immobilized tissue factor (TF) in a parallel-plate perfusion chamber device at blood flow conditions characterized by wall shear rates of 100 S-1 (veins) and 650 S-1 (medium-sized healthy arteries). Native human blood was drawn directly from an antecubital vein by a pump into a heparin-coated mixing device in which DEGR-rFVIIa (0.09 to 880 nmol/L final plasma concentration) or buffer was mixed homogeneously with flowing blood. Subsequently, the blood was passed over a plastic coverslip coated with TF and phospholipids in the parallel-plate perfusion chamber. Fibrin deposition, platelet-fibrin adhesion, and platelet thrombus volume triggered by this surface were measured by morphometry. DEGR-rFVIIa inhibited thrombus formation in a dose-dependent manner, but the efficacy was shear rate dependent. At a wall shear rate of 100 S-1, the IC50 (50% inhibition) was 30 nmol/L, whereas at 650 S-1, the IC50 was 0.6 nmol/L. Binding studies to immobilized TF under flow conditions using surface plasmon resonance revealed a significantly higher on-rate for DEGR-rFVIIa and FVIIa than for FVII, 2.8 x 10(5), 2.6 x 10(5), and 1.8 x 10(5) M-1 S-1, respectively. This indicates that a contributing factor to the shear-dependent efficacy may be a differential importance of on-rates at arterial and venous blood flow conditions.

We have found that synthetic peptides derived from the two epidermal growth factor-like domains of factor VII are inhibitors of tissue factor dependent factor X activation. Inhibition was most pronounced for a constrained sequence of amino acids corresponding to positions 91-102 of factor VII, Cys-Val-Asn-Glu-Asn-Gly-Gly-Cys-Glu-Gin-Tyr-Cys. The biological activity appeared to be localized to the tripeptide 'motif', Glu-Gln-Tyr, within the larger sequence. The cyclic peptide was also an inhibitor of tissue factor induced coagulation of plasma, using lipidated tissue factor or tissue factor expressed on the surface of living cells. However, it did not interfere with intrinsic coagulation. Inhibition of factor X activation was dose-dependent with an IC50 value of 350 microM. Kinetic analyses revealed non-competitive inhibition with respect to factor X and suggested that the peptide sequence interferes with the factor VII/tissue factor/factor X complex formation and function. A pentapeptide analog of the putative pharmacophore was also a dose-dependent inhibitor of factor X activation with an IC50 value of 560 microM, but the tripeptide, Glu-Gin-Tyr, alone was without effect. Our results suggest a direct role for the second epidermal growth factor-like domain of factor VII, and in particular its loop I, in the formation and function of the factor VII/tissue factor/factor X complex.

A novel model is described for characterisation of cell-surface procoagulant activities and their inhibitors. Microcarrier beads were used to present living cells to recalcified blood plasma in the stirred measuring wells of an electromagnetic coagulometer. By this means the procoagulant activity on the surface of the cells could be automatically determined as clotting time. Procoagulant activity was investigated on normal and transformed cells, and representing hemopoietic, endothelial, muscle and connective tissue phenotypes. The procoagulant activity on each cell type was characterised by the use of specifically immunodepleted plasmas and specific inhibitors, including monoclonal antibodies. The predominant cell surface trigger of coagulation found in this series was tissue factor, and only blood monocytes provided some evidence for direct activation of factor X independent of FVII. Human ECV304 transformed endothelial cells were more closely studied as representative of a cell type constitutively expressing procoagulant. Coagulation mediated by ECV304 cells was found to be strictly dependent on tissue factor, as shown by an inhibitory monoclonal antibody, and on coagulation factors V, VII and X. ECV304 procoagulant activity was strongly inhibited by active-site-inactivated FVIIa, a synthetic peptide inhibitor of FXa (Tenstop) and the thrombin inhibitor, hirudin. While not appropriate for routine clinical assessment of coagulation factor function, we have found this model to be valuable in characterising the procoagulant activity on different cell types and particularly useful as a drug discovery tool in the search for new anticoagulants.

