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H Ireland

Publications and source records attributed to H Ireland.

At least 55 records · Page 3Linked to original sources
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

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 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↗
PubMed1990

Assessment of hypercoagulable states by measurement of activation fragments and peptides.

Broad spectrum assays which measure a range of fibrinogen/fibrin derivatives (FDPs) in serum have become an established means of identifying activation of blood coagulation and/or fibrinolysis, such as occurs in disseminated intravascular coagulation (DIC). There is considerable interest in the application of these assays to the diagnosis of other hypercoagulable states, such as recurrent deep venous thrombosis and myocardial infarction. In recent years, more sensitive and specific FDP assays (e.g. for fragment E, fragment E neoantigen, D-dimer, fragment D neoantigen, fibrinopeptide A and fibrin fragment beta 15-42) have been devised, some of which allow measurement in plasma of FDPs without interference from fibrinogen or certain of its derivatives. It was predicted that these assays would both avoid the possibility of artifacts introduced as a consequence of serum preparation and improve detection of hypercoagulable states. In the light of these expectations we have reviewed data published on the use of assays to detect clinical hypercoagulability, giving prominence to assays of crosslinked fibrin derivatives and nothing particularly certain studies that have compared the performance of different assays on the same samples. The accumulating evidence indicates that all of the assays are adequate for detection of DIC. The same cannot be said for other hypercoagulable states. Here much variation is evident between different studies of similar patients in the ability of a particular marker to discriminate between a normal control group and patients determined to be hypercoagulable by an independent method. This variability would seem to be a function of patient group heterogeneity and selection, as assays that detect different antigenic determinants produce results on the same plasma samples that are well correlated. It appears that the precise antigenic determinant does not critically affect detection of hypercoagulability. Additionally, some studies have indicated that use of serum need not introduce artifacts. Despite there being no other obvious advantage, the convenience of some of the plasma assays may well encourage their widespread use. Assays have also been developed for measuring activation fragments of coagulation proteins (e.g. prothrombin fragment F1 + 2 and protein C activation peptide) and for proteinase inhibitor complexes (e.g. thrombin-antithrombin complex) generated during activation of coagulation. The latter assays have been useful in providing a biochemical definition of a 'prethrombotic state'.

Blood Coagulation Disorders↗
PubMed1990

A low-molecular-weight heparin (Kabi 2165, 'Fragmin') in repeated use for haemodialysis: prevention of clotting and prolongation of the venous compression time in comparison with commercial unfractionated heparin.

We have compared a low-molecular-weight heparin (LMWH), Kabi 2165, with commercial unfractionated heparin (UFH) in 21 patients undergoing haemodialysis (1 month with each heparin). The UFH dose regimen comprised a UFH-saline prime of the extracorporeal circuit, an initial bolus of 5000 international units (IU) and an infusion of 1500 IU/h. The LMWH dose regimen comprised a LMWH-saline prime, a bolus of 3000-4000 anti-factor Xa units (aXa U) and an infusion of 750 aXa U/h. Plasma concentrations of the LMWH were slightly less than those of UFH for the first hour of dialysis (0.87 aXa U/ml vs 0.99 IU/ml) but were very similar by the end of the infusion (0.96 vs 1.00) and slightly greater at the end of dialysis, an hour later (0.85 vs 0.69). All haemodialysis sessions were completed uneventfully, with infrequent wispy clot deposits in the drip chamber. The mean frequency of clot deposition was slightly higher with the LMWH (0.20 vs 0.15). Fibrin generation was almost fully suppressed: plasma concentrations of fibrinopeptide A were slightly greater during dialysis with the LMWH (3.31-3.98 vs 2.29-2.75 pmol/ml) but were almost identical by the end of dialysis (5.62 vs 5.49 pmol/ml). The mean 'venous' compression time at the end of dialysis was significantly shorter with the LMWH than with UFH (8.45 min vs 11.12 min). We conclude that the LMWH is effective and safe in repeated use for haemodialysis. It prevents fibrin generation and clot formation to a similar degree as UFH. The shorter venous compression time of the LMWH may reflect a reduced haemorrhagic risk.

