Antenatal Bartter's syndrome: why is this not a lethal condition?

There are four themes in this teaching exercise for Professor McCance. The first challenge was to explain how a premature infant with Bartter's syndrome could survive despite having such a severe degree of renal salt wasting. Second, the medical team wanted to know why there was such a dramatic decrease in the natriuresis in response to therapy, despite the presence of a permanent molecular defect that affected the loop of Henle. Third, Professor McCance was asked why this patient seemed to have a second rare disease, AQP2 deficiency type of nephrogenic diabetes insipidus. The fourth challenge was to develop a diagnostic test to help the parents of this baby titrate the dose of indomethacin to ensure an effective dose while minimizing the likelihood of developing nephrotoxicity. The missing links in this interesting story emerge during a discussion between the medical team and its mentor.

Solid-phase optimisation of achiral amidinobenzyl indoles as potent and selective factor Xa inhibitors.

Starting from the achiral and potent factor Xa inhibitor 1, a new and flexible solid-phase optimisation strategy is described to reduce its cationic character. By replacing one positively charged side chain by a lipophilic substituent, a novel series of highly potent and selective achiral factor Xa inhibitors was discovered. The identified lipophilic replacements in the S4 pocket might be valuable for other approaches towards fXa inhibitors.

Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study.

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.

Discovery of a novel, potent, and specific family of factor Xa inhibitors via combinatorial chemistry.

A series of low molecular weight peptide inhibitors of factor Xa, unrelated to any previously described, was identified by screening a combinatorial peptide library composed of L-amino acids. The minimal inhibitory sequence is a tripeptide, L-tyrosinyl-L-isoleucyl-L-arginyl, which competitively inhibits the hydrolysis of small chromogenic substrates by factor Xa but binds in an orientation which prevents a productive nucleophilic attack by serine 195 of the catalytic triad on the carbonyl carbon of the carboxyterminal arginine. The initial leads identified in an octamer combinatorial peptide library ranged in potency from 4 to 15 microM. These peptides were modified into peptide mimetics with a greater than 1000-fold increase in potency while retaining unusual selectivity for factor Xa over the related serine proteases thrombin, factor VIIa/tissue factor, plasmin, activated protein C, kallikrein, and trypsin. One of the most potent analogues, SEL 2711, with a Ki of 0.003 microM for factor Xa and 40 microM for thrombin, is active in in vitro and ex vivo coagulation assays, suggesting the potential application of these inhibitors in anticoagulant therapy.

High affinity Ca(2+)-binding site in the serine protease domain of human factor VIIa and its role in tissue factor binding and development of catalytic activity.

Factor VIIa, in the presence of Ca2+ and tissue factor (TF), initiates the extrinsic pathway of blood coagulation. The light chain (amino acids 1-152) of factor VIIa consists of an N-terminal gamma-carboxyglutamic acid (Gla) domain followed by two epidermal growth factor-like domains, whereas the heavy chain (amino acids 153-406) contains the serine protease domain. In this study, both recombinant factor VIIa (rVIIa) and factor VIIa lacking the Gla domain were found to contain two high-affinity (Kd approximately 150 microM) Ca2+ binding sites. The rVIIa also contained approximately 6-7 low-affinity (Kd approximately 1 mM) Ca(2+)-binding sites. By analogy to other serine proteases, one of the two high affinity Ca(2+)-binding sites in factor VIIa may be formed involving Glu-210 and Glu-220 of the protease domain. In support of this, a synthetic peptide composed of residues 206-242 of factor VIIa bound one Ca2+ with Kd approximately 230 microM; however, Ca2+ binding was observed only in Tris buffer (pH 7.5) containing 1 M NaCl and not in buffer containing 0.1 M NaCl. In both low or high salt +/- Ca2+, the peptide existed as a monomer as determined by sedimentation equilibrium measurements and had no detectable secondary structure as determined by CD measurements. This indicates that subtle changes undetectable by CD may occur in the conformation of the peptide that favor calcium binding in high salt. In the presence of recombinant TF and 5 mM Ca2+, the peptide inhibited the amidolytic activity of rVIIa toward the synthetic substrate, S-2288. The concentration of the peptide required for half-maximal inhibition was approximately 5-fold higher in the low salt buffer than that in the high salt buffer. From direct binding and competitive inhibition assays of active site-blocked 125I-rVIIa binding to TF, the Kd for peptide-TF interaction was calculated to be approximately 15 microM in the high salt and approximately 55 microM in the low salt buffer containing 5 mM Ca2+. Moreover, as inferred from S-2288 hydrolysis, the Kd for VIIa.TF interaction was approximately 1.5 microM in the absence of Ca2+, and, as inferred from factor X activation studies, it was approximately 10 pM in the presence of Ca2+. Thus, Ca2+ decreases the functional Kd of VIIa.TF interaction approximately 150,000-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

