Antithrombin III in rat hepatocytes

Using specific antibodies and an immunoperoxydase method, the presence of antithrombin III (AT III) in rat hepatocytes was demonstrated. By a specific radioimmunoassay, high levels of immunoreactive AT III were measured in microsomal fractions prepared from rat liver. AT III was extracted from the solubilized microsomes on a heparin affinity column and characterized by crossed immunoelectrophoresis. These data suggest that rat hepatocytes synthesize a protein containing AT III antigenic determinants.     

Antithrombin III in normal pregnancy

Plasma antithrombin III (AT III) was determined in 94 women during and after normal pregnancy employing an automated amidolytic technique. The patients were selected on the following criteria: no toxaemia, spontaneous delivery at term, birth-weight above the 10th percentile and discharged with a healthy baby. AT III levels during pregnancy and early puerperium were not lower than own control values obtained 6–8 weeks after delivery.     

Antithrombin III in mesangial IgA nephritis

Plasma Antithrombin III (A T III) was measured in 97 patients with IgA nephritis, 30 patients with non IgA idiopathic mesangial proliferative glomerulonephritis and 40 healthy subjects. The mean plasma A T III levels in the patients with IgA nephritis (105 +/- 10%) was significantly higher than those of normal controls (96 +/- 5%) (p less than 0.0005). The mean plasma A T III levels in the patients with non IgA nephritis (101 +/- 10%) was not different from those of the normal controls or the patients with IgA nephritis. A T III levels were significantly correlated with proteinuria (p less than 0.0001), segmental sclerosis (p less than 0.001), crescents (p less than 0.01), medial hypertrophy (p less than 0.001) and intensity of IgA staining on IMF (p less than 0.02). Patients with IgA nephritis with raised A T III levels had significantly more proteinuria (p less than 0.003), more segmental sclerosis (p less than 0.007) as well as a greater intensity of IgA staining on IMF (p less than 0.02) when compared to patients with normal A T III levels. The data suggest that raised plasma A T III levels may serve as a prognostic marker in IgA nephritis.     

Antithrombin III antigen in human platelets

Immunoreactive AT III was found in human platelets. AT III antigen was quantified in platelets taken from each of 17 healthy donors by a specific competitive enzyme immunoassay using purified AT III and AT III antibodies. AT III antigen levels in extracts of washed platelets disrupted by freezing and thawing ranged from 32 to 140 ng per 10(9) platelets with a mean value of 70.3 +/- 27.3. When stimulated by arachidonic acid, the platelets released AT III antigen together with immunoreactive fibrinogen. These results show that AT III is present in platelets at a level corresponding to approximately 0.01% of total antithrombin in normal blood, and suggest that platelet AT III, like fibrinogen, is contained in the storage granules.     

Antithrombin III Alger: a new homozygous AT III variant

A qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F IIa or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.     

Antithrombin III in a clinical material

Antithrombin III (AT III) has been determined in 368 patients including 192 with recurrent deep venous thrombosis, 17 women with thrombosis during contraceptives, 28 women on oral contraceptives, 10 patients with myocardial infarction, 49 with renal diseases, 59 with different types of cancer and 13 with liver cirrhosis. In addition 25 patients were examined pre- and post-operatively. Both a biological method (5) and an immunochemical method were used. In addition albumin, orosomucoid, haptoglobin and immunoglobulin were assayed immunochemically.

Normal range (40 healthy persons) was found to be 75–122 % with both methods and no sex differences were seen. All but two of the patients with recurrent deep vein thrombosis or myocardial infarction had normal or elevated values of AT III with both methods. All women with thrombosis during treatment with contraceptives and those examined during such treatment had normal or elevated levels. The same pattern was found among the patients with cancer and renal diseases. All patients with liver cirrhosis had decreased levels of AT III. No significant postoperative decrease of AT III was observed. A positive correlation between AT III and albumin or haptoglobin was found which indicates that the AT III reacts as an acute phase reactant.     

Antithrombin III in renal transplant recipients

Antithrombin III (AT III) is increased in situations where there is increased platelet turnover. Plasma AT III levels measured in 39 renal transplant recipients were significantly higher than in 20 healthy subjects (p less than 0.001) and 20 patient controls (p less than 0.025). AT III levels were significantly correlated with the patients' platelet counts (r = 0.4, p less than 0.02). Transplant patients with less than 1 year follow up had significantly higher AT III levels than patients with more than 1 year follow up (p less than 0.01). All 5 patients with transplant rejection in the study had elevated plasma AT III levels. The data suggest that elevation of plasma AT III may be related to graft rejection.     

