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PubMed1997

Identification of the antithrombin III heparin binding site.

The heparin binding site of the anticoagulant protein antithrombin III (ATIII) has been defined at high resolution by alanine scanning mutagenesis of 17 basic residues previously thought to interact with the cofactor based on chemical modification experiments, analysis of naturally occurring dysfunctional antithrombins, and proximity to helix D. The baculovirus expression system employed for this study produces antithrombin which is highly similar to plasma ATIII in its inhibition of thrombin and factor Xa and which resembles the naturally occurring beta-ATIII isoform in its interactions with high affinity heparin and pentasaccharide (Ersdal-Badju, E., Lu, A., Peng, X., Picard, V., Zendehrouh, P., Turk, B., Björk, I., Olson, S. T., and Bock, S. C. (1995) Biochem. J. 310, 323-330). Relative heparin affinities of basic-to-Ala substitution mutants were determined by NaCl gradient elution from heparin columns. The data show that only a subset of the previously implicated basic residues are critical for binding to heparin. The key heparin binding residues, Lys-11, Arg-13, Arg-24, Arg-47, Lys-125, Arg-129, and Arg-145, line a 50-A long channel on the surface of ATIII. Comparisons of binding residue positions in the structure of P14-inserted ATIII and models of native antithrombin, derived from the structures of native ovalbumin and native antichymotrypsin, suggest that heparin may activate antithrombin by breaking salt bridges that stabilize its native conformation. Specifically, heparin release of intramolecular helix D-sheet B salt bridges may facilitate s123AhDEF movement and generation of an activated species that is conformationally primed for reactive loop uptake by central beta-sheet A and for inhibitory complex formation. In addition to providing a structural explanation for the conformational change observed upon heparin binding to antithrombin III, differences in the affinities of native, heparin-bound, complexed, and cleaved ATIII molecules for heparin can be explained based on the identified binding site and suggest why heparin functions catalytically and is released from antithrombin upon inhibitory complex formation.

Anticoagulants↗

PubMed1997

Heparin-dependent modification of the reactive center arginine of antithrombin and consequent increase in heparin binding affinity.

Antithrombin, the principal plasma inhibitor of coagulation proteinases, circulates in a form with low inhibitory activity due to partial insertion of its reactive site loop into the A-beta-sheet of the molecule. Recent crystallographic structures reveal the structural changes that occur when antithrombin is activated by the heparin pentasaccharide, with the exception of the final changes, which take place at the reactive center itself. Here we show that the side chain of the P1 Arg of alpha-antithrombin is only accessible to modification by the enzyme peptidylarginine deiminase on addition of the heparin pentasaccharide, thereby inactivating the inhibitor, whereas the natural P1 His variant, antithrombin Glasgow, is unaffected, indicating that only the P1 Arg becomes accessible. Furthermore, the deimination of P1 Arg converts antithrombin to a form with 4-fold higher affinity for the heparin pentasaccharide, similar to the affinity found for the P1 His variant, due to a lowered dissociation rate constant for the antithrombin-pentasaccharide complex. The results support the proposal that antithrombin circulates in a constrained conformation, which when released, in this study by perturbation of the bonding of P1 Arg to the body of the molecule, allows the reactive site loop to take up the active inhibitory conformation with exposure of the P1 Arg.

Antithrombin III↗

PubMed1999

Topology of the stable serpin-protease complexes revealed by an autoantibody that fails to react with the monomeric conformers of antithrombin.

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P14 to P9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P1 and P'1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of beta-sheet A, which becomes strand 5 of beta-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P14 to P6 of the RCL are inserted into beta-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex.

Amino Acid Sequence↗

PubMed1980

Antithrombin III patterns in disseminated intravascular coagulation.

Antithrombin III-heparin cofactor has now been recognized as a major inhibitor of thrombin and other serine proteases in the blood coagulation system. Since the reaction between antithrombin III and serine proteases is irreversible, one would expect antithrombin III consumption in the face of pathologic intravascular coagulation and attendant generation of thrombin, IXa, Xa, XIa, XIIa, and plasmin. Using a new assay system for antithrombin III that is unaffected by heparin or fibrino (geno) lytic degradation products, antithrombin III was monitored before and during therapy in 38 patients who had acute or chronic disseminated intravascular coagulation. It was found that early and significant decreases in anththrombin III occur in disseminated intravascular coagulation and thus may serve as a useful diagnostic tool. It was further found that monitoring antithrombin III during therapy reflected a cessation of antithrombin III consumption and, thus, served as an indicator of the efficacy of therapy in stopping the clotting process. Since the assay system is unaffected by fibrino(geno)lytic degradation products and heparin, it proved useful in monitoring the efficacy of heparin therapy for disseminated intravascular coagulation. In addition for this group of patients, it appeared that mini-heparin therapy and large doses of heparin were equally efficacious in correcting other laboratory abnormalities of disseminated intravascular coagulation, and in controlling clinical hemorrhage in disseminated intravascular coagulation.