The effect of two-domain tissue factor pathway inhibitor on endotoxin-induced disseminated intravascular coagulation in rabbits.

Disseminated intravascular coagulation (DIC) is a common complication in sepsis, and may result from endotoxin-induced exposure of tissue factor on the surface of monocytes and endothelial cells. Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent feedback inhibitor of the tissue factor-factor VIIa complex. In the present study the effect on DIC of a two-domain TFPI analogue (2D-TFPI), consisting of the first two Kunitz domains of TFPI but lacking the third domain, was tested. DIC was induced in rabbits by two intravenous bolus injections of endotoxin from Escherichia coli (10 and 50 micrograms/kg) 24 h apart. Simultaneously with the last endotoxin injection an infusion of 2D-TFPI (0, 0.3, 1.0 or 3.0 mg/kg/h) was given. Blood samples were obtained at 0 h, 24 h and 31 h. At 31 h the animals were sacrificed and the kidneys were submitted to histological examination. The degree of fibrin deposition in glomeruli was scored blindly using an arbitrary scale from 0 to 3. Between 24 and 31 h the group receiving endotoxin alone showed a significant decrease in platelet count (65%), plasma fibrinogen (41%), antithrombin III (25%), and factor VIII (63%), and a significant prolongation of the aPTT (14%). Furthermore, massive fibrin deposition was detected in the renal glomeruli at 31 h. Infusions of 2D-TFPI inhibited all the endotoxin-induced changes in a dose-dependent manner. In conclusion, the data demonstrate that inhibition of the TF/FVIIa complex by infusion of 2D-TFPI significantly counteracts endotoxin-induced coagulopathy in rabbits, and might thus be an attractive drug for treatment of endotoxin-induced DIC in humans.

Evidence that the C-terminus of tissue factor pathway inhibitor (TFPI) is essential for its in vitro and in vivo interaction with lipoproteins.

We have previously shown that the C-terminus of TFPI is essential for its anticoagulant activity. In the present study we have assessed the role of this region in the binding of TFPI to lipoproteins. We found that full length TFPI, but not C-terminal degraded TFPI, was capable of coeluting with the plasma lipoprotein fraction on a Superose-6 column. The importance of the TFPI C-terminus in lipoprotein interactions was also assessed using a microtitre plate binding assay. We found that full-length TFPI was capable of binding to VLDL or LDL coated microtitre plates. C-terminal degraded TFPI also bound to VLDL, but with a ten-fold lower affinity than full length TFPI. Interestingly, removal of the C-terminus along with the third Kunitz-type domain resulted in a TFPI form incapable of lipoprotein binding. Since heparin shows strong binding to the C-terminus of TFPI, we also tested its effect on the binding of full length TFPI to VLDL. We found that co-incubation of TFPI with heparin inhibited this binding in a dose-dependent manner. Heparin was also capable of releasing TFPI from a preformed TFPI:VLDL complex, although this reaction required unphysiological amounts of heparin. To assess the physiological function of heparin on FL-TFPI:lipoprotein interactions we also performed gel filtration chromatography of rabbit plasma immediately following i.v. administration of FL-TFPI with and without heparin. Previous experiments indicated that heparin has a protective effect on exogenously added FL-TFPI, increasing its recovery by ten-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of the recombinant factor VIIa in rat bone: importance of the Gla-domain and relevance to factor IX, another vitamin K-dependent clotting factor.

