D-dimer concentration increases with age reducing the clinical value of the D-dimer assay in the elderly.

BACKGROUND: The D-dimer assay is used as an exclusion test in the assessment of suspected venous thromboembolic disease; patients with a negative result have a low probability of thrombosis. We reviewed the D-dimer results from a hospital and community laboratory using the vidas D-dimer test to assess the influence of age on the D-dimer assay. METHODS: D-dimer results from 6631 unselected patients aged more than 16 years were analysed in four age groups and it was shown that the median D-dimer concentration increased with age (16-40 years, 294 ng/mL; 40-60 years, 387 ng/mL; 60-80 years; 854 ng/mL; >80 years, 1397 ng/mL). To test the effect of age on the assay specificity, a cohort of 1897 patients with suspected venous thromboembolic disease was analysed separately. Patients with a negative D-dimer were discharged without further investigation. Patients with a positive result and a clinical suspicion of thrombosis underwent further investigation. One hundred and sixty-five deep vein thrombosis or pulmonary embolus cases were identified. RESULTS: The assay specificity decreased with age from 70% in patients less than 40 years to below 5% in patients more than 80 years. Receiver operator curves were prepared for each age group and the effect of altering the threshold value was analysed. In patients 60-80 years old a threshold value of 1000 ng/mL increased assay specificity to 55% without loss of assay sensitivity. CONCLUSION: The vidas D-dimer assay with a threshold value of 500 ng/mL has little clinical value as an exclusion test in patients more than 80 years old. The assay specificity is poor (26%) in patients aged 60-80 years but could be improved by increasing the threshold value to 1000 ng/mL. We believe that this should be tested in a prospective trial.

Recombinant antitrypsin Pittsburgh undergoes proteolytic cleavage during E. coli sepsis and fails to prevent the associated coagulopathy in a primate model.

During severe sepsis there is dramatic activation of both contact proteases and the coagulation pathway. These processes contribute to the development of shock and disseminated intravascular coagulation (DIC) respectively. The Pittsburgh mutant of antitrypsin (358Met-Arg) is a novel protease inhibitor with activity against both thrombin and the contact proteases and should therefore prove beneficial as a therapeutic agent in the management of septic shock. This hypothesis was supported by an earlier study in a pig model where recombinant antitrypsin Pittsburgh (rAT Pittsburgh) at a concentration of 1 microM alleviated some of the features of shock, but did not improve survival. In order to reduce the lethal effects of E. coli sepsis we postulated that a higher concentration of antitrypsin Pittsburgh would be necessary. To test this hypothesis we used rAT Pittsburgh in a primate model. This was chosen in preference to another species as E. coli sepsis in the primate has been well characterised and closely resembles the changes seen in man. Surprisingly this treatment did not alleviate the features of shock and unexpectedly appeared to exacerbate the associated coagulopathy. We propose two possible mechanisms for this unforeseen outcome. The first results from the broad spectrum of activity of antitrypsin Pittsburgh. As well as inhibiting thrombin and the contact proteases, the Pittsburgh mutant also inhibits activated protein C. Inhibition of the protein C system is known to exacerbate septic shock. Secondly, a significant quantity of inactive antitrypsin Pittsburgh, cleaved at the reactive centre, was detected in the plasma of the treated animals. Proteolytically altered serpins, including antitrypsin. have been shown to enhance the inflammatory process. Therefore the accumulation of cleaved rAT Pittsburgh might be expected to exacerbate septic shock.

Antithrombin cambridge II (Ala384Ser): clinical, functional and haplotype analysis of 18 families.

Thirty-one individuals from 18 unrelated families with antithrombin deficiency have been identified as having a single point mutation within codon 384 (13268 GCA-->TCA) resulting in an alanine to serine substitution. Six families (11 individuals) were identified by the screening of individuals with thromboembolic disease or with a family history of thromboembolic disease, whilst the remaining 12 families (20 individuals) were identified by screening of asymptomatic blood donors. Four individuals had a history of venous thrombotic disease, a further 2 gave a history of superficial thrombophlebitis but the remaining 25 individuals were asymptomatic. Affected individuals demonstrated normal immunological levels of antithrombin but a decrease in anti-IIa activity in the presence of heparin. Haplotype analysis was used to examine the possibility of a founder effect to explain the high frequency of this non-CpG mutation. 29/31 individuals showed a single common "core" haplotype, the only variation existing in the number of copies of an (ATT)n repeat polymorphism--13, 14, 15 or 17. The results suggest that at most there are four independent origins for this mutation.