Anticoagulants↗

PubMed2003

Effects of argatroban as an anticoagulant for haemodialysis in patients with antithrombin III deficiency.

BACKGROUND: In congenital or acquired antithrombin III-deficient patients undergoing haemodialysis, coagulation or residual blood in the blood circuit and dialyser is commonly observed under anticoagulation with heparin. Argatroban, a synthetic thrombin antagonist, directly inhibits thrombin activity in a manner that is different from that of heparin, thereby displaying an anticoagulating effect without the activation of antithrombin III. For this reason, the anticoagulating effect of argatroban in haemodialysis patients with antithrombin III deficiency was investigated. METHODS: A retrospective nationwide survey was conducted among patients with congenital or acquired antithrombin III deficiency who had undergone haemodialysis with argatroban as an anticoagulant from April 1996 to April 2000. Inclusion criteria were patients with antithrombin III activity <70% of normal, and patients in whom blood coagulation or residual blood in the extracorporeal circuit could not be prevented by the use of heparin during haemodialysis. RESULTS: Of 80 patients who underwent haemodialysis with argatroban, 59 met the inclusion criteria. Compared with the data before the administration of argatroban, significant improvements of residual blood in the dialyser and arterial and venous drip chambers were observed at the last administration of argatroban. A significant rise in antithrombin III activity was also observed. Among 80 safety analysis cases, no adverse events were reported in 66 patients (82.5%). As severe adverse events, one showed bleeding tendency and one had prolongation of prothrombin time. CONCLUSION: Argatroban was an effective and safe anticoagulant for haemodialysis in patients with congenital or acquired antithrombin III deficiency.

Aged↗

PubMed1984

Anticoagulant properties of heparin preparations from different animal sources with equivalent high affinity for antithrombin III.

Three kinds of heparins from different animal sources--hog-intestinal heparin (167 USP units/mg), bovine-lung heparin (149 USP units/mg) and whale-intestinal heparin (Balaenoptera borealis L., 174 USP units/mg)--were fractionated through an antithrombin III-Sepharose column and high-affinity (HA) subfractions (1.2-1.8 M NaCl eluted fractions) were separated. These HA heparin specimens, which were confirmed to be equivalent in strength of binding to antithrombin III, were compared with respect to the anticoagulant properties by means of [1] whole blood clotting assay and [2] thrombin or [3] factor Xa inhibition assay. The activities of whale HA heparin were [1] 82%, [2] 38%, and [3] 45%, respectively, of those of the hog and bovine HA heparins (which showed approximately identical activities). Since it was confirmed that the affinity for antithrombin III of whale heparin is of the same nature as those of hog and bovine heparins by (a) fluorometric titration and (b) affinity chromatography on an antithrombin III-Sepharose column saturated with hog HA heparin, the above difference in anticoagulant properties between the whale HA heparin and hog and bovine HA heparins may be considered to be due to a difference in undescribed saccharide-sequence structures at sites other than the well-characterized antithrombin III-binding sites, suggesting strongly that some heparin saccharide structures other than antithrombin III-binding oligosaccharide participate in the anticoagulant activity dependent on antithrombin III-heparin binding--at least in the manifestation of anti-thrombin activity and the enhancement of anti-Xa activity.

Animals↗

PubMed2001

Hemostatic effects of antithrombin III supplementation during cardiac surgery: results of a prospective randomized investigation.