Bleeding Time↗
PubMed1990

A comparative evaluation of assays for markers of activated coagulation and/or fibrinolysis: thrombin-antithrombin complex, D-dimer and fibrinogen/fibrin fragment E antigen.

Measurements were made of levels of D-dimer in plasma and serum, thrombin-antithrombin complex (TAT) in plasma and fibrinogen/fibrin fragment E antigen (FgE) in serum in a normal healthy control group and in patients with a range of disorders associated with hypercoagulability. Levels were determined in 31 normal healthy controls, 30 patients with disseminated intravascular coagulation (DIC), 21 patients with deep venous thrombosis (DVT), 27 patients with myocardial infarction (MI), 26 patients with acute leukaemia and 56 patients with liver disease. Considering all subjects, significant correlations were established between the results of all assays. Notably high correlations (r greater than 0.9) were established between plasma and serum levels of D-dimer, between plasma levels of D-dimer and serum levels of FgE, and between serum levels of D-dimer and FgE. All assays showed very high discrimination (sensitivity) between the normal control group and patients with DIC (97-100%), but there were marked differences between the assays in sensitivity for DVT and MI. In general, the FgE assay was more sensitive than the D-dimer assay, whilst both the FgE and D-dimer assays were more sensitive than the TAT assay. The same trends were apparent in the capability of the assays to discriminate between the normal control group and patients with acute leukaemia and liver disease: disorders with an unknown prevalence of activation of coagulation/fibrinolysis. Our results indicated that measurements of fibrinogen/fibrin degradation products (FDPs) in serum were almost unaffected by artefacts. The data further suggested that the broad-spectrum FgE assay was better than the more specific D-dimer assay in detecting clinical hypercoagulability. Our study showed that, in the clinical conditions examined, FDPs were more effective markers of hypercoagulability than TAT.

Acute Disease↗
PubMed1989

Plasma D dimer: a useful marker of fibrin breakdown in renal failure.

D dimer and other large fragments produced during the breakdown of crosslinked fibrin may be measured by enzyme immunoassay using monoclonal antibodies. In 91 patients with renal disease and varying degrees of renal dysfunction, plasma D dimer showed no correlation with renal function, whereas FgE antigen, a fibrinogen derivative which is known to be cleared in part by the kidney, showed a significant negative correlation with creatinine clearance. Plasma concentrations of D dimer were, however, increased in patients with chronic renal failure (244 +/- 31 ng/ml) (mean +/- SEM) and diabetic nephropathy (308 +/- 74 ng/ml), when compared with healthy controls (96 +/- 13 ng/ml), and grossly elevated in patients with acute renal failure (2,451 +/- 1,007 ng/ml). The results indicate an increase in fibrin formation and lysis, and not simply reduced elimination of D dimer by the kidneys, and are further evidence of activated coagulation in renal disease. D dimer appears to be a useful marker of fibrin breakdown in renal failure.

Biomarkers↗
PubMed1989

A low molecular weight heparin ("fragmin") for routine hemodialysis: a crossover trial comparing three dose regimens with a standard regimen of commercial unfractionated heparin.

In 20 hemodialysis patients using mainly flat plate dialyzers, we have conducted a controlled randomized crossover trial of three dose regimens of a low molecular weight heparin (LMWH), "Fragmin" (Kabi 2165), in comparison with a standard dose regimen of commercial unfractionated heparin (UFH) that had previously been shown to provide effective anticoagulation. The aim of the present study was to find the lowest dosage regimen of the LMWH which would be as effective as the standard UFH regimen. The UFH regimen comprised a prime with heparinized saline, an initial intravenous bolus of 5,000 international units (IU) and an infusion of 1,500 IU/h. The three LMWH regimens comprised a LMWH-saline prime, an infusion of 750 anti-factor Xa (aXa) U/h and three different bolus doses: 1) LMWH-low: 3,000 aXa U. 2) LMWH-medium: 4,000 aXa U. 3) LMWH-high: 5,000 aXa U. With the UFH regimen, plasma heparin levels of around 1.0 IU/ml were maintained during the heparin infusion, declining to 0.71 IU/ml an hour after the infusion was terminated. The LMWH-medium regimen produced very similar plasma aXa levels. The LMWH-high regimen also produced similar plasma aXa levels: therefore, it had no advantage over the LMWH-medium regimen. The LMWH-low regimen produced significantly lower levels than the other regimens during the heparin infusion (0.81-0.85 aXa U/ml, p less than 0.025). Dialysis proceeded uneventfully at all times and plasma levels of fibrinopeptide A (FPA) were suppressed well with all 4 regimens (2.77-5.74 pmol/ml) but tended to rise after the infusion was switched off (5.52-8.45 pmol/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical Protocols↗
PubMed1988