Commercial antithrombin concentrate contains inactive L-forms of antithrombin.

The preparation of antithrombin concentrate for clinical use requires a viral inactivation step. In most commercial preparations this is achieved by heat pasteurisation. This process would be expected to alter the conformation of antithrombin from the active native species to an inactive latent (L-form) state (1, 2). To determine if this occurs during commercial preparation and to identify the proportion of the product in the inactive state, we examined the various antithrombin conformations within a therapeutic concentrate. The antithrombin concentrate was separated into five fractions by heparin-Sepharose chromatography. The fraction with the highest heparin affinity retained full activity, whereas the four fractions with reduced heparin affinity (approximately 40% of the total antithrombin) had lost their inhibitory function. These inactive antithrombins were intact, monomeric, thermostable and resistant to unfolding in 8 M urea. Moreover, the protein patterns on isoelectric focusing and non-denaturing-PAGE showed that there were at least two different L-forms with isoelectric points separate from the native active species. Our findings demonstrate that approximately 40% of the antithrombin preparation examined exists as inactive L-forms. The clinical significance of administering this altered material is uncertain.

Type I antithrombin deficiency: five novel mutations associated with thrombosis.

The genetic basis of Type I antithrombin deficiency has been investigated in six unrelated kindred with positive histories of thrombosis using a PCR amplification/direct sequencing approach. Four frameshift mutations, all introducing premature translation termination codons were identified. Thus, deletions, of a C at nucleotide position 2599 or 2600, a G at position 2601-2602 and a CT dinucleotide at position 7428-7429 were detected in three kindred and confirmed by restriction enzyme analysis. The identical insertion, of a T at nucleotide 2770, was observed in two apparently unrelated families. This finding may have been due to a founder effect since antithrombin gene polymorphism analysis showed all affected individuals to share a common haplotype. An in frame deletion of 6 bp at nucleotide position 2690-2696 causing the removal of codons 76 and 77 encoding Ile 76 and Phe 77 was also detected indicating that these amino acids are essential for stability of the mature antithrombin.

The plasma turnover of transfused antithrombin concentrate in patients with acquired antithrombin deficiency.

Antithrombin concentrate, prepared from human plasma, has been used as replacement therapy in 35 patients with acquired antithrombin deficiency. The inhibitory activity of the concentrate, measured by chromogenic assay, correlates well with the manufacturer's quoted activity. The mean in vivo recovery of the product was 0.0124 iu mL-1 per iu of antithrombin (AT) concentrate administered by kilogram body weight. The recovery was similar in all diagnostic groups studied and did not vary during the course of treatment. Consumption of the antithrombin concentrate was monitored by measuring the production of thrombin-antithrombin complexes and the loss of plasma antithrombin activity. The mean concentration of thrombin-antithrombin complexes was elevated (23 ng mL-1) at the time of admission to the intensive care unit and fell progressively over the next 4 days. The mean time for the decay of half the antithrombin activity was 23 h during the first 24 h of therapy and rose to 42.1 h after day 1. The recovery and half-life measurements are necessary to plan an appropriate dosage regimen for the administration of this antithrombin concentrate in acquired deficiency states.

The influence of isotretinoin upon fibrinolysis in patients with acne.

A patient with haemophilia A and acne was recently reported to have experienced increased bleeding during therapy with isotretinoin. The aim of the present study was to investigate the influence of isotretinoin upon fibrinolysis, and more specifically upon tissue plasminogen activator (tPA) and tissue plasminogen activator inhibitor (PAI) levels, in haemostatically normal individuals. Thirteen patients with severe acne received a 4-month course of isotretinoin at a dose of 1 mg/kg per day. In all cases, the acne responded to therapy, and the patients did not show any evidence of unexpected bleeding or bruising throughout the study. Although all investigations remained within the normal range, tPA measurements rose significantly (P < 0.001) after 3 months' therapy compared with pretreatment values. Retinoids have been shown to stimulate tPA production in vitro from human endothelial cells. Our results confirm that this can be demonstrated in vivo. Prior to therapy with isotretinoin, when all patients had inflammatory acne, PAI measurements were elevated in 12 of 13 individuals. The mean measurement of PAI decreased from 98.38 +/- 63.30 ng/ml prior to treatment, to 24.63 +/- 15.2 ng/ml (P < 0.01) in the eight patients who returned for blood tests 6 weeks after completing therapy. The decline in PAI levels appears to reflect the resolving cutaneous inflammation following treatment with isotretinoin, rather than a direct effect of isotretinoin on the synthesis or release of PAI. This study provides further evidence that tPA production is stimulated by isotretinoin, and that this, together with falling PAI levels, may accelerate fibrinolysis. There was no clinical evidence that haemostasis was impaired in these haematologically normal individuals.(ABSTRACT TRUNCATED AT 250 WORDS)

The clinical use of antithrombin concentrate in septicaemia.

