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Biomedical subjects

D A Lane

Publications and source records attributed to D A Lane.

At least 19 recordsLinked to original sources
PubMed1992

Antithrombotic properties of dermatan sulphate (MF 701) in haemodialysis for chronic renal failure.

The therapeutic potential of the glycosaminoglycan (GAG), dermatan sulphate (DS), as an antithrombotic agent in humans has yet to be established. We have performed dose ranging studies of DS to determine its effectiveness as an antithrombotic agent in patients (n = 6-8) undergoing haemodialysis for chronic renal failure. In an initial study, Study 1, i.v. bolus doses of 2-4 mg/kg and 5-6 mg/kg DS were given to patients dialysing with polyacrylonitrile hollow fibre (PAN HF) membranes. In a second crossover study, Study 2, performed using cuprophane hollow fibre (CHF) membranes, i.v. bolus doses of 3 mg/kg and 6 mg/kg DS were compared to a standard unfractionated heparin (UFH) regime that has been shown previously to inhibit fibrin formation. Further infusion studies, Study 3 and Study 4 evaluated the antithrombotic efficacy of an i.v. DS bolus of 3 mg/kg plus an i.v. infusion of DS 0.6 mg kg-1 h-1 and a DS bolus of 5 mg/kg plus an infusion of 1 mg kg-1 h-1 over 5 h, respectively. These studies were compared to standard UFH regimes in a randomised crossover design. Plasma levels of fibrinopeptide A (FPA) and thrombin-antithrombin (TAT) were used as markers of fibrin formation and thrombin generation during dialysis using both membranes. The changes in DS concentration following administration of the different doses were similar in Studies 1 and 2. However, the effectiveness of DS as an anticoagulant appeared to depend markedly on the different dialyser types used in the two studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1992

Antithrombin Budapest 3. An antithrombin variant with reduced heparin affinity resulting from the substitution L99F.

The molecular basis and functional properties of a variant antithrombin (AT) protein. AT Budapest 3, were studied. A single base substitution was identified in codon 99, CTC----TTC, altering the normal leucine to phenylalanine. The proband presented with a history of venous thrombotic disease and was found to be homozygous for the mutation. The variant protein demonstrated reduced heparin affinity and reduced antiproteinase activity in the presence of either unfractionated heparin or the AT-binding heparin pentasaccharide, when compared to normal AT. A small change in the isoelectric point was also identified. The substituted amino acid residue of AT Budapest 3 is located near to the proposed AT heparin binding site, and it is suggested that reduced heparin affinity of the variant protein may result from substitution-induced distortion of positive charge geometry in the binding site and/or changes in its position relative to the rest of the inhibitor molecule.

Amino Acid Sequence↗
PubMed1992

Antithrombin III Budapest: a single amino acid substitution (429Pro to Leu) in a region highly conserved in the serpin family.

Antithrombin III (AT) is a major plasma serine protease inhibitor and a member of the serpin family of proteins. We have characterized the molecular and genetic basis of AT Budapest, an inherited variant of AT that is associated with thrombotic disease in affected family members. A single amino acid substitution, 429Pro to Leu, was identified, occurring in a region of the molecule that is highly conserved in members of the serpin family. Two forms of variant protein were present in approximately equal amounts in the plasma of the propositus, who is homozygous for the mutation. One form, which had apparently normal Mr, bound heparin strongly and retained some residual thrombin inhibitory activity. The other form had only weak heparin affinity and no antiproteinase activity, and had slightly decreased mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions; this normalized in the presence of a reducing agent, suggesting it was caused by a change in conformation. Additional support for a difference in conformation of the two forms of variant was provided by the finding that the fraction that bound heparin-Sepharose was recognized by a monoclonal antibody raised against normal AT, whereas the weak-affinity fraction was not.

Amino Acid Sequence↗
PubMed1992

Dermatan sulphate in haemodialysis.

Experimental work suggests that dermatan sulphate has potential as an antithrombotic agent: it can inhibit venous thrombi yet has less effect upon bleeding than heparin. While heparin functions as an anticoagulant primarily by its ability to accelerate the action of the plasma protein inhibitor antithrombin III, dermatan sulphate acts selectively through a structurally related inhibitor, heparin co-factor II, to inhibit thrombin. We have done a series of dose-finding studies of the use of dermatan sulphate as an anticoagulant/antithrombotic agent in patients on maintenance haemodialysis. Dermatan sulphate proved to be an effective anticoagulant in this setting.

Dermatan Sulfate↗
PubMed1992

Causal propositions in clinical research and practice.

