Familial Functional Antithrombin III Deficiency

A family with a tendency to thrombosis and decreased antithrombin III (AT III) activity in plasma, but normal immunoreactive AT III is reported. 7 members of the family had the AT III defect, 4 of whom have had thrombotic episodes. The importance of biological determination of AT III when studying patients with recurrent thrombotic episodes is emphasized.     

Antithrombin III kumamoto II; A single mutation at Arg393-his increased the affinity of antithrombin III for heparin

Abnormal antithrombin III (AT III) was found in a 30-year-old woman who suffered from recurrent thrombosis during pregnancy and the postpartum period. Among her family members, only her father had recurrent episodes of deep vein thrombosis of the lower extremities, from his youth. The antithrombin and antifactor Xa heparin cofactor activities of the proposita's plasma were 61% and 42% of normal, respectively. The progressive antithrombin and antifactor Xa activities were also decreased to 55% and 58% of normal, respectively. The immunoreactive level of AT III was within the normal range (23.1 mg/dl). Analysis of the proposita's plasma by crossed immunoelectrophoresis in the presence or absence of heparin and by affinity chromatography on heparin-Sepharose revealed that the proposita's AT III had apparently normal affinity for heparin. Nucleotide sequencing of 7 exons of the proposita's AT III gene amplified by polymerase chain reaction (PCR) disclosed that the second base of codon 393 comprised both G and A, indicating Arg393-His conversion. The base sequences of exons 1,2,3a, 3b, 4, and 5 were normal, excluding any other mutation. These findings indicated that the proposita's AT III was a variant of AT III at the thrombin binding site and that the proposita was a heterozygote for the abnormality. Heparin affinity of purified abnormal AT III from the proposita's plasma was demonstrated to be increased upon affinity chromatography using heparin-Sepharose, suggesting that the mutation (Arg393-His) per se could possibly increase the affinity of antithrombin III for heparin. For this variant AT III (Arg393-His), the name AT III Kumamoto II is proposed. ©1995 Wiley-Liss, Inc.     

Hereditary antithrombin iii deficiency and thromboembolic disease

Two teenage brothers with recurrent thromboembolic disease were found to have antithrombin III deficiency. A family study spanning four generations revealed a total of 10 members with antithrombin III deficiency. Five of the 10 affected family members have had thrombotic problems. Antithrombin III deficiency was documented by coagulation assays measuring heparin cofactor, anti-Factor Xa, and progressive antithrombin activity; the level of antithrombin III antigenic material measured by immunoelectrophoresis was low in subjects with abnormal coagulation assays. The clinical features which may lead one to suspect the hereditary hypercoagulable condition of antithrombin III deficiency are reviewed.     

Choice of anticoagulant in a congenital antithrombin III (AT III)-deficient patient with chronic renal failure undergoing regular haemodialysis

Summary We describe a 43-year-old male patient with congenital antithrombin III deficiency requiring haemodialysis due to extension of venous thrombus from recurrent deep vein thrombosis. During dialysis with adequate heparinization, the patient often revealed clot formation in the extracorporeal circuit resulting in unexpected discontinuation of dialysis. When either a combination of antithrombin III concentrate plus heparin or the newly developed synthetic antithrombin preparation, MD805, was infused during dialysis, he could be uneventfully dialysed with either of the two regimens. The functional antithrombin III activity with MD805 increased to the same level as that obtained with antithrombin III concentrate, and it was possible to achieve an antithrombotic effect, as measured from the APTT and thrombin-antithrombin III complex with MD805 during and after dialysis. We thus found that MD805 could be used as an anticoagulant drug when an AT III-deficient patient required anticoagulation in the extracorporeal circulation.     

