Thrombin generation and formation of thrombin-antithrombin III complexes in congenital antithrombin III deficiency

Two families with quantitative congenital AT III deficiency and a high incidence of thromboembolism are reported. In two unrelated patients (one from each family) thrombin generation in whole blood occured more rapidly than in the control, as demonstrated by the kinetics of prothrombin consumption, AT III disappearance and thrombin-antithrombin III complexes formation. Similar results were obtained in plasma and can be experimentally reproduced with a plasma depleted of AT III by immunoadsorption using a rabbit anti-AT III antiserum. The addition of purified AT III in vitro leads to a complete correction of the abnormalities when the level of AT III is greater than 0.8 unit/ml.     

Abolition by dextran sulfate of the heparin-accelerated antithrombin III/thrombin reaction

Dextran sulfate did not inhibit the amidolytic activity of thrombin on Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide, but abolished inhibition of the enzyme with antithrombin III (AT III) in the presence of heparin.

Dextran sulfate did not bind to AT III and had less affinity than immobilized heparin for the protein. Dextran sulfate bound strongly to thrombin and had higher affinity than immobilized heparin for the enzyme.

These findings indicate that binding of dextran sulfate to a site other than the active site of thrombin to prevent the approach of AT III in the presence of heparin.     

Isolation and properties of high molecular weight heparin

A heparin fraction comprising the highest molecular weight components of beef-lung heparin preparations was isolated by chromatography on Biogel P-100. This fraction, all of which is highly active in accelerating the inhibition of thrombin by antithrombin III, includes material with considerably greater activities than previously reported. All of the high-molecular weight fraction binds to antithrombin-Sepharose. The fraction has a molecular weight of 3.5 × 10⁴ and binds to antithrombin III in a 1:1 molar ratio. The dissociation constant of the complex is 1.38 × 10^?8 M ± 0.26 × 10^?8.     

Systemic haemostatic alterations during no-flow ischaemia and reperfusion of the ascending colon in ponies

Naturally occurring and experimentally induced ascending colon volvulus (ACV) result in transmural compression, increased microvascular permeability and thrombosis and necrosis of the intestinal mucosa analysis of haemostatic variables in horses with naturally occurring ACV have identified changes in plasma antithrombin III (AT III) activity as the most reliable for diagnosing hypercoagulation and predicting survival. In an experimental pony model of no-flow ACV, which results in microvascular injury and thrombosis, systemic haemostasis was evaluated during ischaemia and reperfusion to determine whether selected haemostatic assays consistently yield results suggestive of altered haemostasis (hypercoagulation). Ten normal adult ponies were randomly divided into two equal groups (Group 1, control; Group 2, experimental volvulus). All experiments were performed under general anaesthesia and were terminal. Haemostatic variables (prothrombin time, PT; activated partial thromboplastin time, APTT, platelet count, fibrinogen concentration, and plasma AT III activity) were measured prior to general anaesthesia, immediately before colonic volvulus, at 60 and 120 min of ischaemia, and at 60 and 120 min of reperfusion. Mean plasma AT III activity decreased in both the control and experimental groups after induction of general anaesthesia. Mean plasma AT III activity continued to decrease after reperfusion in Group 2 ponies and differed significantly (p<0.05) from control ponies at 120 min of reperfusion. Prothrombin time, APTT, platelet count and fibrinogen concentration values were not significantly different between groups. The decreased plasma AT III activity following the experimental procedures in this model of no-flow colonic ischaemia and reperfusion was suggestive of systemic hypercoagulation.This study was supported by The Graduate School, University of Wisconsin-Madison.     

ANTITHROMBIN IN INFANCY AND CHILDHOOD

ABSTRACT: Teger-Nilsson, A-C. (Department of Clinical Chemistry, Department of Pediatrics, and Department of Blood Coagulation Research, Karolinska sjukhuset, Stockholm). Antithrombin in infancy and childhood. Acta Pediatr Scand, 64:624, 1975.–Anti-thrombin III was measured immunologically, and antithrombin activity was measured with aid of a new synthetic tripeptide substrate for serine proteases in plasma of healthy infants and children, 0–14 years of age. Both methods gave decreased values in the youngest infants as compared with adults. The antithrombin increased with age and reached adult values about 6 months of age. In infants up to one month of age, antithrombin III measured immunologically was significantly lower than antithrombin activity, whereas there was no difference between the antithrombin methods in the higher ages. Antithrombin III concentration and antithrombin activity as measured were poorly correlated.     

Plasma elimination of antithrombin III (heparin cofactor activity) is accelerated in term newborn infants

Antithrombin III (AT III) levels are markedly increased in newborn infants following exchange transfusion with adult blood, and subsequently return to pre-exchange values. This transient rise in AT III (heparin cofactor activity), was used to estimate its plasma elimination half-life. AT III activities were measured serially, before and after double-volume exchange transfusions with heparinised blood in newborn infants requiring therapy for severe hyperbilirubinaemia.The plasma elimination half-life of AT III activity was calculated to be 3.9±1.4 h ( ±SEM). Compared with published data on the kinetics of AT III infusions in adults, the neonate has a considerably accelerated turnover. This finding has important implications for the design of future therapeutic trials of AT III concentrates and provides further evidence that plasma proteins, including components of the coagulation system, appear to have different kinetics in the neonatal period.     