Background: The aims of the present study were to investigate whether ionic and nonionic contrast media (CM) affect: 1) the procoagulant and fibrinolytic activities of cultured human vessel endothelium; and 2) early events of tissue-factor-induced arterial thrombus formation under conditions which may follow a percutaneous transluminal coronary angioplasty (PTCA) procedure. The following 3 CM were studied: iohexol (nonionic monomer, Omnipaque); iodixanol (nonionic dimer, Visipaque); and ioxaglate (ionic dimer, Hexabrix). Saline (0.9%) and glucose (40 vol%) were used as control., Methods and Results: Exposing endothelium to 40 vol% CM for 10 min did not affect the selected parameters of cellular procoagulant (tissue factor), anticoagulant (thrombomodulin), fibrinolytic (tissue plasminogen activator) or antifibrinolytic (plasminogen activator inhibitor-1) activity or antigen. However, ioxaglate had a profound impact on the cell morphology, which was noted already after one minute of exposure. The cells contracted and rounded, exposing large areas of extracellular matrix. Iohexol showed this phenomenon to a considerably lesser extent, whereas iodixanol induced a slight swelling of the cells without detectable exposure of extracellular matrix. The effect of the respective CM on tissue-factor-driven thrombus formation at an arterial shear rate of 2600 s-1 was studied in an ex vivo parallel-plate perfusion chamber device. In this model, human native blood was passed over a tissue factor/phospholipid-rich surface following 30 s exposure to 100% CM. The CM was washed out by nonanticoagulated blood drawn directly from an antecubital vein by a pump positioned distal to the perfusion chamber. Such a pre-exposure of the procoagulant surface to iodixanol reduced the fibrin deposition around the platelet thrombi by 50% (p<0.01). However, iohexol and ioxaglate did not affect fibrin deposition. None of the 3 CM affected the recruitment of platelets in the thrombi, since similar values were obtained with pre-exposure to 40 vol% of saline., Conclusion: Iodixanol appears to be most biocompatible with endothelium, and has a moderate inhibitory effect on fibrin deposition in flowing blood. This differs from iohexol, and in particular from ioxaglate, which induce endothelial changes in morphology with no effect on fibrin deposition. Since none of the CM affected the platelet aggregate formation, and since ioxaglate has been reported to have stronger anticoagulant and antithrombotic properties than iodixanol or iohexol in in vitro assays, it is apparent that these properties were not reflected in thrombus formation under the experimental conditions of high arterial shear.

Factor VII (FVII) and tissue factor (TF) form a binary complex which initiates the extrinsic pathway of the blood coagulation cascade. The infrequent tripeptide motif Trp-Lys-Ser (WKS) is found three times in TF. It has been suggested that the motif is involved in binding of TF to FVII(a). Also. Lys165 and Lys166 of TF have been reported to be important for factor X activation. To elucidate the molecular interactions between TF and FVIIa, and the interactions between the binary complex and FX, we examined the inhibitory effect of synthetic TF and FVII peptide analogs. One- and two-stage chromogenic assays were employed, as well as one-stage coagulation assay. The peptide analogs of TF possessed the WKS motif, the double lysine residues or other regions of TF. Synthetic peptides of FVII encompassing sequences of the FVII285-305 region were included for comparative purposes. TF154-167 and FVII300-305 significantly inhibited both FX activation and plasma coagulation. FVII285-294 acted synergistically, increasing that effect observed by FVII300-305 on FX activation. However, TF163-175 possessing the double lysine residues did not inhibit FX activation, indicating that inhibition of FXa formation and coagulation by TF154-167 is due to the region 154-162 of TF. None of the peptides, including the WKS tripeptide, interfered with the FVIIa activity of the TF/FVIIa complex. Thus, the results do not suggest that the WKS motifs are necessary for binding of TF to FVIIa but that the third WKS motif may be of importance for the activation of FX.