Hepatocarcinoma in cirrhosis

It has been reported that hepatoma (HCC) cells produce abnormal proteins such as erytropietin, fibrinogen, prothrombin, and recently, antithrombin III (AT III). In a preliminary report, we reported increased AT III levels in patients bearing HCC independent of their clinical liver status. The present study was performed to assess antithrombin III levels and other serological data present in patients with cirrhosis and in patients with cirrhosis and clinical findings of neoplastic disease. In 70 well-matched patients (47 with cirrhosis and 23 with cirrhosis and proven HCC) serum total cholesterol, albumin, prothrombin, alkaline phosphatase, AFP, aminotransferases, and AT III were determined. Together with AFP and alkaline phosphatase, patients with HCC had higher values of AT III (88±7%) and total cholesterol (184±17 mg/100 ml), as compared with cirrhotic patients (AT III 56±3.6%; total cholesterol 113±5 mg/100 ml) (P<0.001). No difference was observed between these two groups for albumin, prothrombin, and aminotransferases. In HCC patients, AT III levels were related to the total cholesterol level (R ²=0.317), whereas in the cirrhotic patients it correlated with the prothrombin level (R ²=0.274). These data suggest that in HCC patients a greater rate of synthesis of AT III occurs, whereas in cirrhotic patients lower levels of AT III occur due to impaired synthesis or increased catabolism of the protein. The serial determination of AT III in cirrhotic patients as a means of detecting neoplastic transformation is suggested.Presented at the Proceeding of the International Meeting on Normal and Neoplastic Growth in Hepatology, Bari, Italy, June 1989.     

Severe Antithrombin III Deficiency in a Patient with Pre-Eclampsia.

Severe acquired antithrombin III (AT III) deficiency was observed in a patient with severe pre-eclamptic toxaemia. Plasma AT III concentration of 0.25 U/ml was found in both functional and immunological assays. The patient was treated with human AT III concentrate as a result of the development of progressive disseminated intravascular coagulation (DIC), the further deterioration of renal function, the risk for thromboembolic complications and the possible adverse effects of heparin therapy. The selective correction of AT III activity resulted in a rapid disappearance of coagulation abnormalities. The patient underwent uncomplicated caesarian section. This observation indicates that acquired severe AT III deficiency may occur as an early feature of DIC in severe pre-eclamptic toxaemia.     

Simplified Assay for Antithrombin III Activity Using Chromogenic Peptide Substrate

A simplified method for the assay of antithrombin III (AT) with the highly reactive thrombin substrate 2AcOH H-D-CHG-Ala-Arg-pNA (substrate Th-1) is described. The assay may be performed at either 30°C or 37°C, and alternatively with the substrate H-D-Phe-Pip-Arg-pNA (S-2238). The standard curve is linear in the 12.5–150% range. For routine assays, 3 standard dilutions of plasma are sufficient, and these may be stored at -20°C for 3 weeks. As only the test plasma must be diluted prior to the assay procedure, the test is more rapidly performed than previous manual assays. In 80 patients plasma samples, with AT in the 19–108% range, there was a high correlation with the results of immunoquantification (r = 0.96). There was also a high correlation betweeen the results obtained with the manual method and the automated version described using the Cobas-Bio® Centrifugal Analyser and substrate Th-1 (r = 0.96). Low AT levels in hereditary deficiency (particularly during heparin treatment), in liver cirrhosis, in disseminated intravascular coagulation (DIC), and heparin-treated thrombosis were confirmed.     

Incidence and clinical characteristics of hereditary disorders associated with venous thrombosis.

At present, different congenital defects in several proteins--antithrombin III (AT III), protein C (PC), protein S (PS), and plasminogen (PLG)--are known to be causes of hereditary predisposition to thrombosis (thrombophilia). The incidence of these hereditary disorders in our 204 patients (106 males and 98 females) with venous thromboembolism were 4% (three cases deficient in PC, three in PS, two in PLG, and one patient in AT III). Their families were studied. In all cases the disorders were inherited as an autosomal dominant trait. The first thrombotic episodes occurred at a age of below 40 years. There was no relationship between protein levels and the occurrence of thrombosis, although a significant relationship was observed between a positive history of thromboembolic disease and a diagnosis of protein deficiencies. We evaluated the differences between primary thrombosis and secondary thrombosis. The most common thrombotic sites were the deep veins. There were no differences between males and females. Evaluation of PC, PS, AT III, and PLG in patients with thromboembolic disease should be considered.     

