Antithrombin Heparin Cofactor Research Articles

Antithrombin-p.Ala416Pro: The Second Reported Case in Japan

A 42-year-old man was referred to our department due to recurrent deep venous thrombosis. He, his father and his aunt had low antithrombin (AT) heparin cofactor activity and progressive AT activity levels with normal AT antigen levels. A single nucleotide substitution of G to C was found at nucleotide position c.1246 in exon 7 of the patient's AT gene, resulting in a p.Ala416Pro mutation of AT. The same mutation was identified in his father and aunt, but not his sister, who had a normal AT level. These results show that the AT-p.Ala416Pro mutation was responsible for type IIa AT deficiency in this family.

In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

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.

Changes in plasma antithrombin (heparin cofactor activity) during intravenous heparin therapy: observations in 198 patients with deep venous thrombosis.

Plasma antithrombin (AT) measured as heparin cofactor activity decreased 0.16 +/- 0.13 U/ml (mean +/- SD) in 198 patients who received heparin infusion during 1 wk for deep venous thrombosis (DVT). The decrease was weakly, but significantly correlated to heparin dose (r = 0.15, p = 0.02) and to heparin plasma concentration (r = 0.12, p = 0.05). In patients with subnormal AT at start of heparin treatment, AT decreased less and tended to normalize at the end of heparin infusion, suggesting increased synthetic rate. The decrease in AT was apparently unrelated to the extension or the fate of the thrombus, and also unrelated to other patient and disease characteristics apart from a significantly higher decrease in diabetics. 5 out of 9 patients with AT values below 0.60 U/ml had serious disease, and 1 died from pulmonary embolism (PE) shortly after cessation of heparin (AT 0.22 U/ml). We conclude that the main decrease in AT occurs during the initial 3 d of heparin treatment. If AT stays above 0.70 U/ml after 3 d of treatment, further AT monitoring is hardly indicated.

Antithrombin III “Northwick Park”: A Variant Antithrombin with Normal Affinity for Heparin but Reduced Heparin Cofactor Activity

Further studies have been carried out in a previously reported family with congenital antithrombin III (AT III) deficiency due to an abnormal variant of AT III (AT III Northwick Park). The variant has been identified in five members of the family, three of whom had a history of venous thrombosis. Inheritance followed an autosomal dominant pattern. The affected family members have reduced levels of antithrombin heparin cofactor (41-67%) and progressive antithrombin activity (44-62%) but normal levels of immunoreactive AT III (91-162%). Two dimensional immunoelectrophoresis (2 DIE) of AT III in the absence of heparin revealed an abnormal fast-moving peak in addition to the normal peak but 2 DIE in the presence of heparin appeared normal. Further studies confirmed that the abnormal AT III binds completely to heparin but has no heparin cofactor or progressive antithrombin activity. These results would be consistent with a mutation affecting the binding site for thrombin.

Heparin cofactor activities in a family with hereditary antithrombin III deficiency: evidence for a second heparin cofactor in human plasma

Plasma levels of antithrombin-heparin cofactor, determined by heparin- dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (+/- 7%) and 49% (+/- 5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 M NaCl and did not cross- react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 M NaCl, was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 x 10(-7) M), while the concentration of antithrombin III in patient plasma (8.0 x 10(-7) M) was only 50% of normal (1.6 x 10(-6) M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin- heparin cofactor concentrating measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin- heparin cofactor.

