Antifactor Xa activity measured with amidolytic methods.

Methods for the assay of antifactor Xa activity in the presence and absence of heparin are described. Diluted plasma is incubated with bovine, activated factor X (Xa) in stage I, and remaining Xa is measured with the chromogene substrate Bz-Ile-Glu-Gly-Arg-pNA in stage II. In the presence of heparin, the inactivation is completed in 30 sec, and this method measures total Xa-inactivating capacity in diluted plasma (Method I). In a clincal material, this capacity showed a strong positive correlation (r=0.85) to the thrombin-inactivating capacity of diluted heparinized plasma (heparin cofactor activity) and apparently reflects antithrombin III (At-III) concentration. In the absence of heparin, the inactivation of factor xa occurs slowly. With an incubation of 5 min, about 25% of Xa is inactivated, and this assay reflects initial inactivation of Xa (Method II). With this method, a positive, but less strong correlation to the thrombin-inactivating capacity was found (r=0.58), indicating that inhibitors different from At-III accounts for a minor part of the initial inactivation. Determinations in plasma, in which At-III was removed by immunoadsorption, indicated that At-III accounts for about 80% of the initial inactivation. The results of the assays are not significantly influenced by varying concentrations of fibrinogen, fibrinogen degradation products or heparin in the test plasma.     

Inhibition of Thrombin-Induced Aggregation of Human Platelets by Heparin and Antithrombin III

Heparin aggregated washed platelets in the presence of divalent cations. The aggregation was not significantly influenced by antithrombin III, albumin, or by the presence of serum. The significance of this finding is discussed. Heparin, together with antithrombin III, inhibited the release of platelet adenine nucleotides and also the platelet aggregation induced by thrombin. The inhibition was rapid and progressive. Much higher thrombin concentrations were inhibited than with antithrombin III alone. The inhibition was more pronounced at 37° C than at 19° C, and could not be reversed by addition of polybrene.     

Thrombin-induced Proteolysis of Human Antithrombin III: an Outstanding Contribution of Heparin

S ummary. Products obtained in reaction of excess antithrombin III (AT III) with human α-thrombin and heparin were found to contain markedly less of residual AT III than products of similar reactions without heparin. The increased utilization of AT III was primarily due to limited proteolysis of a portion of unbound inhibitor, associated with the release of a 50 000-dalton protein fragment. Thrombin-induced release of this fragment was promoted by polydispersed heparin preparation and, to a variable degree, by all heparin fractions obtained in gel filtration. The optimum amount of heparin required to facilitate AT III proteolysis was of 5 μg/ml (0.8 u/ml). Excessive reduction of the residual inhibitory activity and changes in two-dimensional immunoelectrophoresis suggested that AT III in plasma is also subjected to nonproductive proteolysis and formation of a modified AT III derivative following addition of thrombin and heparin. These data indicate that the effect of heparin on AT III is more complex than generally recognized. On the one hand, heparin interacting with AT III and thrombin contributes to a rapid binding and neutralization of the enzyme; on the other, heparin facilitates proteolytic degradation of unbound inhibitor even in the presence of small quantities of thrombin accounting for excessive reduction of the overall inhibitory potential of AT III.     

Antifactor Xa Activity in Thrombophilia Studies in a Family with At-III Deficiency

Antithrombin III (At-III) concentration and amidolytic assays reflecting functions of the At-III molecule were studied in members of a family with a strong tendency to thrombosis. Antifactor Xa and heparin cofactor activity closely paralleled the At-III concentration. In all individuals with a history of thrombosis At-III concentration was about 50% of normal. The clinical features and causes of death are briefly reviewed. Mean age in deceased family members with a tendency to thrombosis was 55 years, in members with no history of thrombosis it was 65 years.     