The vitamin K-dependent clotting factors II, VII, IX, and X are proteins which undergo gamma-carboxylation of specific glutamic acid residues prior to secretion from the liver. These unique Ca2+ binding amino acids allow the interaction of the proteins with cell surface phospholipids, a function that is crucial for expression of full procoagulant activity of the proteins. The N-terminal region of the molecule contains the gamma-carboxylation sites and is termed the Gla-domain. A preliminary observation in rats suggested that mineralized bone accumulated activated recombinant FVII (rFVIIa: NovoSeven) as well as the non-activated, single chain rFVII. The present study investigated the role of the Gla-domain in the accumulation of rFVII in bone, as well as the influence of the activation state of FVII on this phenomenon. Rats were treated with 125I-labelled rFVII, rFVIIa, Gla-domainless rFVIIa, factor IX, iodide, or recombinant human growth hormone (rhGH). Following sacrifice, radioactivity was measured in mineralized bone, among other tissues. Following administration of 125I-radiolabelled rFVII, rFVIIa and factor IX, but not Gla-domainless rFVIIa, iodide or rhGH, extensive sequestration occurred in endochondrally as well as intramenbranously ossified bones. The results indicate that the proteins containing a Gla-domain, and only these, are sequestered in bone. Additionally, the normally occurring form of FVII in the circulation, the single-chain FVII, exhibited similar kinetics in rat bone and plasma, as the two-chain rFVIIa. The half-life of rFVII/rFVIIa in mineralized bone was between 3 and 4 days, implying that significant bone accumulation of the factor will take place at steady state.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa.

The interaction of circulating factor VII/VIIa with tissue factor presented by cells in extravascular tissues represents the initial event in the extrinsic pathway of blood coagulation. To determine the tissue factor binding domains in human factor VIIa, we have subjected recombinant human factor VIIa to tryptic digestion and isolated two proteolytic fragments (molecular mass = 32 and 20 kDa) by a combination of immunoaffinity chromatography and reversed phase high performance liquid chromatography (HPLC) which strongly inhibits the tissue factor-enhanced amidolytic activity of factor VIIa and inhibits the activation of factor X by factor VIIa in the presence of tissue factor. The 32-kDa factor VIIa fragment consisted of residues 1-137/143 from the light chain of factor VIIa connected by a disulfide bond to residues 153-277 from the heavy chain. The 20-kDa factor VIIa fragment consisted of residues 1-137 of the light chain of factor VIIa in disulfide linkage with residues 248-266 of the heavy chain. The 32- and 20-kDa factor VIIa fragments inhibited the tissue factor apoprotein-enhanced factor VIIa amidolytic activity with Ki values of 35 and 65 nM, respectively. The Ki values for the inhibition of relipidated tissue factor apoprotein-enhanced factor VIIa amidolytic activity by the 32- and 20-kDa factor VIIa fragments were 70 and 610 nM, respectively. Factor X activation by factor VIIa-relipidated tissue factor was inhibited half-maximally by the 32- and 20-kDa factor VIIa fragments at 65 and 680 nM concentrations, respectively. Equilibrium binding studies indicated that the 32- and 20-kDa factor VIIa fragments interacted with cell surface tissue factor expressed on J82 cells in a specific and saturable manner with Kd values of 30 and 64 nM, respectively. In addition, a peptide consisting of residues 1-109 from the light chain of factor VIIa obtained by reduction and HPLC of the 20-kDa factor VIIa fragment retained inhibitory activity, but the selective removal of the gamma-carboxyglutamic acid domain from the 20-kDa factor VIIa fragment by cathepsin G cleavage resulted in the complete loss of inhibitory activity in this fragment. Our data strongly suggest that the epidermal growth factor-like domains covalently linked to the gamma-carboxyglutamic acid domain in factor VIIa constitute the high affinity tissue factor binding domain in this molecule.