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 activity and concentration in diabetes mellitus

Antithrombin III (AT III) levels have been reported to be low, normal, and high in diabetes mellitus. Furthermore, a discrepancy between AT III activity and antigen concentration was reported. We have evaluated the behaviour of AT III activity and antigen level both in type 1 and type 2 diabetes, either in uncontrolled or in well controlled patients. AT III activity and antigen levels showed values similar to normal. No difference was seen between type 1 and type 2 diabetes. Similar results were observed in the group of well controlled diabetic patients. AT III activity and antigen did not correlate with blood glucose and glycosylated haemoglobin (HbA1). No difference was observed between AT III activity and antigen levels in any group. Therefore the hypercoagulable state found in diabetes mellitus does not depend on AT III modifications. A discrepancy between AT III activity and antigen was not confirmed. A dysantithrombinaemia, explained on the basis of an inactivation of protein glycosylation in diabetes mellitus has not been confirmed.     

Antithrombin III synthesis in rat liver parenchymal cells

We have previously demonstrated by immunoperoxydase the presence of immunoreactive antithrombin III (AT III) in rat hepatocytes. We now present direct evidence that rat hepatocytes in culture synthesize AT III like immunoreactive material : ³⁵S-methionine was added to the culture medium and incubated with hepatocytes. After incubation, AT III was immunologically characterized in the medium. We found significant amounts of ³⁵S-AT III among the radioactive proteins synthesized and secreted by the cells.     

Antithrombin III Geneva: a hereditary abnormal AT III with defective heparin cofactor activity

A 43-year-old man presented a pulmonary embolism. The unusual circumstances of apparition, the age and the increased heparin requirements suggested an antithrombin III (AT III) deficiency. AT III activity was low in the propositus and seven other members of his family (mean 55%), but immunologic levels were normal (mean 110%). Crossed immunoelectrophoresis in absence of heparin showed a normal pattern, but in presence of heparin showed an abnormal peak as compared with controls. Kinetics experiments showed a normal inhibition of thrombin and Xa in absence of heparin, but abnormal in presence of heparin. Affinity chromatography on heparin-Sepharose revealed two populations of AT III, one of which was devoid of heparin cofactor activity. The toponym AT III Geneva is proposed for this new familial abnormal AT III with defective heparin cofactor activity.     

Antithrombin III and heparin inactivation in thrombin involving reactions

The inhibitory capacity of antithrombin III (AT III) was measured by a quantitative method independent of the velocity of inhibition. When AT III was in excess of thrombin in plasma or in purified system the capacity of inhibitor decreased quantitatively in proportion to the amount of thrombin neutralized. Heparin present in reaction together with thrombin invariably induced a more extensive utilization of inhibitor than thrombin alone. The extent of this additional loss of inhibitory capacity was to a limited degree related to the concentration of heparin. Heparin itself was neutralized in thrombin-AT III reaction losing its anticoagulant property in proportion to the amount of thrombin bound by inhibitor. This quantitative neutralization of heparin occurred not only when the anticoagulant participated in thrombin-AT III binding but also when heparin was added to a medium containing a preformed thrombin-AT III complex. These results suggest that acceleration of binding and increased utilization of binding capacity are the two regular effects of heparin on thrombin-involving reactions of AT III. Both of these effects may be abolished by quantitative binding of heparin to thrombin-AT III complex.     

Antithrombin III I. Evaluation of an automated antithrombin III method

An automated method for the determination of antithrombin III activity (as heparin cofactor activity) is described using the chromogenic substrate Chromozym® TH. A linear relationship is obtained between 10–160% of the AT-III activity of normal plasma and there are good correlations with both antigen level as well as with progressive antithrombin activity. The effect of anticoagulant has been studied and reveals that K₂EDTA is preferred. It is found that in normals there is no distinct age group with decreased antithrombin III activity. In addition, normal activities were found in a group of pregnant women, but activities were low in newborns and even lower in premature newborns. Antithrombin III activity was found to be normal in liver disease except for liver cirrhosis where activities were decreased. The method was used in the screening of patients in a large hospital over a 14 day period. From 1,037 assays performed on plasmas from 634 patients, 148 patients with decreased antithrombin III levels were found.