To date no single agent has emerged as the treatment of choice for the management of septic shock and it is becoming increasingly clear that in future it will prove necessary to combine treatments to provide the optimum therapy. One group of agents that should theoretically prove beneficial in sepsis are the protease inhibitors, antithrombin being the one most widely investigated to date. This article discusses the evidence supporting its use in the treatment of septicaemia.

Insertions/deletions in the antithrombin gene: 3 mutations associated with non-expression.

We have investigated the molecular basis of antithrombin deficiency in 3 individuals, 2 of whom had a proven family history of thromboembolic disease. An approximate 50% reduction in functional and immunologic levels of antithrombin was detected in plasma from the propositi indicating an allelic deficiency of antithrombin. In each case direct sequencing of amplified DNA revealed a novel mutation involving single bases: two being insertions, of a T in codon 48 and an A in codon 208, and the third being the deletion of an A in codon 370. The three mutations, which were confirmed by cloning and sequencing the normal and variant alleles, all caused frameshifts leading to premature termination of protein translation. In no case could a truncated antithrombin be detected in plasma from the propositus suggesting either that it fails to be secreted, or is rapidly degraded.

Antithrombin Cambridge II, 384 Ala to Ser. Further evidence of the role of the reactive centre loop in the inhibitory function of the serpins.

Four unrelated individuals have been identified with an identical antithrombin variant, associated in one of them with episodes of recurrent venous thromboses. In each case, the plasma antithrombin concentration was normal and the only function abnormality was a minor but consistent decrease in the heparin-induced thrombin inhibition suggesting a mutation at or near the reactive centre of the molecule. Amplification and direct sequencing of exon 6 showed a G----T mutation at nucleotide 1246, which corresponds to a substitution of a serine for an alanine at residue 384. This is one of a series of conserved alanines that form the stalk to the reactive centre loop. The observed changes in this variant are compatible with recent structural studies that infer that mobility of this stalk with partial re-entry into the A-sheet of the molecule is necessary for optimal inhibitory activity.

Screening for heparin binding variants of antithrombin.

A chromogenic assay for use as a screening test for the identification of antithrombin deficiency is described. The heparin concentration and the incubation time in the assay were optimised specifically to permit the detection of heparin binding defects of antithrombin. The sensitivity of antithrombin assays for the detection of this type of variant was significantly impaired when an incubation time of more than 30 seconds was used. Several commercially available assays recommend a longer incubation time than 30 seconds and therefore some patients with heparin binding defects of antithrombin may not be identified. The assay described here allows heparin binding variants of antithrombin to be identified and distinguished from other types of antithrombin deficiency in a simple two stage procedure.

A pilot study of antithrombin replacement in intensive care management: the effects on mortality, coagulation and renal function.

A prospective, randomized, controlled trial to examine the effects of antithrombin supplementation on mortality, coagulation and renal function has been carried out on 132 intensive care patients. Antithrombin activity was measured in all patients on admission to the intensive care unit (ICU). Patients with an antithrombin activity of less than 70% were randomized to either receive antithrombin replacement or to act as controls. Antithrombin activity was maintained above 70% in the treated patients throughout their stay on ICU. Ninety-three patients had an antithrombin activity of less than 70% and 35 received replacement therapy. Patients with antithrombin activity below 70% remained on the ICU significantly longer and had a significantly higher mortality rate than patients with antithrombin activity above 70%. Antithrombin supplementation neither reduced mortality nor shortened the intensive care stay. Fifty patients with reduced antithrombin activity remained on the ICU for at least 4 days, 25 received antithrombin and 25 acted as controls; coagulation parameters and renal function have been monitored in these patients. Fibrinogen concentration and platelet count were unaffected by antithrombin replacement. Antithrombin supplementation did not appear to reduce the incidence of impaired renal function in sepsis, trauma and postoperative patients. The creatinine clearance fell below 20 ml/min in eight patients in the no-treatment arm while by comparison only three patients in the treatment arm developed impaired renal function. Our study does not demonstrate a clear role for the use of antithrombin supplementation in intensive care, however the finding that antithrombin reduced renal impairment is encouraging and a larger study to confirm this finding is at present underway.