Low antithrombin III: accurate predictor of idiopathic respiratory distress syndrome in premature neonates

A prospective study was performed in premature neonates to determine the predictive values of antithrombin III (AT III) deficiency immediately after birth, for the subsequent development of idiopathic respiratory distress syndrome (IRDS), intraventricular haemorrhage (IVH) and death. Of the 81 premature infants studied, 24 developed IRDS (30%). Of these 24 premature infants, 8 also developed IVH and 9 infants died within the follow-up period of 7 days. The mean plasma AT III level was significantly lower in the infants developing IRDS (0.23 U/ml vs 0.35 U/ml,P<0.0005). Within this study group 33 neonates of less than 30 weeks' gestation showed a prevalence for IRDS of 48%. In this group, AT III activity levels below 0.30 U/ml were 8.5 times as likely to result from infants with IRDS than from infants without IRDS. The diagnostic accuracy indices of criteria for the development of IRDS were: a sensitivity of 100%, a specificity of 88%, a positive predictive value of 89% and a negative predictive value of 100%. The predictive values for the development of IVH and occurrence of death were insignificant. Therefore, in premature neonates the combination of less than 30 weeks' gestation and an AT III below 0.30 U/ml is highly suggestive of IRDS and may facilitate the evaluation of early treatment.Abbreviations AT III antithrombin III - DIC disseminated intravascular coagulation - IRDS idiopathic respiratory distress syndrome - IVH intraventricular haemorrhage - NPV negative predictive value - PPV positive predictive value     

Clinical multicenter evaluation of a new FXa-based Antithrombin assay

Introduction: The determination of functional Antithrombin is a central part of thrombophilia screening. In this multicenter study, a new FXa‐based method (INNOVANCE^® Antithrombin) was evaluated on four different analyzers. Methods: The INNOVANCE Antithrombin method was evaluated by precision and reference interval studies and by comparing the new method with established methods through parallel measurement of samples from 249 patients and 151 apparently healthy individuals. Results: The INNOVANCE Antithrombin assay demonstrated on all analyzers repeatability coefficients of variation (CVs) ≤ 3.2% and within‐device and between‐run CVs ≤ 6.9%. The reference intervals of all analyzers are comparable with 2.5th percentiles between 80% and 85% of normal. The INNOVANCE Antithrombin and the FIIa‐based Berichrom^® AT III (A) methods demonstrated good concordance with correlation coefficients of r = 0.908 or higher. The INNOVANCE Antithrombin method demonstrated furthermore an excellent comparability with the STA^® Antithrombin III assay and an acceptable comparability with the Coamatic^® LR Antithrombin assay. The patients with congenital deficiency (n = 31) were identified with all assays except for the patients carrying the P41L heparin‐binding site mutation, which was only identified with the INNOVANCE Antithrombin and the STA Antithrombin III methods. Conclusion: The INNOVANCE Antithrombin assay has high sensitivity for Antithrombin deficiencies and is reliable, precise and suitable for routine clinical use.     

Thrombophilic screening in young patients (< 40 years) with idiopathic ischemic stroke: a controlled study

Introduction

Despite extensive clinical and laboratory investigations, the etiology of ischemic stroke remains unknown in approximately one third of patients.

Materials and Methods

Thirty-four consecutive patients less than 40 years old (Males 13, Females 21, mean age 26.6 years, range 2-39) with documented ischemic stroke underwent, one year after the acute event, laboratory evaluation of antithrombin, protein C, free and total protein S, activated protein C resistance, fibrinogen, factor VII:C, homocysteine levels and antiphospholipid antibodies (APA). Moreover, prevalence of F5 R506Q, F2 G2021A and homozygosis for thermolabile variant C677T of the methylenetetrahydrofolate reductase (MTHFR) were also evaluated and compared to the results obtained in 120 normal controls.

Results

Antithrombin and protein C levels resulted normal in all cases. One patient (2.9%) showed free protein S deficiency and 3 patients (8.8%) had activated protein C resistance. Homocysteine levels above 15 μmol/L were found in one patient (2.9%). APA were found in 21 patients (61.7%) and in only 2 out of 120 (1.66%) controls (OR = 95.31; 95% C.I.: 18.22-667.81). The multivariate analysis selected that the presence of APA was significantly associated with an increased risk of stroke (OR = 156.60; 95% C.I.: 25.99-943.47) in this cohort of patients. The combination between APA and cardiovascular risk factors determined a risk of 29-fold (OR = 29.31; 95% CI: 3.28-261.69).

Discussion

Our data suggest that the presence of APA is associated with an increased risk of idiopathic ischemic stroke in young patients. Furthermore, also the combination of APA and cardiovascular risk factors is significantly associated with development of idiopathic ischemic stroke.