Antithrombin III and liver cirrhosis

Antithrombin III (AT III) levels in 37 healthy people and in 103 patients diagnosed of hepatic cirrhosis (75 due to ethylism, 26 cryptogenic and 7 post-hepatitis) have been studied. Forty seven patients presented a compensation in their cirrhosis and 56 an unbalance. AT III concentration was decreased in cirrhotic patients (14.9 + 1.09 mg/dl), being p less than 0.0005 in relation to healthy patients (24.3 + 0.87 mg/dl). Concentration resulted lesser in patients with unbalance (13.9 + 1.8 mg/dl) than in patients with compensation (18.1 + 1.6 mg/dl). Moreover, statistical study between them showed significant results. AT III, though is a protein whose hepatic synthesis is not clear, decreases in diffuse hepatic disease and so much as more severe is the hepatic damage. Cirrhotic patients did not present thromboembolic phenomena, perhaps because of depression of coagulation factors.     

Heparin Induced Thrombocytopenia: Diagnosis and Contemporary Antithrombin Management

Heparin-induced thrombocytopenia (HIT) may be complicated by severe thrombotic complications and death. Currently no specific laboratory test is available to make the diagnosis. When HIT is clinically suspected, heparin should be discontinued immediately. While no specific therapy for HIT exists, there is increasing evidence that acute antithrombin therapy may significantly reduce morbidity and mortality. Among several agents, the direct antithrombins, such as r-hirudin and argatroban, look the most promising for acute treatment.     

Protein C,Protein S,and Antithrombin III Deficiencies

Journal of Thrombosis and Thrombolysis -     

Antithrombin III and the Nephrotic Syndrome

Plasma and urinary antithrombin III (AT-III) was measured in 15 cases of nephrotic syndrome. Plasma AT-III correlated well with serum albumin, but poorly with pro-teinuria, whereas urinary AT-III correlated well to proteinuria. The plasma AT-III level had a mean similar to 25 healthy controls, but the range was significantly wider. A case with nephrotic syndrome and left renal vein thrombosis is reported. The urinary output of AT-III rose and the plasma level fell with the activity of the disease. Although AT-III and albumin have similar molecule weight, their renal clearance was found to be different. It is suggested that urinary loss of AT-III plays a role in the hypercoagulable state sometimes found in the nephrotic syndrome.     

Factor-Xa Inactivation by Antithrombin III

S ummary . The inactivation of the procoagulant activity of bovine factor Xa by antiproteinase present in antithrombin III preparation follows the pseudo-first-order kinetics with respect to factor Xa. The rate of inactivation is devoid of the dimension of factor Xa concentration when antiproteinase in excess is held constant. In the presence of physiological concentrations of factor V, phospholipids and ionic calcium, the affinity of factor Xa for antithrombin III drastically decreases, and the half-life of the enzyme is very significantly prolonged. This biological stabilization of factor Xa requires the presence of all three substances: factor V, phospholipid and calcium. However, it is quantitatively dictated only by factor V concentrations. Calcium ions and phospholipid required for factor Xa protection from inactivation by antithrombin III have a rather low optimum concentration range, beyond which the original fast rate of inactivation tends to be restored. A direct interaction between antithrombin III and factor V or phospholipid was not found. Thus binding of factor V mediated by phospholipid and calcium ions, which brings about the increase in factor Xa proteolytic activity, also decreases the susceptibility of this enzyme to a biological inactivator in blood.     

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.     

Microheterogeneity of Human Antithrombin III

Antithrombin purified from normal human plasma has been separated into two fractions by isoelectric focusing in a pH 4-6 gradient. These fractions were homogeneous by polyacrylamide gel electrophoresis, had similar amino acid composition and the same specific activity. Both of them cross reacted with antiserum against antithrombin. They were found to contain different amounts of sialic acid and aminosugars. After neuraminidase treatment only a single, homogeneous peak was found by isoelectric focusing--with unchanged antithrombin activity--suggesting that the microheterogeneity is due to a difference in glycosylation.     