Albumins from several species activated the bifunctional, Zn2+ metalloenzyme amino-peptidase/leukotriene A4 hydrolase (EC 3.3.2.6). Bovine serum albumin, 1 mg/mL, increased hydrolysis of L-proline-p-nitroanilide and leucine-enkephalin by 12-fold and 7-fold, respectively. The apparent Km for L-proline-p-nitroanilide was inversely proportional to the albumin concentration from 0 to 1 mg/mL, declining from 9.4 to 0.7 mM without an appreciable change in apparent Vmax. These data imply a random activation process in which the enzyme-activator complex is catalytically dominant. Hill plots indicated a 1:1 stoichiometric relationship between albumin and enzyme. Secondary plots of slope versus the reciprocal of albumin concentration indicated that it binds to the enzyme with an affinity constant of 0.9 microM. The pH optimum of the nonactivated enzyme occurred at pH 8; the albumin-activated enzyme had an optimum near pH 7. Neither ultrafiltration nor dialysis of albumin altered its activating effect, but boiling abolished it. Albumin did not affect other cytosolic or microsomal leucine aminopeptidases, or gamma-glutamyltransferase. Albumin functions as a nonessential activator, since enzymatic activity was always detectable in its absence. Chloride ions, which activate other Zn2+ metalloenzymes, also activated leukotriene A4 hydrolase/aminopeptidase with an EC50 = 50 mM, increasing its initial velocity 2.2-fold in the absence of albumin. Zn2+ activated the enzyme, increasing its apparent Vmax but not its apparent Km, suggesting it replaced Zn2+ lost from the active site, especially at acidic pH. At concentrations greater than 30-50 microM, Zn2+ was inhibitory. Albumin mitigated the effect of chloride, but not the effect of Zn2+ or that of the competitive inhibitor, captopril.(ABSTRACT TRUNCATED AT 250 WORDS)

Leukotrienes were transported into rat hepatocytes by a temperature- and energy-dependent mechanism. The uptake was saturable with high- and low-affinity sites (Km values approx. 1 and 17 microM). Competition and kinetic experiments indicated that leukotrienes C4, D4 and E4 were transported by a common mechanism. The maximal velocity of transport was about 50% higher for leukotrienes D4 and E4 than for leukotriene C4. Leukotriene B4, glutathione disulfide, and the glutathione-S-conjugate of acetaminophen did not interfere with the transport of leukotriene C into hepatocytes. This suggests that the process is specific for cysteine-containing leukotrienes. It is likely that the transport mechanism described here participates in biliary excretion of leukotrienes. This route was previously found to be a major one for elimination of leukotriene C3 in mice and guinea-pigs.

Leukotriene E4 was metabolized to two polar products by rat liver microsomes. These products were characterized by physico-chemical and chemical techniques. The chemical structures, (5S, 6R)-5,20-dihydroxy-6S-cysteinyl-7,9-trans-11,14-cis-icosatetraenoic acid (omega-hydroxy-leukotriene E4) and (5S, 6R)-5-hydroxy-6S-cysteinyl-7,9-trans-11,14-cis-icosatetraen-1,20-d ioic acid (omega-carboxy-leukotriene E4) suggested that leukotriene E4 was transformed by an omega-hydroxylase and omega-hydroxyleukotriene E dehydrogenase in sequence. N-Acetyl-leukotriene E4 was also transformed by these enzymes, but at a rate six times lower than leukotriene E4. The products formed from N-acetylleukotriene E4 were characterized as being N-acetyl-omega-hydroxy-leukotriene E4 and N-acetyl-omega-carboxy-leukotriene E4. Other substrates were 11-trans-leukotriene E4 and N-acetyl-11-trans-leukotriene E4. In contrast, leukotrienes C4 and D4 were not converted into omega-oxidized metabolites. The leukotriene E omega-hydroxylase reaction required NADPH and molecular oxygen as cofactors, and was most rapidly catalyzed by liver microsomes. Liver cytosol, fortified with NAD+, converted omega-hydroxyleukotriene E4 and N-acetyl-omega-hydroxy-leukotriene E4 into omega-carboxy metabolites. Microsomes contained at least 18 times less omega-hydroxy-leukotriene E dehydrogenase activity than did cytosol. Liver microsomes supplemented with acetyl-coenzyme A converted omega-hydroxy and omega-carboxy-leukotriene E4 into the corresponding N-acetyl derivatives. The novel enzyme, leukotriene E omega-hydroxylase, which is described here is distinct from a previously described leukotriene B omega-hydroxylase based on substrate competition and kinetic data.