Changes in endothelial-related coagulation proteins in response to venous occlusion.

One hundred patients with a history of thrombophilia were divided into two groups based on fibrinolytic response to venous occlusion. Good responders with a euglobulin clot lysis time less than or equal to 105 min showed significant release of tPA, PAI and vWF. Of the poor responders with an ECLT greater than 105 min, 24% showed a subnormal increase in tPA, and a significant proportion of these also showed a reduced or absent rise in vWF. We have shown poor fibrinolytic response was related to either raised levels of PAI, poor release of both tPA and vWF, or poor release of tPA or vWF alone suggesting different mechanisms of fibrinolytic impairment. Protein S levels were not significantly changed in either group following occlusion.

The incidence of dysfunctional antithrombin variants: four cases in 210 patients with thromboembolic disease.

210 patients, with a history of venous thrombosis, have undergone prothrombotic investigations. In nine cases a consistent deficiency of antithrombin was identified. In five there was a reduction in the plasma antigenic concentration of antithrombin and in a further four cases deficiency was due to the presence of a dysfunctional antithrombin variant. The variants have all been characterized by DNA analysis and in three the mutations have been confirmed by peptide sequencing (antithrombin Basel (41 Pro to Leu). Hamilton (382 Ala to Thr). Cambridge I (384 Ala to Pro) and Cambridge II (384 Ala to Ser). The incidence of antithrombin deficiency in patients with a history of venous thrombosis has previously been quoted at between 2% and 3%: there is no published data available on the incidence of antithrombin variants. In our series 5% of patients who presented before the age of 40 years had antithrombin deficiency, and 2% of the total number of patients investigated had a dysfunctional variant. Our figures indicate that a significant number of cases of antithrombin deficiency are due to dysfunctional variants and that the true incidence of antithrombin deficiency in patients with a history of venous thrombosis is in the order of 5%.

Antithrombin Dublin (-3 Val----Glu): an N-terminal variant which has an aberrant signal peptidase cleavage site.

Antithrombin Dublin is an electrophoretically fast variant of antithrombin which has normal heparin affinity. Direct sequencing of amplified exon 2 revealed a Val----Glu substitution at position -3. N-terminal sequencing of antithrombin from two individuals, heterozygous for the Dublin mutation, showed the presence of a truncated antithrombin in which the N-terminal dipeptide is absent. We propose that the prepeptide mutation redirects signal peptidase cleavage to a site two amino acids downstream into the mature protein.

Changes in the natural anticoagulants following bone marrow transplantation.

The natural anticoagulants, protein C, protein S and antithrombin, were measured in 21 patients following bone marrow transplantation (BMT). The patients were divided into two groups, those with normal protein C concentration post-BMT and those with significantly reduced protein C concentrations; 12 cases fell into the second group. In addition there was an associated fall in protein S in this group of patients. In spite of the presumed hypercoagulable state only one patient developed overt veno-occlusive disease (VOD) of the liver and no other thrombotic events occurred. The fall in protein C and protein S was coincident with the peak incidence of VOD and is likely to be a contributing factor to the genesis of this condition. Only protein S measured pre-BMT was of predictive value in identifying patients likely to develop reduced levels of these anticoagulants post-BMT. Age, sex, diagnosis, type of conditioning and the pre-BMT measurement of protein C and antithrombin had no predictive value.

Coagulation changes following hepatic revascularization during liver transplantation.

The coagulation changes during liver transplantation have been studied in 14 selected patients. Blood usage in all cases was limited to 8.5 liters, and the preoperative coagulation results were only minimally deranged. Bleeding during the operative procedure was easily managed in all cases. Nonetheless, even in this selected group of "low risk" patients, we have demonstrated that during the anhepatic phase and particularly following hepatic revascularization there is activation of both coagulation and fibrinolysis. These findings imply that if bleeding occurs following revascularization, in addition to the use of replacement blood products, treatment should be directed at reducing the consumptive coagulopathy and inhibiting fibrinolysis. We suggest as a first step antithrombin supplementation to maintain activity above 70%, and an antifibrinolytic agent, such as aprotonin, should be considered as adjuncts to therapy at revascularization.