Identification of a calcium binding site in the protease domain of human blood coagulation factor VII: evidence for its role in factor VII-tissue factor interaction.

Previous studies have identified a putative calcium binding site involving two glutamic acid residues located in the protease domain of coagulation factor IX. Amino acid sequence homology considerations suggest that factor VII (FVII) possesses a similar site involving glutamic acid residues 210 and 220. In the present study, we have constructed site-specific mutants of human factor VII in which Glu-220 has been replaced with either a lysine (E220K FVII) or an alanine (E220A FVII). These mutants were indistinguishable from wild-type factor VII by SDS-PAGE but only possessed 0.1% the coagulant activity of factor VII. Incubation of E220K/E220A FVII with factor Xa resulted in a slower than normal activation rate which eventually yielded a two-chain factor VIIa molecule possessing a coagulant activity of approximately 10% that of wild-type rFVIIa. Amidolytic activity measurements indicated that E220K/E220A FVIIa, unlike wild-type factor VIIa, possessed no measurable amidolytic activity toward the chromogenic substrate S-2288, even at high CaCl2 concentrations. Addition of tissue factor apoprotein, however, induced the amidolytic activity of the mutant molecule to a level 30% of that observed for wild-type factor VIIa. This tissue factor dependent enhancement of E220K/E220A FVIIa amidolytic activity was calcium dependent and required a CaCl2 concentration in excess of 5 mM for maximal rate enhancement. This was in sharp contrast to wild-type factor VIIa which required CaCl2 levels of 0.5 mM for maximal enhancement of tissue factor dependent amidolytic activity. Competition binding experiments suggest that the decrease in amidolytic and coagulant activity observed in the factor VII mutants is a direct result of impaired tissue factor binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Measurement of basal levels of factor VIIa in hemophilia A and B patients.

Previous results, presented in abstract form, indicate that replacement of thromboplastin with a mixture of phospholipid and truncated soluble tissue factor apoprotein results in a coagulation assay that can directly measure plasma factor VIIa levels without interference from zymogen factor VII (Atherosclerosis Thromb 11:1544a, 1991 [abstr]). We have exploited the specificity and sensitivity of such a factor VIIa specific coagulation assay to directly assess the in vivo relationship of factor VIII and factor IX on the production of factor VIIa levels under nonthrombotic and nonstimulatory conditions. Normal individuals (n = 20) were found to possess an average circulating factor VIIa level corresponding to 4.34 +/- 1.57 ng/mL, or approximately 1% of their total factor VII antigen. Severe factor VIII deficient patients (n = 13) possessed a slightly lower but statistically significant (P less than .01) decrease in their basal factor VIIa levels (2.69 +/- 1.52 ng/mL), corresponding to approximately 60% of that observed in normal individuals. On the other hand, severe factor IX deficient patients (n = 7) were found to possess even lower levels of factor VIIa corresponding to 0.33 +/- 0.15 ng/mL, or less than 10% of that observed in normal individuals. Measurement of total factor VII antigen levels shows that the variation in basal factor VIIa levels stems from differences in the degree of factor VII activation as opposed to differences in factor VII antigen levels. Our present data are consistent with the hypothesis that factor IXa is the principal in vivo activator of factor VII under basal conditions.

The role of phospholipids and the factor VII Gla-domain in the interaction of factor VII with tissue factor.