Antithrombin Milano, single amino acid substitution at the reactive site, Arg393 to Cys.

Antithrombin Milano is an unusual antithrombin variant, exhibiting an abnormal, fast moving component on crossed immunoelectrophoresis (in the absence of heparin). Antithrombin isolated from the propositus could be resolved into two peaks on anion-exchange chromatography; antithrombin Milano peak 1 of Mr approximately 60,000 which could inhibit thrombin, and antithrombin Milano peak 2 of Mr approximately 120,000 which was inactive. The latter component also reacted with antisera to both antithrombin and albumin on immunoblotting. Under reducing conditions, the approximately 120,000 Mr component migrated on SDS-PAGE as two distinct bands with Mr approximately 60,000, one of which reacted with antiserum to antithrombin and the other (of slower mobility) of which reacted with antiserum to albumin only. These and other results established the approximately 120,000 Mr component to be an inactive, disulphide-linked variant antithrombin and albumin complex. The variant antithrombin was isolated, following reduction and S-carboxymethylation, by reverse-phase HPLC and then it was fragmented with CNBr. A major CNBr pool containing the sequence Gly339-Met423 was treated with trypsin, followed by V8 protease. The resulting peptides were analysed by fast atom bombardment mass spectrometry (Fab-MS) mapping. A peptide of molecular mass 1086, corresponding to the normal sequence Ala382-Arg393, was almost absent from the mass spectrum, but an additional peptide of mass number 1772 was present. These results are almost identical to those found in another variant antithrombin, Northwick Park (Erdjument et al., J Biol Chem, 262: 13381, 1987; Erdjument et al., J. Biol Chem, 263: 5589-5593, 1988), indicating the same single amino acid substitution of Arg393 to Cys.

Amino Acid Sequence↗
PubMed1988

Low molecular weight heparin in haemodialysis for chronic renal failure: dose finding study of CY222.

A dose finding study of the very low molecular weight heparin CY222 (MW 2500) in patients (n = 8) with chronic renal failure undergoing dialysis has been carried out to (i) establish an effective dose and (ii) determine the relationship between ex vivo anti-factor Xa levels in plasma and the anticoagulant effect (in vivo suppression of FPA levels). Doses of CY222 were compared to a dose (5000 iu bolus + 1500 iu/hr) of unfractionated heparin (UFH) that has been shown to suppress FPA levels during prolonged (greater than 5 hr) dialysis (Ireland et al., J Lab Clin Med 103, 643, 1984). CY222 given iv in increasing doses produced a dose related increase in anti-factor Xa levels (measured as Institute Choay u/ml, with CY222 itself as standard) and suppression of FPA levels. When given in its highest dose, 20,000 Institute Choay u bolus + 1500 Institute Choay u/hr, there was little effect upon KCCT, FPA levels were statistically indistinguishable from those of the UFH regime (indicating comparable anticoagulant effect), but anti-factor Xa levels (expressed in Institute Choay u/ml) were 2-3 times those of UFH (expressed in iu/ml). All samples were also assayed for anti-factor Xa level against the proposed low MW Heparin Standard. Plasma levels of CY222 were then found to be 2.78 times lower, so that the anti-factor Xa levels of CY222 required to produce comparable anticoagulant effect were then indistinguishable from those of UFH.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug Evaluation↗
PubMed1988

Relationship between ex vivo anti-proteinase (factor Xa and thrombin) assays and in vivo anticoagulant effect of very low molecular weight heparin, CY222.