The concept of causation is central to clinical research and practice. The health science literature on causality, largely contributed by epidemiologists, has examined the population-based question of whether an exposure can cause a given health outcome. Most of this literature has focused on criteria for assessing causality, rather than attempting to define it. Moreover, the population-based approach is rather distant from the individual persons in whom causes must act, which has led to different perspectives on causality among epidemiologists and health policy markers, on the one hand, and clinical practitioners and the lay public, on the other. We attempt to bridge the gap between these perspectives by defining three probabilistic causal propositions based on the locus (individual vs population) and time frame (past vs future outcome) to which they refer, beginning with the individual in whom a health outcome has already occurred ("retrodictive" causal propositions, i.e. It Did) and proceeding to "potential" causal propositions (It Can) for populations and "predictive" causal propositions (It Will) for individuals or populations. We conclude by showing how attention to these distinctions may help avoid common pitfalls that can impair clinical or public health decision-making.

Causality↗
PubMed1992

Antithrombin and its deficiency states.

Antithrombin is the most important physiological proteinase inhibitor of thrombin and other coagulation proteinases. It is a single chain glycoprotein of MW 58,200 which has sequence homology with alpha 1-antitrypsin and other members of the serpin superfamily of inhibitors. Two functional domains of importance have been identified, the reactive centre that interacts with the proteinase and a heparin binding domain. Failure to maintain an adequate level of functional antithrombin in plasma results in an increased risk of thromboembolism: deficiency can be inherited or acquired. There is still uncertainty regarding the prevalence of inherited deficiency and the prevalence of thrombosis in affected individuals. The production of antithrombin is under the control of a single gene which is localized on chromosome 1q 23-25. Characterization of the coding sequence, which is distributed over seven exons, has allowed the analysis of the molecular basis for inherited antithrombin deficiency. To date more than 100 cases have been successfully investigated at the gene and/or protein sequence level and 40 novel mutations have been identified. Mutations causing amino acid substitutions solely affecting the heparin binding site have thus far been located primarily at the N-terminal region of the molecule, residues 7-129; this region has been postulated to align as a positive groove in the molecule that forms the primary contact region for the essential antithrombin binding pentasaccharide of heparin. Not all the residues in which substitutions have been found are basic and some serve to maintain the conformation of nearby basic regions. Examples of this are provided by the Pro-41 to Leu mutation and a recently investigated mutant, Leu-99 to Phe. The reactive site defects are an interesting group, including those that alter P1, P1' and P12-P10 residues. Perhaps more remote mutations can also be included such as Pro-429 to Leu. The P1 and P1' mutations directly block interaction of the proteinase with anti-thrombin, while P12-P10 mutants (which have mutations affecting serpin strand s4A) enable the substrate reaction to proceed to completion, i.e. the antithrombin-thrombin complex is not stabilized and the mutant inhibitor is transformed into a substrate. The effect of the Pro-429 to Leu substitution is impairment of the reactive site and heparin binding, and the finding that this variant is not completely recognized by some MAbs implies a conformational change at the C terminus. Another group (nine cases) of interesting mutations is emerging, that has its primary defect in or near serpin strand 1C, amino acid sequence 402-407.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

A microtitre plate ELISA to measure thrombin-antithrombin complex using pan-specific antibodies.

A sandwich enzyme-linked immunosorbent assay (ELISA) has been developed to measure plasma levels of thrombin-antithrombin complex (TAT). The assay is performed in a microtitre plate using polyclonal antibodies specific for antigenic determinants on prothrombin and antithrombin. Antibody to prothrombin was immobilized on a solid phase, using a titre predetermined to optimize capture of TAT. The performance of the microtitre plate ELISA for TAT has been extensively investigated and compared with the performance characteristics of a tube-based ELISA for TAT which is available commercially (Enzygnost-TAT, from Behringwerke, Marburg, Germany). Studies with plasma containing various levels of prothrombin showed that the zymogen competed with TAT for capture antibody in both assays. Variations in prothrombin levels between plasma samples present a potential source of artifact, but one which does not critically affect the performance of either assay in detecting large elevations in TAT. A high correlation (r = 0.88) was established between the results of plasma samples assayed by both assays, whether citrate or EDTA anticoagulant was used to prepare plasma. High correlations (r > 0.90) were also established for each assay between the results of plasma prepared with EDTA as compared to citrate anticoagulant. Both assays were able to discriminate completely between a group of 16 normal controls and a group of 31 patients with disseminated intravascular coagulation (DIC).

Antibody Specificity↗
PubMed1992

Pleiotropic effects of antithrombin strand 1C substitution mutations.