Whether or not the factor VII Gla-domain is involved in the high-affinity interaction of factor VII and tissue factor via calcium-dependent interactions with surrounding phospholipids is unknown. To investigate this, we have purified the factor VII Gla-peptide (FVII-GP) from digested recombinant human factor VIIa and assessed its effect on factor VII:tissue factor interactions. FVII-GP inhibited the activation of factor X by factor VIIa in the presence of either soluble or cell surface tissue factor half-maximally at 0.5 microM and 2.7 microM, respectively. However, FVII-GP failed to inhibit the specific binding of factor VIIa to cell-surface tissue factor, and did not inhibit the ability of tissue factor to stimulate the amidolytic activity of factor VIIa. Unrelipidated tissue factor apoprotein stimulated the amidolytic activity of factor VIIa to the same extent as relipidated tissue factor apoprotein. These findings suggest that the factor VII Gla-domain does not directly interact with tissue factor, but rather is important for calcium binding and concomitant expression of other factor VII epitopes necessary for tissue factor recognition and binding. To test this hypothesis, we have prepared a monoclonal antibody against a putative factor VII epitope that participates in the interaction of factor VII with cell-surface tissue factor (peptide 195-206) and assessed its ability to bind to factor VII in the presence and absence of calcium. Binding of this monoclonal antibody (PW-4) to intact factor VIIa was calcium-dependent and could be inhibited in a dose-dependent manner by peptide 195-206.(ABSTRACT TRUNCATED AT 250 WORDS)

The C-terminus of tissue factor pathway inhibitor is essential to its anticoagulant activity.

Tissue factor pathway inhibitor (TFPI) from different cell lines shows up to 15-fold differences in the ratio of anticoagulant to chromogenic activity. The anticoagulant activity was dependent on the purification procedure used and it was possible to isolate two fractions of recombinant TFPI. Only one of these fractions showed anticoagulant activity comparable with TFPI from normal human plasma, and Western blotting showed that the low-activity fraction did not react with an antibody raised against a peptide of TFPI located near the C-terminal. Analysis by mass spectroscopy of peptides from V8 protease digests showed that C-terminal amino acids could only be identified from the high-activity form, while heterologous fragmentation had taken place in the form with low anticoagulant activity. Previously published studies on TFPI have been performed using material of low anticoagulant activity compared with plasma TFPI, and we suggest that these studies have been performed with material degraded in the C-terminus.

The importance of residues 195-206 of human blood clotting factor VII in the interaction of factor VII with tissue factor.

Previous studies indicated that human and bovine factor VII exhibit 71% amino acid sequence identity. In the present study, competition binding experiments revealed that the interaction of human factor VII with cell-surface human tissue factor was not inhibited by 100-fold molar excess of bovine factor VII. This finding indicated that bovine and human factor VII are not structurally homologous in the region(s) where human factor VII interacts with human tissue factor. On this premise, we synthesized three peptides corresponding to regions of human factor VII that exhibited marked structural dissimilarity to bovine factor VII; these regions of dissimilarity included residues 195-206, 263-274, and 314-326. Peptide 195-206 inhibited the interaction of factor VII with cell-surface tissue factor and the activation of factor X by a complex of factor VIIa and tissue factor half-maximally at concentrations of 1-2 mM. A structurally rearranged form of peptide 195-206 containing an aspartimide residue inhibited these reactions half-maximally at concentrations of 250-300 microM. In contrast, neither peptide 263-274 nor peptide 314-326, at 2 mM concentration, significantly affected either factor VIIa interaction with tissue factor or factor VIIa-mediated activation of factor X. Our data provide presumptive evidence that residues 195-206 of human factor VII are involved in the interaction of human factor VII with the extracellular domain of human tissue factor apoprotein.

Synthesis, purification, and characterization of an Arg152----Glu site-directed mutant of recombinant human blood clotting factor VII.