Heparin Cofactor Activities in a Family With Hereditary Antithrombin III Deficiency: Evidence for a Second Heparin Cofactor in Human Plasma

Plasma levels of antithrombin-heparin cofactor, determined by heparin-dependent antithrombin assay, and antithrombin III antigen were measured in 22 members of a large kindred predisposed to venous thrombosis. While 11 members had reduced plasma levels of both antithrombin-heparin cofactor and antithrombin III antigen, the levels of antithrombin-heparin cofactor were always greater than the levels of antithrombin III antigen: 66% (+/- 7%) and 49% (+/- 5%) of normal plasma, respectively. Pooled normal plasma and plasma from one of the affected family members (60% antithrombin-heparin cofactor and 47% antithrombin III antigen) were fractionated by heparin-agarose affinity chromatography. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 0.4 M NaCl and did not cross-react with antibody specific for antithrombin III and did not inhibit factor Xa at an appreciable rate in the presence of heparin, was designated heparin cofactor A. Antithrombin-heparin cofactor, which eluted from heparin-agarose with buffer containing 2.0 M NaCl, was functionally and antigenically identified as antithrombin III. The concentrations of heparin cofactor A in normal and patient plasma were similar (4.5 x 10(-7) M), while the concentration of antithrombin III in patient plasma (8.0 x 10(-7) M) was only 50% of normal (1.6 x 10(-6) M). The functional properties of both heparin cofactor A and antithrombin III obtained from patient plasma were normal. From the results of the present study it would appear that the antithrombin-heparin cofactor concentrating measured in patient plasma reflects the combined concentrations of heparin cofactor A and antithrombin III. Since heparin cofactor A does not cross-react with antibody to antithrombin III, the concentration of antithrombin III antigen in patient plasma is thus lower than the concentration of antithrombin-heparin cofactor.

Inhibition of activated factor XII by antithrombin-heparin cofactor.

The activation of Factor XII occurs via fragmentation of this zymogen into a diverse spectrum of enzymatically potent molecular species. To study the interaction of antithrombin-heparin cofactor and heparin with activated Factor XII, we have employed two forms of this enzyme with widely differing physical characteristics and biologic potencies. Antithrombin-heparin cofactor was found to be a progressive, time-dependent inhibitor of both forms. The addition of heparin dramatically accelerated the rates of these interactions. Furthermore, sodium dodecyl sulfate gel electrophoresis of reduced proteins has indicated that antithrombin-heparin cofactor functions by forming an undissociable complex with either species of the enzyme. This complex represents a 1:1 stoichiometric combination of activated Factor XII and inhibitor. In the presence of heparin, both species undergo virtually instantaneous complex formation with antithrombin-heparin cofactor without exhibiting alterations in dissociability or stoichiometry.

Antithrombin-heparin cofactor: An inhibitor of plasma kallikrein

Human antithrombin-heparin cofactor is a naturally occurring plasma inhibitor of serine proteases generated during activation of the coagulation and fibrinolytic systems. We have demonstrated that purified preparations of this inhibitor also neutralize the esterolytic activity of human kallikrein as well as the ability of the enzyme to release kinins. When an excess of inhibitor is present, the inactivation process follows pseudo-first-order kinetics. Furthermore, the addition of heparin to mixtures of kallikrein and antithrombin-heparin cofactor results in a doubling of the rate and extent of enzyme neutralization. Disc gel analysis of incubation mixtures of kallikrein and excess antithrombin-heparin cofactor, with and without heparin, revealed that the enzyme band had vanished in conjunction with the emergence of two new electrophoretic species. These two new components probably represent stoichiometric complexes of kallikrein and antithrombin-heparin cofactor since a twofold increase in the concentration of enzyme doubled the concentration of these new molecular species. In plasmas which contain adequate levels of other protease inhibitors, antithrombin-heparin cofactor does not appear to be a quantitatively important inactivator of kallikrein. This is suggested by our finding that the addition of heparin at concentrations as high as 50 units/ml did not increase the inhibitory capacity of normal plasma directed against this enzyme. However, plasma from patients with hereditary angioedema had little neutralizing activity directed against kallikrein and revealed a marked increase in this inhibitory capacity when therapeutic concentrations of heparin were added. Our observation suggests that this acidic mucopolysaccharide may prove useful in controlling acute attacks of this disorder.