There is uncertainty as to which activities of unfractionated heparin (UFH) and low MW heparin are responsible for their anticoagulant and antithrombotic properties. We have sought to answer this question by examining plasma samples taken during a recently conducted dose-finding study of the low MW heparin, CY222, in haemodialysis for chronic renal failure. In this study, in vivo anticoagulant effect was assessed by measurement of plasma FPA levels. UFH was administered as a dose of 5000 iu bolus + 1,500 iu/h maintenance infusion, while the effects of three doses of CY222 were studied (10,000, 15,000 and 20,000 Institute Choay anti-factor Xa u bolus, all with 1,500 Institute Choay anti-factor Xa u/h maintenance infusion). Anti-factor Xa levels were determined by chromogenic substrate assay. Anti-thrombin levels were determined by chromogenic substrate assay and by quantitation of catalysed thrombin-inhibitor complexes (using autoradiography). Analysis of the results indicate that plasma fibrinopeptide A (FPA) levels correlate with anti-factor Xa (r = -0.45) and anti-thrombin (substrate) (r = -0.63) levels of UFH, but only with the anti-factor Xa levels (r = -0.41) of CY222. These results suggest that the anti-factor Xa assay is currently the most suitable assay for monitoring low MW heparins such as CY222 in humans.

Anticoagulants↗
PubMed1988

Equivalent effective doses of heparin and low molecular weight heparin(oid)s in haemodialysis for chronic renal failure.

In an attempt to provide an objective means of comparing the anticoagulant action of heparin with those of newly developed low MW heparin-like anticoagulants we have utilized the fibrinpeptide A assay as a method of determining fibrin formation during haemodialysis. In vivo suppression of FPA levels by increasing doses of administered anticoagulant enables equivalent effective doses of anticoagulants to be determined without making prior assumptions about which ex vivo activities of these compounds are responsible. In this communication we summarize results of studies with heparin and three low MW heparin(oid)s.

Blood Coagulation↗
PubMed1987

Formation of a covalent disulfide-linked antithrombin-albumin complex by an antithrombin variant, antithrombin "Northwick Park".

Antithrombin is a major proteinase inhibitor of the blood coagulation system. Its inherited deficiency or abnormality is often associated with thromboembolism. Antithrombin "Northwick Park," a functionally inactive variant antithrombin, has recently been shown by us (Lane, D.A., Flynn, A., Ireland, H., Erdjument, H., Samson, D., Howarth, D., and Thompson, E. (1987) Br. J. Haematol. 65,451-456) to be present in plasma, in part, as a high Mr (approximately 120,000) component which has a characteristic electrophoretic mobility in agarose gels in the absence of denaturing agents. In this communication, we present evidence that this Mr approximately 120,000 variant component is comprised of an antithrombin-albumin covalent disulfide-linked complex. This proposal is supported by results of: (a) fast atom bombardment mass spectrometry of the isolated reduced, S-carboxymethylated, trypsin-digested Mr approximately 120,000 complex; (b) sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this complex and its reduced and S-carboxymethylated constituents; (c) immunoblotting of these polyacrylamide gels with antisera specific for antithrombin and albumin; (d) NH2-terminal sequence analysis of one of the isolated, S-carboxymethylated proteins that comprise the Mr approximately 120,000 complex; and (e) fast atom bombardment mass spectrometry of its tryptic peptides.

Antithrombin III↗
PubMed1987

Antithrombin III Glasgow: a variant with increased heparin affinity and reduced ability to inactivate thrombin, associated with familial thrombosis.