Antithrombin III. Physiologic, physiopathologic and laboratory aspects]

Hemostatic control is based in a delicate balance between the activities of activator enzymes and their inhibitors, each one depending on a large number of proteins. Plasma Antithrombin III (ATIII) is one of the most important coagulation inhibitors and the fundamental enzyme for the therapeutical action of heparin. In the last years it was well established that ATIII deficiency accounts for a thrombotic state and inefficiency of heparin therapy. In this work, the authors review the biology of ATIII including its biochemical nature, its physiology, physiopathology and mechanism of action, analysing the implications of its deficiency. The authors draw the attention on clinical and laboratory studies that analyse the prevalence and importance of congenital and acquired deficiency of ATIII, in relation to the prevalence of venous thrombosis. Finally, the laboratory methods applied to the study of ATIII and to the biological control of heparin therapy are described with emphasis on the importance of the ATIII concentrates on this type of treatment. Also the fundamental aspects of heparin resistance are specially mentioned.     

Antithrombin III in systemic lupus erythematosus

Plasma antithrombin III (AT III) was studied in 39 patients with systemic lupus erythematosus (SLE) and in 12 patients with other connective tissue disorders. AT III was measured immunologically by the Mancini method as well as by functional assay using thrombin and the chromogenic substrate, chromozyn TH (Boehringer). Reduced AT III activity was found in 17 patients; 8 had thrombosis. In 6 patients low AT III correlated with disease exacerbations and 2 had systemic vasculitis. No significant correlation could be demonstrated between low AT III levels and thromboembolic disease. A marked variation of functional AT III activity was observed in 30 patients in whom the presence of the lupus anticoagulant was demonstrated. The significance of this association is discussed.     

Immunological Studies on Human Antithrombin III

Immunoelectrophoresis of antithrombin III (At-III) resulted in a single precipitation arc in the α₂-region. At-III was quantitated by electrophoresis or single radial diffusion in agarose gels containing antibodies. Addition of bovine thrombin apparently reduced the concentration of At-III in serum by about 20%. Addition of At-III antibodies to human serum or plasma reduced their progressive antithrombin activity by about 70% and abolished their heparin cofactor activity. The At-III concentration in serum from healthy males decreases moderately with increasing age up to 60 years. In females below 45 years of age the concentration was significantly lower than in males. In females between 45 and 55 years a slight but significant increase was found bringing the concentration above that in males in the higher age groups. Use of oral contraceptives appeared to lower the At-III concentration by about 15%.     

Immunochemical Analysis of Active and Inactive Antithrombin III

Antithrombin III (AT) levels from normal and AT deficiency persons were measured by electroimmunoassay (EIA) and the rewlts compared with a chromogenic assay (S2238). Discrepant results were obtained when plasma and serum were compared using one antiserum, and therefore did not always relate to functional activity. Another antiserum, however, when used was capable of differentiating active AT from inactive AT complexed with its proteases and demonstrated close correlation with all samples tested ( r=0.97 ). The specificity of the antisera and consequent anomalous results were elucidated when purified human thrombin was added to plasma samples and subsequently reanalysed. Quantitative differences observed when serum samples were compared by single radial immuno-diffusion and electroimmunoassay with one antiserum, illustrates the fundamental principle differences between the two methods. These results give some insight as to why previous anomalies with AT immunoassays have occurred. They also indicate that plasma and not serum should be used as clinical test material. AT antisera should be capable of recognizing and distinguishing free AT from AT/protease complexes if the results obtained by electroimmunoassay are to correlate with functional activity.     

New Potential Therapeutic Modalities: Antithrombin III

The role of the disordered coagulation in the pathogenesis of the microcirculatory failure that frequently accompanies sepsis has been clearly established in a multitude of preclinical animal systems. There is consistent and reliable evidence of activation of the coagulation pathways resulting in a net procoagulant state in human septic shock. Despite this experimental and clinical information, there in no compelling evidence that the therapeutic administration of anticoagulants actually benefits patients with severe sepsis or septic shock.Antithrombin III has been used clinically for over twenty years for the prevention and treatment of disseminated intravascular coagulation and multi organ failure in patients with sepsis. A wealth of experimental evidence now supports the potential clinical utility of Antithrombin III in sepsis. This endogenous plasma protein has both anticoagulant and anti-inflammatory properties that may prove to be of therapeutic value. The practical clinical value of Antithrombin III in septic shock is currently being tested in a large phase III multinational study.