Failure to suppress thrombin generation during cardiac surgery promotes fibrin generation, fibrinolysis, and a consumptive coagulopathy. Acquired deficiencies of antithrombin III may play a contributory role. We hypothesized that antithrombin III supplementation to normal physiologic concentrations would decrease thrombin generation and potentially reduce peri-operative bleeding. Twenty patients undergoing coronary artery bypass graft surgery were randomized for this prospective, double-blind, placebo-controlled study. Ten patients received antithrombin III supplementation (50 U/kg) by intravenous infusion prior to incision, and 10 patients received a placebo. Blood samples were obtained pre-operatively, at 1 and 2 h following initiation of cardiopulmonary bypass (CPB), and at 1, 3, and 24 h after completion of CPB. Samples were analyzed for antithrombin III, thrombin-antithrombin III (TAT) complex, and D-dimer concentrations. Cumulative blood loss was recorded at 6 and 12 h after CPB. No statistically significant differences in patient demographics or total heparin dose administered were observed between groups. As expected, plasma antithrombin III concentrations were maintained near pre-operative values in the treatment group, but not in the placebo group. Despite this difference, no statistically significant alterations in generation of TAT complex, D-dimer, or blood loss occurred between groups. Antithrombin III supplementation to maintain normal physiologic concentrations during CPB did not alter significantly thrombin generation, fibrinolytic activity, or blood loss in adults undergoing elective cardiac surgery.

Aged↗

PubMed2001

Antithrombin treatment in patients with traumatic brain injury: a pilot study.

This study will determine if early administration of antithrombin concentrate to patients with traumatic brain injury (TBI) can inhibit or significantly shorten the time of coagulopathy. The progress of brain injury monitored by computed tomographic scan (CT) was also assessed, as was the time needed for intensive care and outcome related to Glasgow outcome scale (GOS). Twenty-eight patients with isolated brain trauma verified with CT were included in either of two parallel groups. The Glasgow coma score (GCS) was mean 7.5, and median 7.0; signifying a moderate to severe traumatic brain injury but with a mortality of only 3.5%. The patients randomized to antithrombin treatment received a total of 100 U/kg BW during 24 hours. To measure hypercoagulability, soluble fibrin (SF), D-dimer (D-d), and thrombin-antithrombin complex (TAT) were assessed together with antithrombin (AT) and routine coagulation tests. Before treatment, SF, D-d, and TAT were markedly increased in both groups. Soluble fibrin and D-dimer (measured after treatment began) appeared to decrease faster in the AT group, and there was a statistically significant difference between the groups at 36 hours for SF and at 36 hours, 48 hours, and at Day 3 for D-d. Thrombin-antithrombin complex levels were very high in both groups but, surprisingly, showed no significant difference between the groups. The authors conclude that antithrombin concentrate administered to patients with severe TBI resulted in a marginal reduction of hypercoagulation. We could not detect any obvious influence by antithrombin on brain injury progress, on CT, or on outcome or time needed for intensive care.

Adolescent↗

PubMed2001

There is no effect on remodeling of vascular wall 4 weeks after local delivery of antithrombin in a porcine model of coronary overstretching.

BACKGROUND: Antithrombin III is an effective endogenous inhibitor of thrombin with antiproliferative and anti-inflammatory capabilities. Systematic administration of direct thrombin inhibitors as well as of antithrombin has proven effective at reducing formation of neointima after injury to vessel wall in various animal models. Local delivery of antithrombin attenuates deposition of platelets after balloon injury to porcine coronary vessels. OBJECTIVE: To test our hypothesis that local delivery of antithrombin affects remodeling of vessel wall after balloon injury to the left anterior descending artery (LAD) in pigs. METHODS: With a balloon:vessel diameter ratio of 1.5 deep vessel-wall injury was performed with conventional balloon angioplasty in the LAD in 16 pigs. After balloon injury the pigs were administered locally two doses of 2.5 ml antithrombin (250 U) or, as a control, two doses of 2.5 ml albumin (10 mg). All pigs were administered 200 U/kg bodyweight heparin before catheterization. The animals were then kept in stalls and fed normal grain. After approximately 4 weeks the animals were killed and lesions were assessed by computer-assisted image analysis. The areas of each vessel-wall layer and the percentage area stenosis were calculated. As a measure of injury, the length of rupture of the lamina elastica interna was measured. RESULTS: The injury was found to be equally profound in pigs of these groups. There was no significant difference between the groups concerning the areas of vessel-wall layers. The percentage area stenosis was similar for pigs in these two groups (36.5 +/- 14.9% for pigs in the antithrombin group versus 35.4 +/- 16.2% for pigs in the control group, NS). CONCLUSIONS: Local delivery of 250 U antithrombin to the LAD in pigs did not affect remodeling of the vessel wall 4 weeks after balloon injury.