A functional antithrombin III (AT III) deficiency has been identified in two generations of a family with a high incidence of thrombosis. The deficiency presented as approximately 50% reduction in heparin cofactor activity compared to its antigen concentration. No abnormality was detected by crossed immunoelectrophoresis in the presence or absence of heparin. Plasma from the propositus was precipitated with dextran sulphate, applied to heparin-Sepharose and the AT III stepwise eluted with NaCl. The AT III had a reduced ability to inactivate thrombin, when this was monitored by substrate hydrolysis or by SDS polyacrylamide gel electrophoresis. Its mobility was normal by the latter technique using 10-20% gradient gels under reducing and non-reducing conditions. AT III from the patient was reapplied to heparin-Sepharose and eluted with a NaCl gradient. An active pool eluted in the same NaCl concentration range used to purify normal AT III, while predominantly inactive AT III eluted at higher NaCl concentrations. It is concluded that this variant, designated AT III Glasgow, has increased affinity for heparin but reduced ability to inactivate thrombin.

Antithrombin III↗
PubMed1987

Antithrombin III Northwick Park: demonstration of an inactive high MW complex with increased affinity for heparin.

It has been shown previously that antithrombin III Northwick Park has reduced ability to inactivate thrombin and is characterized by an additional anodal component on crossed immunoelectrophoresis (Howarth et al, 1985). We have applied plasma from an affected family member to heparin-Sepharose and eluted the antithrombin III with a salt gradient. Evidence is presented that a variant component has slightly higher affinity for heparin than normal antithrombin III. Furthermore, this variant component is present in plasma as an approximately 120,000 MW inactive antithrombin III complex that can be reduced with dithiothreitol to MW approximately 60,000, indicating disulphide bridging. Using ion-exchange chromatography, the inactive complex has been isolated and shown to migrate in the same position as the anodal peak on crossed immunoelectrophoresis.

Antithrombin III↗
PubMed1986

The anticoagulant effect of heparinoid Org 10172 during haemodialysis: an objective assessment.

The heparinoid of natural origin Org 10172 has anti-factor Xa activity but minimal anti-thrombin activity, and little effect upon broad spectrum assays such as the KCCT in vitro. Its anticoagulant effects have been compared to those of commercial heparin in 7 patients undergoing haemodialysis for chronic renal failure. Commercial heparin was administered in a dose (5,000 iu bolus + 1,500 iu/hour continuous iv infusion) previously shown to inhibit fibrin formation during haemodialysis. This produced mean anti-factor Xa levels in plasma between 0.7-1.0 iu/ml and largely suppressed fibrin formation for 5 h dialysis measured as mean FPA levels in plasma. Administration of Org 10172 as a bolus of 1,350 anti-factor Xa u or 2,000-2,400 anti-factor Xa u produced plasma anti-factor Xa levels of less than 0.5 u/ml and allowed fibrin clot and FPA generation during dialysis. Org 10172 administered as a bolus dose of 4,000-4,800 anti-factor Xa u produced mean anti-factor Xa levels of greater than 0.5 u/ml, allowed dialysis of 6 patients for 5 h and appreciably suppressed FPA generation during dialysis, with little effect on the KCCT. It is concluded that the anti-factor Xa activity of Org 10172 may reflect its ability to inhibit fibrin during dialysis and that single bolus injection of Org 10172 may be a useful alternative method of achieving anticoagulation.

Anticoagulants↗
PubMed1986

On the evaluation of heparin and low molecular weight heparin in haemodialysis for chronic renal failure.

Anticoagulation during haemodialysis for chronic renal failure can be assessed by measurement of plasma fibrinopeptide A (FPA) levels as an objective method of monitoring the initial step in fibrin formation, in conjunction with visual inspection of the dialyser circuit for fibrin clot deposition. Employing this approach, unfractionated commercial heparin administered as an intravenous bolus followed by an intravenous maintenance dose (5,000 IU + 1,500 IU/h) was found to suppress almost completely fibrin formation and deposition during prolonged dialysis. Comparison of a low molecular weight heparin, Kabi 2165, revealed that it can inhibit fibrin formation in the extracorporeal circulation, that this property is largely reflected in its anti-factor Xa activity in plasma, and that a useful and effective dose of Kabi 2165 for haemodialysis may be 4,000 anti-factor Xa U intravenous bolus + 750 anti-factor Xa U/h intravenous maintenance infusion. This dose only minimally alters the KCCT and corresponds to approximately 60% of that of unfractionated heparin, which may be important in the long-term use of heparin in these patients.

Biological Availability↗