The separation of active and inactive forms of heparin

Heparin has been fractionated into two distinct forms. The isolation of these species was accomplished by sucrose density gradient centrifugation of heparin mixed with antithrombin-heparin cofactor. Approximately 1 3 of this mucopolysaccharide was bound to antithrombin-heparin cofactor and had potent anticoagulant activity. This component was clearly separated from the remaining 2 3 of the heparin which could not form a stable complex with antithrombin-heparin cofactor and had minimal anticoagulant activity.

Synthesis of heparin-sepharoses and their binding with thrombin and antithrombin-heparin cofactor

Heparin was linked covalently to Sepharose by a reaction between a uronic acid-carboxyl group of heparin and aminohexyl-Sepharose. The characteristics of this material in binding thrombin and antithrombin-heparin cofactor, was compared with those of heparin-aminoethylbromide method (heparin-Sepharose I). Although all the preparations bind thrombin and antithrombin-heparin cofactor they differ in their binding properties and purification efficiency. Highly purified antithrombin heparin cofactor was isolated by adsorption on heparinaminohexylsepharose and desorption with 1.0 M NaCl. The greatest purification of thrombin is obtained by absorption on heparin-Sepharose I and successive elution with 0.3 M and 0.4 M NaCl. Heparin-Sepharose prepared from partially degraded heparin has a relatively low affinity for thrombin and antithrombin-heparin cofactor.

53] Antithrombin-heparin cofactor

Publisher Summary At the beginning of this century, Contejean, Morawitz, and others recognized that thrombin gradually lost activity when added to defibrinated plasma or serum. On the basis of these data, they surmised that a specific inactivator of the proteolytic enzyme antithrombin must be present under physiological conditions. In 1916, McLean isolated heparin from the liver, as well as the heart, and demonstrated its potent anticoagulant properties. Confusion about its inhibitory effect on purified proeoagulants was resolved in 1939 by Brinkhous et al., who showed that heparin was effective as an anticoagulant only in the presence of a plasma component, which they termed heparin cofactor. In the 1950s, the kinetic studies of Waugh and Fitzgerald and Monkhouse et al indicated that plasma antithrombin activity and plasma heparin cofactor activity are intimately related. These investigators suggested that heparin acts to accelerate the rate at which antithrombin neutralizes thrombin. This chapter surveys current methods of assaying for antithrombin–heparin cofactor and provides a variety of techniques for isolating this inhibitor from human, rabbit, and canine plasma. In addition, this communication describes, in some detail, the specificities and properties of this plasma protease inhibitor. It elaborates on several assay methods, as well as the purification procedures.

Inhibition of human factor IXa by human antithrombin

A procedure is presented for the purification of Factor IX from human plasma. The final product is homogeneous as judged by disc gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Furthermore, it is completely free of other coagulation component activities. Factor IX is converted to its enzymatically active form by the addition of small quantities of Factor IXa in the presence of calcium ions. This activated species is added to purified antithrombin-heparin cofactor and the interaction is studied in the presence and absence of heparin. Antithrombin-heparin cofactor is found to be a progressive, time-dependent inhibitor of Factor IXa and neutralizes approximately 57% of this enzyme's proteolytic activity within 30 min. The addition of heparin dramatically accelerates the rate of this interaction with virtually complete inhibition of Factor IXa occurring within 15 s. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin-heparin cofactor functions as a potent inhibitor of Factor IXa by forming an undissociable complex with the enzyme which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.