Six different substitution mutations were identified in four different amino acid residues of antithrombin strand 1C and the polypeptide leading into strand 4B (F402S, F402C, F402L, A404T, N405K, and P407T), and are responsible for functional antithrombin deficiency in seven independently ascertained kindreds (Rosny, Torino, Maisons-Laffitte, Paris 3, La Rochelle, Budapest 5, and Oslo) affected by venous thromboembolic disease. In all seven families, variant antithrombins with heparin-binding abnormalities were detected by crossed immunoelectrophoresis, and in six of the kindreds there was a reduced antigen concentration of plasma antithrombin. Two of the variant antithrombins, Rosny and Torino, were purified by heparin-Sepharose and immunoaffinity chromatography, and shown to have greatly reduced heparin cofactor and progressive inhibitor activities in vitro. The defective interactions of these mutants with thrombin may result from proximity of s1C to the reactive site, while reduced circulating levels may be related to s1C proximity to highly conserved internal beta strands, which contain elements proposed to influence serpin turnover and intracellular degradation. In contrast, s1C is spatially distant to the positively charged surface which forms the heparin binding site of antithrombin; altered heparin binding properties of s1C variants may therefore reflect conformational linkage between the reactive site and heparin binding regions of the molecule. This work demonstrates that point mutations in and immediately adjacent to strand 1C have multiple, or pleiotropic, effects on this serpin, leading ultimately to failure of its regulatory function.

Adolescent↗
PubMed1991

Antithrombin III: a database of mutations.

Elucidation of the molecular defects responsible for antithrombin III deficiency is proceeding rapidly. In order that a record is kept of the new and duplicated mutations that are found, we have compiled a database that we plan to update annually. In this, the first report of the database, we list 6 antithrombin III locus sequence polymorphisms and 94 recorded mutations causing functional deficiency of the protein, 38 of which are novel. As is the case with mutations affecting other protein genes, most mutations of antithrombin III involve a CG to TG or CA change.

Antithrombin III↗
PubMed1991

Dose finding study of a low molecular weight heparin, Innohep, in haemodialysis.

A pilot investigation was performed with Innohep, a low molecular weight (LMWH) preparation (peak maximum molecular mass 3,000-6,000), to determine possible dose regimens for patients undergoing regular maintenance haemodialysis for chronic renal failure. Results from this study suggested that suppression of macroscopic clot formation and fibrinopeptide A (FPA), a marker of fibrin formation, could be achieved following bolus injections rather than bolus injections and an infusion. On the basis of these preliminary findings, a randomised crossover study was performed in eight patients undergoing regular maintenance haemodialysis for 5-7 h to determine the effective antithrombotic dose of this LMWH. Single i.v. bolus doses of 1,250 AFXa u, 2,500 AFXa u and 5,000 AFXa u (n = 7-8) were compared to an UFH regime of 5,000 iu + 1,500 iu/h. Excessive clot formation in the dialyser bubble trap, necessitating additional UFH to enable completion of a prolonged (up to 7 h) dialysis, was observed in all patients on the 1,250 AFXa u dose (mean duration of dialysis prior to UFH, 3 h) but in a single patient only receiving the other LMWH doses. A dose-related response in the AFXa activity, measured by chromogenic substrate (CS) assay was seen in the three LMWH groups, with levels declining significantly (p less than 0.05) from 1-7 h. This contrasted with the constant levels maintained during dialysis with UFH. FPA levels were significantly elevated after 2 h following the 1,250 AFXa u bolus and after 4 h following the 2,500 AFXa u bolus. There was no significant difference in FPA levels between the 5,000 AFXa u bolus and UFH.(ABSTRACT TRUNCATED AT 250 WORDS)

Dose-Response Relationship, Drug↗
PubMed1991

P1 variant antithrombins Glasgow (393 Arg to His) and Pescara (393 Arg to Pro) have increased heparin affinity and are resistant to catalytic cleavage by elastase. Implications for the heparin activation mechanism.

The heparin affinity of normal and two P1 variants of antithrombin-III (AT) was studied by gradient elution with NaCl in Tris buffer on heparin-Sepharose. At pH 7.4 normal AT eluted at [Na+] 0.78 mol/l and the variants both showed increased affinity with AT Pescara eluting at [Na+] 0.86 mol/l and AT Glasgow at [Na+] 0.92 mol/l. We have earlier proposed a model for heparin activation in which the native state of AT maintains a salt bridge involving the P1 Arg-393 residue. Binding of heparin induces a higher heparin affinity conformation in which the salt bridge is disrupted to reveal the reactive centre for inhibition of thrombin. The Glasgow and Pescara variants, lacking a reactive centre P1 basic residue, would be unable to form this salt bridge, and we suggested that the high affinity conformation which they adopt as their native state would resemble the heparin induced conformation. To examine this model, we measured the heparin induced fluorescence of two P1 variants and tested the susceptibility of their reactive loops to catalytic cleavage. Both variants had fluorescence spectra indistinguishable from normal AT. In the absence of heparin, neither variant was more susceptible than normal to catalytic cleavage by human neutrophil elastase. These findings suggest that the conformation of these P1 variants is different to that of fully heparinized normal AT.