Coagulation factor VII circulates in blood as a single-chain zymogen of a serine protease and is converted to its activated two-chain form, factor VIIa, by cleavage of an internal peptide bond located at Arg152-Ile153. Previous studies using serine protease active-site inhibitors suggest that zymogen factor VII may possess sufficient proteolytic activity to initiate the extrinsic pathway of blood coagulation. In order to assess the putative intrinsic proteolytic activity of single-chain factor VII, we have constructed a site-specific mutant of recombinant human factor VII in which arginine-152 has been replaced with a glutamic acid residue. Mutant factor VII was purified in a single step from culture supernatants of baby hamster kidney cells transfected with a plasmid containing the sequence for Arg152----Glu factor VII using a calcium-dependent, murine anti-factor VII monoclonal antibody column. Purified mutant factor VII was indistinguishable from plasma-derived or recombinant wild-type factor VII by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and migrated as a single band with an apparent molecular weight of 50,000. The average specific activity of several mutant factor VII preparations was 0.00025 unit/micrograms, or 0.01% of that observed for recombinant wild-type factor VII preparations. The clotting activity of mutant factor VII was, however, completely inhibited following incubation with dansyl-Glu-Gly-Arg chloromethyl ketone, suggesting that the apparent clotting activity of mutant factor VII was due to a contaminating serine protease.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of human factor VII by factors IXa and Xa on human bladder carcinoma cells.

Single chain factor VII is converted by limited proteolysis to its activated form, factor VIIa, by a number of blood coagulation proteases including factor IXa and factor Xa. We have determined the relative rate of human factor VII activation by human factors IXa and Xa in two different systems: one containing Ca++ and human bladder carcinoma (J82) cells, and the other containing Ca++ and mixed brain phospholipids. The rate of factor VII activation was determined by a one stage coagulation assay, and proteolytic cleavage of factor VII was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. On a molar basis, factor Xa was sixfold more efficient than factor IXa beta in activating factor VII when the activation reaction occurs on J82 cell surfaces. In contrast, when incubation takes place in a suspension of mixed phospholipids, factor Xa was 18-fold more efficient in activating factor VII than factor IXa beta. In addition, factor IXa alpha activated factor VII at a rate approximately one-half that observed using factor IXa beta. In the absence of cells or phospholipids, no activation of factor VII by either factors IXa or Xa was observed. The addition of stoichiometric amounts of either recombinant human factor VIII (des B-domain) or plasma-derived factor VIIIa failed to augment the rate of factor VII activation by either factors IXa alpha or IXa beta. Likewise, purified human factor Va failed to influence the rate of factor VII activation by factor Xa in either system. Collectively, our studies reveal that J82 cells possess procoagulant phospholipid capable of readily supporting the activation of factor VII by either factors IXa beta or Xa. Our data also demonstrate that the relative ability of factor IXa beta and Xa to activate factor VII is significantly different when these reactions occur on tumor cell surfaces as compared with suspensions of mixed phospholipids.

Inhibition of prothrombin activation by factor X and factor IX Gla-peptides.

In the present study, human factor X and factor IX were each digested with chymotrypsin, and the Gla-peptide from each protein was purified by QAE-Sephadex chromatography. The effect of each Gla-peptide on the activation of human prothrombin by a complex of factor Xa, phospholipid, and calcium was studied using an amidolytic assay for generated thrombin. Prothrombin activation was half-maximally inhibited by factor X Gla-peptide at a concentration of 0.7 microM. Factor IX Gla-peptide was markedly less inhibitory and inhibited this reaction half-maximally at a concentration of 3.7 microM. Kinetic analyses revealed that the factor X Gla-peptide inhibited this reaction in an apparent competitive manner, whereas the factor IX Gla-peptide yielded an exponential Dixon plot. Heat decarboxylation experiments revealed that 3-4 gamma-carboxyglutamic acid residues are critical for the expression of inhibitory activity in each peptide. These studies indicate that, in spite of their structural homology, the ability of each of these Gla-peptides to act as a prothrombinase inhibitor is markedly different.