Evaluation of a new immunoturbidimetric test (Liatest® antithrombin III) for determination of antithrombin antigen

Antithrombin (AT) is a glycoprotein mainly synthesised in the liver, and is one of the most important inhibitors of coagulation. Although AT activity determination is more important in clinical practice and anti‐IIa and anti‐Xa chromogenic tests are widely available, sometimes AT antigen measurement is preferred. Immunoradiometric and enzyme linked immunoabsorbent assay (ELISA) tests have been the only methods available so far for AT antigen determination, but both of them are relatively complicated, time consuming, and the ELISA test cannot be used for single sample assessment. A new immunoturbidimetric test (Liatest® antithrombin III) is now available. We have adapted it for the Cobas Mira® biomedical analyser. The intra‐ and inter‐assay and total coefficients of variation for normal and low control plasma, as well as for normal pool plasma were low (< 3%). Blood samples from 32 patients with different clinical conditions were assessed both by the standard ELISA method and by the immunoturbidimetric method. The correlation coefficient was 0.95. According to our findings it seems that the immunoturbidimetric test (Liatest® antithrombin III) for determination of AT antigen automated for the Cobas Mira® biochemical analyser is easy to perform, time sparing and precise, with good comparability to other assays. It is suitable for measuring of AT antigen in routine laboratory work.     

Hereditary antithrombin III deficiency: case report and review of recent therapeutic advances

We report on a newly diagnosed family with hereditary antithrombin III deficiency, with thromboembolic complications in the propositus. Both the propositus and his asymptomatic sister had decreased plasma levels of antithrombin III antigen and activity (28-52% of normal with good agreement between functional and immunologic assays). The propositus developed deep venous thrombosis, followed by massive pulmonary emboli despite heparin therapy and was treated with streptokinase and heparin with excellent results. Shortly thereafter, small bowel obstruction required surgical intervention, and antithrombin III concentrate, recently available in the United States as an investigational new drug (I.N.D.), was administered with no postoperative thrombotic complications. He was subsequently asymptomatic while on warfarin prophylaxis but twice developed venous thrombosis when he failed to take warfarin. The addition of danazol therapy led to a sustained rise in the antithrombin III level. Each of these therapeutic approaches is discussed and the literature reviewed with emphasis on the newer agents--streptokinase, antithrombin III concentrate, and danazol.     

Antithrombin III contribution to cholestasis differential diagnosis: its correlation with one stage prothrombin time and factor V

Levels of antithrombin III (AT III) measured by two different methods, Factor V (FV) and One Stage Prothrombin Time, have been studied in two groups of patients: 55 with chronic liver disease and 22 with extra hepatic cholestasis. Results show a statistically significant correlation between the decrease of AT III and FV in the first group, and the normality of these parameters (unrelated to One Stage Prothrombin Time) in the second group. Levels at AT III and FV correlate directly with the functional integrity of hepatic parenchyma, so they may help in the differential diagnosis of different types of cholestasis.     