Antithrombin III↗
PubMed1991

The pharmacokinetics of dermatan sulphate MF701 in healthy human volunteers.

1. The pharmacokinetics of dermatan sulphate MF701 were studied in 12 healthy males after administration of single intravenous bolus (200 mg), intramuscular (100 and 300 mg) and oral (1 g) doses. The study was conducted according to a within-subject crossover design in two paired blocks. 2. Plasma drug concentrations were measured using a competitive binding assay and a range of biological activity assays, including a sensitive catalysed thrombin inhibition test. 3. Following intravenous administration, plasma concentrations of dermatan sulphate determined by competitive binding assay were described by a two-compartment open model with an initial t1/2, in of 0.6 h and a t1/2,z of 7.5 h. Biological activity assays were insufficiently sensitive to detect the second phase, and therefore yielded apparent monoexponential kinetics. 4. After intramuscular injection the apparent bioavailability of dermatan sulphate was 16-20%. Plasma drug concentrations increased in proportion to dose when measured by competitive binding assay. Low concentrations persisted for more than 24 h at the higher dose, and these may prove therapeutically relevant on chronic administration. 5. We confirm that dermatan sulphate is the only glycosaminoglycan known to generate significant plasma concentrations following oral administration. Oral bioavailability was estimated to be 7%.

Adolescent↗
PubMed1991

Novel point mutations leading to type 1 antithrombin deficiency and thrombosis.

Direct sequencing of antithrombin III (AT) gene fragments specifically amplified by the polymerase chain reaction was utilized to identify the molecular basis of type 1 AT deficiency in two unrelated kindreds, both with thrombotic disease. Two novel point mutations were identified, deletion of a T from the second position of codon 81 in one propositus and insertion of a G in codon 424 in the second kindred. The AT 81(-T) frameshift mutation leads to a premature stop signal in codon 89, while the AT 424(+G) allele has a premature stop only one codon short of the normal gene. The latter mutation changes the eight carboxy terminal residues of AT, including 429Cys, and increases the proportion of polar amino acids in this region. We suggest that altered folding of the mutant protein may explain the AT deficiency.

Adult↗
PubMed1991

Antithrombin Vicenza, Ala 384 to Pro (GCA to CCA) mutation, transforming the inhibitor into a substrate.

Antithrombin (AT) Vicenza has been previously identified as a functionally abnormal antithrombin associated with familial thrombosis (Finazzi et al, 1985). It binds normally to heparin, but loses its affinity following interaction with thrombin: it is a poor inhibitor of thrombin. AT Vicenza was isolated from plasma by heparin-Sepharose and thrombin-Sepharose chromatography, fragmented with cyanogen bromide (CNBr) and its tryptic peptides were analysed by fast atom bombardment mass spectrometry mapping. An abnormal peptide mass 1112 was identified. Edman degradation confirmed a substitution of Ala to Pro in the sequence Ala 383-Arg 393. Polymerase chain reaction amplification of exon 6 of the gene followed by genomic sequencing, localized the mutation to codon 384, GCA to CCA. The same mutation has recently been reported in AT Charleville (Mohlo-Sabatier et al, 1989). Sodium dodecyl-sulphate polyacrylamide gel electrophoresis of AT Vicenza (/Charleville) under non-reducing conditions revealed an apparent increase in mol. wt following interaction with thrombin: under reducing conditions the mol. wt was less than that of normal AT. This indicated cleavage and unfolding of the molecule. The site of cleavage was determined by incubation of AT Vicenza (/Charleville) with thrombin-Sepharose, reduction and S-carboxymethylation and reverse phase FPLC. A peptide was identified with the NH2-terminal sequence beginning Ser-Leu-Asn, demonstrating the cleavage had occurred at the reactive site of the variant. It is concluded that the Ala 384 to Pro substitution transforms AT Vicenza (/Charleville) from an inhibitor into a substrate.

Amino Acid Sequence↗
PubMed1991

Antithrombin Glasgow II: alanine 382 to threonine mutation in the serpin P12 position, resulting in a substrate reaction with thrombin.