Enhancement of migration inhibitory factor activity by plasma esterase inhibitors

The plasma esterase inhibitors alpha2-macroglobulin, alpha1-antitrypsin, C1-inhibitor, antithrombin-heparin cofactor, and, as previously described, soybean trypsin inhibitor (Kunitz) and diisopropylphosphorofluoridate (9) enhance the response of guinea pig macrophages to migration inhibitory factor. To obtain this effect, macrophages are incubated with inhibitors prior to assay. The data suggest that (a) the enhancement of migration inhibitory factor response is due to the inhibition of esterases associated with the macrophage through a distinct active site on the inhibitors, and (b) that the active sites of antithrombin-heparin cofactor and soybean trypsin inhibitor, which interact with the macrophage enzymes, are different from the active sites of these inhibitors which interact with thrombin and trypsin respectively. Chemical modification of the active site of antithrombin-heparin cofactor for thrombin and of soybean trypsin inhibitor for trypsin does not affect their capacity to enhance the migration inhibitory factor response. From studies with thrombin, it was known that antithrombin-heparin cofactor has a heparin binding site. Addition of heparin was found to prevent the migration inhibitory factor-enhancing effect of antithrombin-heparin cofactor. The present results suggest that plasma esterase inhibitors may play a regulatory role in the response of macrophages to mediators of cellular immunity.

Effect of sodium and potassium ions on the activity of human antithrombin-heparin cofactor

A method was developed for partial purification of human antithrombin-heparin cofactor and the effect of sodium and potassium ions on its activity was determined. It was found that the thrombin-antithrombin complex formation facilitated with heparin was further accelerated by sodium or potassium chloride. The rate of thrombin inactivation showed a maximum between 0.1 and 0.2 M ion concentration in the presence of 0.012 units heparin per ml.

The Inhibition of Human Plasmin by Human Antithrombin-Heparin Cofactor

Abstract The interaction of purified human antithrombin-heparin cofactor and human plasmin was studied in the presence and absence of heparin. Antithrombin is a progressive, timedependent inhibitor of the proteolytic and esterolytic activities of plasmin. Incubation of plasmin with antithrombin for 15 to 30 min results in 90 to 100% inhibition of both activities of the enzyme. The presence of heparin dramatically accelerates the rate of interaction of antithrombin and plasmin, with nearly complete inhibition within 30 s of incubation. Sodium dodecyl sulfate gel electrophoresis of reduced and nonreduced proteins indicates that antithrombin functions as a potent antiplasmin by forming an undissociable complex which is stable in the presence of denaturing or reducing agents (or both). This complex represents a 1:1 stoichiometric combination of enzyme and inhibitor. Heparin increases the rate of formation of this complex without affecting its dissociability or stoichiometry.

The Purification and Mechanism of Action of Human Antithrombin-Heparin Cofactor

A procedure is presented for purifying antithrombin-heparin cofactor from human plasma. The final product is homogeneous as judged by disc gel electrophoresis, sodium dodecyl sulfate gel electrophoresis, and immunoelectrophoresis. The final yield averages 12%. A specific antibody directed against pure inhibitor preparations precipitates virtually all of both antithrombin and heparin cofactor activity from defibrinated plasma. The purification, the immunoprecipitation, and other data, indicate that both activities are properties of a single molecular species. The inhibitor and thrombin form a 1:1 stoichiometric complex which cannot be dissociated with denaturing and reducing agents. Addition of heparin, a widely used anticoagulant which specifically accelerates the action of our inhibitor, increases the rate of formation of this complex without altering its stoichiometry or its dissociability. Interaction of thrombin with antithrombin-heparin cofactor requires the presence of the active center serine of the enzyme and arginine residue(s) on the inhibitor, since chemical modification of either of these critical residues inhibits complex formation both in the presence and absence of heparin. We suggest that, in analogous fashion to trypsin-trypsin inhibitor systems, a specific interaction occurs between the active center serine of thrombin and a unique arginine-x reactive site on the antithrombin-heparin cofactor. Furthermore, lysyl residues of the inhibitor probably serve as a binding site for heparin, since chemical modification of these residues virtually eliminates heparin cofactor activity with only minimal reduction of antithrombin activity. We postulate that heparin binds to the inhibitor and causes a conformational change which results in a more favorable exposure of the arginine reactive site, allowing a rapid interaction with thrombin.