Antithrombin III progressive function: a biochemical analysis

Antithrombin III (AT III) was isolated by two procedures using polyethylene glycol-400 (PEG) precipitation as the first stage. The PEG supernatant (PEG-sup) was applied to a heparin-affinity chromatographic system and AT III-heparin cofactor (AT III-HCF) was isolated. The PEG precipitate (PEG-ppt) was separated by a Sephacryl S-200 column. Fractions were collected and those demonstrating maximum AT III antigen and progressive thrombin inhibition were pooled and reapplied to the washed Sephacryl S-200 column. Fractions were again collected and assayed via specific antisera for AT III, alpha 1-antitrypsin (alpha 1 AG), alpha 2-macroglobulin (alpha 2 M), and alpha 1-acid glycoprotein (alpha 1 AT). AT III antigen (AT III AGN) and progressive function were confined primarily to one peak containing virtually no alpha 2 M, a low level of alpha 1 AT, and moderate quantities of alpha 1 AG. The PEG-sup, PEG-ppt, AT III-HCF, and the fraction obtained after two passes across Sephacryl S-200 (S#2) were similar in that they showed reactivity with specific AT III antisera and demonstrated heparin cofactor activity. They differed, however, in that the PEG-sup and AT III-HCF demonstrated considerably reduced progressive antithrombin function assessed over 30 min. This function was present in the PEG-ppt and S#2 fractions and this progressive activity was potentiated by EDTA. AT III two-dimensional immunoelectrophoresis (2-DIE) in the presence of heparin of both the Sephacryl and heparin-affinity purified components were very different, with the Sephacryl-purified AT III AGN showing both a fast peak and a very prominent slow-moving hump. The AT III heparin affinity fraction showed primarily a fast component. Dilution of the S#2 and PEG-ppt fractions resulted in considerable loss of the progressive activity and also the slow-moving component on 2-DIE. On the basis of these observations, it is postulated that AT III purified by PEG precipitation is in an aggregated form and that aggregate formation and dissolution is associated with AT III progressive activity.     

The antithrombin III content of cryoprecipitate prepared from blood collected with and without heparin

Antithrombin III (AT III) is a plasma protein which acts as the principal inhibitor of thrombin and is a major modulator of intravascular coagulation. Hereditary deficiency of AT III leads to recurrent episodes of thromboembolism. Acquired deficiency of AT III occurs in persons with a variety of conditions, including severe liver disease and disseminated intravascular coagulation. Replacement of AT III may be important in some deficient persons. To determine if cryoprecipitate is a useful source of AT III, we measured the AT III content of cryoprecipitate prepared from citrate phosphate dextrose blood using coagulation and fluorogenic assays and immunoassays. Using the fluorogenic assay, we also determined the effect of adding heparin to blood on the cryoprecipitation of AT III. Functional and antigenic AT III levels were similar to those of normal plasma in all citrate phosphate dextrose blood units tested, indicating that AT III is not concentrated in cryoprecipitate. Heparin had no effect on the cryoprecipitation of AT III.     

Low‐molecular‐weight heparin in patients with advanced cirrhosis

Background & aims: The use of low‐molecular‐weight heparins (LMWH) in patients with advanced liver diseases is frequently avoided because of the enhanced risk of bleeding complications. However, many patients with impaired liver function are at a high risk of thrombosis or have an indication for therapeutic anticoagulation. Therefore, the aim of this study was to evaluate the pharmacokinetics of LMWH in patients with cirrhosis. Methods: Eighty‐four consecutive patients with cirrhosis and a clinical indication for prophylactic or therapeutic anticoagulation were included. The LMWH doses were chosen according to current guidelines. Antifactor Xa activity (anti‐Xa) was assessed on two consecutive days, 4 h after drug administration. The severity of liver disease was quantified using Child–Turcotte–Pugh score, the MELD score and clinical features and was correlated with the anti‐Xa value and the occurrence of complications. Results: Antifactor Xa activity was negatively correlated with the severity of the liver disease, and a positive correlation was observed between antithrombin‐III (AT) levels and anti‐Xa value. AT itself was negatively correlated with the severity of liver disease. Seven patients had an episode of variceal bleeding. No patient died during the observation interval and no thromboembolic events occurred. Conclusion: Prophylactic use of LMWH in patients with cirrhosis appears to be safe. A decreased anti‐Xa value in cirrhotic patients and a negative correlation with liver function challenge the unconditional use of anti‐Xa assays in LMWH monitoring in cirrhotic patients and reveals a potential limitation of anti‐Xa analysis in these patients. Low levels of AT, because of reduced hepatic synthesis, are the most likely cause of this phenomenon.     