A female with recurrent thrombosis was found to have a functional abnormality of antithrombin, with a ratio of functional to immunological activity in plasma of approximately 50%. Crossed immunoelectrophoresis in the presence of heparin was normal, indicating an abnormality of the reactive site, rather than the heparin binding domain. Accordingly, the antithrombin was isolated by heparin-Sepharose chromatography: this produced a mixture of normal and variant antithrombin, as the patient was heterozygous for the abnormality. To remove the normal component, the antithrombin was passed through a column of thrombin-Sepharose. On sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), prior to its application to thrombin-Sepharose, the antithrombin migrated as a single band with identical mobility to that of normal antithrombin. After thrombin-Sepharose, the purified variant component was proteolysed, and migrated as two components, one with a reduced and one with enhanced mobility under non-reducing conditions. This demonstrated that the variant was unable to form stable inhibitor-thrombin complexes and was cleaved in a substrate reaction with thrombin. One site of cleavage was unambiguously ascertained to be the Arg 393-Ser 394 reactive site bond, by NH2 terminal sequencing of the cleaved variant antithrombin: 10 steps beginning at the P1' position, Ser-Leu-Asn-Pro-Asn-Arg,..., were clearly identified. The mutation responsible for this defect was studied by polymerase chain reaction (PCR) amplification of exon 6 of the antithrombin gene and direct sequencing of the amplified product. The presence of both a G and A in the first position of codon 382, identified the mutation GCA to ACA, which results in the substitution of Ala 382 to Thr. This is identical to that reported for antithrombin Hamilton (Devraj-Kizuk et al, 1988), although antithrombin gene polymorphism analysis suggests that the antithrombin Glasgow II mutation has arisen independently. We have recently shown (Caso et al, 1991) that mutation at a nearby position, Ala 384 to Pro, also transforms another variant, antithrombin Vicenza/Charleville, into a substrate for thrombin. The present results with antithrombin Glasgow II suggest that all the alanine residues at the base of the reactive site loop in positions P12-10 may be important for the formation of a stabilized inhibitor-thrombin complex.

Adult↗
PubMed1991

Hemodialysis and heparin. Alternative methods of measuring heparin and of detecting activation of coagulation.

A bolus dose of heparin was administered pre-dialysis to patients (n = 6) undergoing regular maintenance hemodialysis with cuprophane flat plate and hollow fiber membranes. Blood samples were withdrawn at hourly internals for measurement of a) heparin and b) activation markers of coagulation, fibrinolysis and platelets. Two assay methods for heparin were employed; amidolytic assay of anti-factor Xa activity in plasma and a simple whole blood clotting time based upon factor Xa inhibition (Heptest). Results from these heparin assays correlated well with each other (r = 0.89) and both showed similar negative correlations (r = -0.72, amidolytic and r = -0.66, Heptest) with levels of a marker of fibrin clot formation, fibrinopeptide A (FPA). Large differences in levels of FPA were observed during dialysis with the two dialyzer types, when similar levels of heparin were present. Heparin levels declined from 1-5-h dialysis and were associated with rises in plasma levels of FPA, thrombin-antithrombin complex (TAT) and beta thromboglobulin (BTG), but not of D-dimer. Regression analysis revealed the best correlation was between FPA and TAT (r = 0.94), followed by FPA and BTG (r = 0.81). FPA and D-dimer exhibited significant, but lower (r = 0.42), correlation. TAT levels, like FPA levels, showed good correlation with heparin (r greater than 0.65). It is concluded that the Heptest assay may be a useful bedside measurement of heparin levels and the TAT assay may be a simplified means of evaluating coagulation system activation during dialysis.

Antithrombin III↗
PubMed1990

A frameshift mutation leading to type 1 antithrombin deficiency and thrombosis.

Type 1 antithrombin III (ATIII) deficiency, which is the commonest form of inherited ATIII defect, is characterized by a quantitative reduction in both immunologically and functionally detectable protein. This condition is associated with a high incidence of thromboembolic disorder. Previous investigations have shown that the ATIII genes in the majority of cases are grossly intact, but the precise underlying molecular defects remain unknown. We have investigated the molecular basis of a type 1 ATIII deficiency in an Italian kindred by enzymatic amplification of the ATIII gene sequences in affected family members and direct sequencing of the amplified genomic DNA. A novel mutation, the deletion of a single T in the second position of codon 119, was identified in each of the affected individuals. The resulting frameshift leads to a premature termination in codon 126, effectively resulting in a null allele.

Adult↗