Six major basic cytosolic glutathione transferases from rat liver catalyzed the conversion of leukotriene A4 methyl ester to the corresponding leukotriene C4 monomethyl ester. Glutathione transferase 4-4, the most active among these enzymes, had a Vmax of 615 nmol X min-1 X mg protein-1 at 30 degrees C in the presence of 5 mM glutathione. It was followed in efficiency by transferase 3-4 which had a Vmax of 160 nmol X min-1 X mg-1 under the same conditions. Transferases 1-1, 1-2, 2-2 and 3-3 had at least 30 times lower Vmax values than transferase 4-4.

A polar metabolite of leukotriene C4 was formed by sequential conversions with soybean lipoxygenase I and liver peroxidase. The structure of this product was found to be 5(S), 15(S)-dihydroxy-6(R)-S-glutathionyl-7,9,13-trans-11-cis-eicosatetraenoic acid (15-hydroxy-delta 13-trans-leukotriene C3. The HPLC behaviour, the molar extinction coefficient and the biological activity of the metabolite are reported. Preliminary evidence suggests that this product is formed by mammalian tissues.

Previous investigations have demonstrated metabolism of leukotriene (LT) C4 in vivo involving transformations of the tripeptide, but not the fatty acid part, yielding N-acetyl LTE4 as a main biliary metabolite in the rat. In addition, several polar metabolites were detected in the same studies. The present report describes the characterization of a metabolite of N-acetyl LTE4 formed during incubations with rat liver microsomes. The structure, 5,20-dihydroxy-6-s-(2-acetamido-3-thiopropionyl)-7,9-trans-11, 14-cis-eicosa-tetraenoic acid, of this metabolite showed that it is formed by hydroxylation of the fatty acid part. Preliminary evidence indicates that it is one of several polar metabolites formed in vivo.

Five - 20 nmoles of [5,6,8,9,11,12,14,15-3H8]leukotriene C4 was injected into three male volunteers. Forty-eight percent of the administered 3H was recovered from urine and 8% from feces, within a 72 hr period. Of the total urinary radioactivity 44% was excreted during the first hour after injection. This activity was mainly found in one compound, designated "I". The radioactivity excreted into urine later than one hour after injection, consisted partly of Compound I and two additional components, and partly of polar, non-volatile material. Compound I was identified as leukotriene E4 by UV-spectroscopy and cochromatographies in three high performance liquid chromatography systems with synthetic reference compounds. A total of 13% of administered radioactivity was excreted in urine as leukotriene E4.

The in vitro metabolism of leukotriene B4 is initiated by omega-hydroxylation. This reaction is followed by oxidation of the omega-hydroxyl group to a carboxyl group. In vivo extensive beta-oxidation occurs and the main excreted products after administration of leukotriene B4 are water and carbon dioxide. Experiments performed in vitro and in vivo have demonstrated that a major pathway of metabolism of the glutathione containing leukotrienes involves modifications of the tripeptide substituent. The metabolic alterations are initiated by enzymatic elimination of the N-terminal gamma-glutamyl residue, catalyzed by the enzyme gamma-glutamyl transferase. This reaction is followed by hydrolysis of the remaining peptide bond resulting in elimination of the C-terminal glycine residue. The enzyme catalyzing the latter reaction is a membrane bound dipeptidase which occurs in kidney and other tissues. The product formed by these reactions, leukotriene E4, has been tentatively identified as a urinary metabolite in man following intravenous administration of leukotriene C4. In rats, the two major fecal metabolities of leukotriene C4 were characterized as being N-acetyl leukotriene E4 and N-acetyl 11-trans leukotriene E4. These compounds are formed in reactions between leukotriene E4 or 11-trans leukotriene E4 and acetyl coenzyme A. The reactions are catalyzed by a membrane bound enzyme present in liver, kidney and other tissues.