Treatment of venous thrombosis in antithrombin III deficient patients with concentrates of antithrombin III

Summary Two patients with familial antithrombin III deficiency developed deep venous thrombosis of the lower limb. The diagnosis of venous thrombosis was made by the indium labelled platelet technique which also allowed for the daily assessment of thrombus size. Each patient received treatment with Warfarin, subcutaneous heparin, and infusions of antithrombin III concentrates. The authors conclude that infusions of antithrombin III concentrates may be of value in limiting the extent of acute thrombosis in patients with a severe deficiency of this protein and may help prevent pulmonary embolism. The haemorrhagic risk of continuing modest doses of heparin with high dose ATIII therapy appears small. In addition to its value in the diagnosis of venous thrombosis the indium platelet technique may give an early indication of thrombus extension and may thus indicate the effectiveness of treatment.     

Treatment of venous thrombosis in antithrombin III deficient patients with concentrates of antithrombin III

Summary Two patients with familial antithrombin III deficiency developed deep venous thrombosis of the lower limb. The diagnosis of venous thrombosis was made by the indium labelled platelet technique which also allowed for the daily assessment of thrombus size. Each patient received treatment with Warfarin, subcutaneous heparin, and infusions of antithrombin III concentrates. The authors conclude that infusions of antithrombin III concentrates may be of value in limiting the extent of acute thrombosis in patients with a severe deficiency of this protein and may help prevent pulmonary embolism. The haemorrhagic risk of continuing modest doses of heparin with high dose ATIII therapy appears small. In addition to its value in the diagnosis of venous thrombosis the indium platelet technique may give an early indication of thrombus extension and may thus indicate the effectiveness of treatment.     

Laboratory and clinical aspects of inherited thrombotic disorders.

The laboratory evaluation of patients with recurrent thrombosis is frequently frustrating, with a low diagnostic yield obtained despite extensive testing. The likelihood of reaching a diagnosis in these patients can be increased by considering diagnostic possibilities usually overlooked and by using assays optimal for their detection. This review summarizes clinical and laboratory issues important in inherited thrombotic disease and discusses practical aspects and a strategy for laboratory testing. New information is provided on the fibrinolytic disorders that may be a common cause of recurrent thrombosis.     

FAMILIAL DEFICIENCY OF ANTITHROMBIN 111: EVIDENCE FOR HETEROGENEITY EXISTING WITHIN THE FAMILY

An Australian family with familial antithrombin Ill (AT III) deficiency is described. The deficiency inherited in an autosomal co-dominant manner is characterised by proportionate reduction in antigenically and biologically measured AT III. Some members with AT Ill deficiency have had major venous thromboses, and the deficiency has possibly been the cause of death in two individuals in the family. Heterogeneity was observed in laboratory and clinical findings in this family.     

Coagulation abnormalities in idiopathic portal venous thrombosis

BACKGROUND: Portal vein thrombosis, usually idiopathic, is the cause of portal hypertension in 46% of Indian patients, who present with a variceal bleed. The presence of lupus anticoagulant (LA) and antithrombin III deficiency have been reported to be associated with an increased tendency to venous thrombosis. METHODS AND RESULTS: We studied 30 patients with portal venous thrombosis diagnosed by ultrasound. Two patients were positive for a lupus anticoagulant and both had very prolonged partial thromboplastin time with kaolin. None of the patients had antithrombin III deficiency.     

Coronary artery bypass surgery in patients with inherited antithrombin deficiency

Summary. Antithrombin (AT) replacement in coronary artery bypass grafting procedures in three individuals with inherited antithrombin deficiency is described. All three had a significant personal or family history of thrombotic disease. All patients achieved satisfactory AT levels throughout bypass and in the postoperative period. All received heparin prophylaxis in the postoperative period. None suffered thrombotic or bleeding complications.