Angioplasty, Balloon↗

PubMed2001

Antithrombin reduces mesenteric venular leukocyte interactions and small intestine injury in endotoxemic rats.

We examined the hypothesis that recombinant human antithrombin would reduce mesenteric venule leukocyte adhesion and small intestine injury in endotoxemic rats. Endotoxemic (endotoxin 10 mg/kg, intravenously) rats were treated either with saline or recombinant human antithrombin (250 and 500 U/kg). In some rats, indomethacin (100 mg/kg, intraperitoneally) was injected 60 min prior to endotoxin and recominant human antithrombin (500 U/kg) treatment. Compared to controls, intravital videomicroscopy of the mesentric venule showed an increase of leukocyte rolling (55+/-17 versus 70+/-19 leukocytes/min; P < 0.05) and firm adhesion (1.1+/-0.3 versus 5.8+/-0.8 leukocytes/100 microm; P < 0.05) in endotoxemic rats. Recombinant human antithrombin attenuated endotoxin-induced venular endothelium leukocyte adhesive cascade. The beneficial effects of recombinant human antithrombin on leukocyte adhesion were inhibited by indomethacin (100 mg/kg, intraperitoneally) in endotoxemic rats. Endotoxin treatment increased fluorescein isothiocyanate (FITC)-labeled dextran 4,000 (FD4) gut lumen to plasma ratio and wet weight/dry weight ratio. Recombinant human antithrombin (500 U/kg) attenuated endotoxin-induced gut injury. These observations suggest that recombinant human antithrombin reduces endothelium-leukocyte interactions in endotoxemic rats by interacting with local prostacyclin production.

Animals↗

PubMed2004

Inhibition of coagulation and inflammation by activated protein C or antithrombin reduces intestinal ischemia/reperfusion injury in rats.

OBJECTIVE: To examine whether administration of activated protein C or antithrombin reduces local splanchnic derangement of coagulation and inflammation and attenuates intestinal dysfunction and injury following intestinal ischemia/reperfusion. DESIGN: Randomized prospective animal study. SETTING: University research institute. SUBJECTS: Adult male Wistar rats, weighing 300-325 g (n = 72). INTERVENTIONS: Rats were subjected to superior mesenteric artery occlusion consisting of 20 or 40 mins of ischemia and 3 hrs of reperfusion. A randomized intravenous administration of vehicle (0.9% NaCl), heparin, antithrombin, or activated protein C was performed during ischemia, 15 mins before reperfusion. Coagulation and fibrinolysis variables obtained from portal blood were correlated with mucosal fibrin deposition (determined by anti-rat fibrin antibody staining), intestinal function (glucose/water clearance), and intestinal injury (histologic evaluation by Park/Chiu score). MEASUREMENTS AND MAIN RESULTS: Activated protein C- or antithrombin-treated animals demonstrated less ischemia/reperfusion-induced intestinal dysfunction and histologic changes compared with control animals, whereas intravenous administration of heparin only showed less histologic derangement. Activated protein C- or antithrombin-treated animals showed less thrombin generation, fibrin degradation products, and fibrin deposition compared with control animals, as confirmed by histologic examination, whereas heparin administration showed only a limited reduction of portal fibrin degradation product concentrations. Furthermore, activated protein C or antithrombin administration markedly inhibited the inflammatory response, as reflected by reduced interleukin-6 plasma concentrations to baseline values, whereas heparin had no effect. CONCLUSIONS: Administration of activated protein C or antithrombin inhibited local and systemic derangement of coagulation and inflammation following intestinal ischemia/reperfusion, diminished mucosal fibrin deposition, and attenuated ischemia/reperfusion-induced intestinal injury. These observations suggest that activated protein C or antithrombin reduces ischemia/reperfusion-induced intestinal injury, both through their anticoagulant and anti-inflammatory effects.

Animals↗

PubMed2006

High-dose antithrombin III in the treatment of severe sepsis in patients with a high risk of death: efficacy and safety.

OBJECTIVE: To explore if patients with severe sepsis and with a predicted high risk of death (according to the Simplified Acute Physiology Score II) might have a treatment benefit from high-dose antithrombin III. DESIGN: Subgroup analysis of a randomized, placebo-controlled, double-blind, prospective phase III study. SETTING: Unifactorial and multifactorial reanalysis of prospectively defined populations from the KyberSept trial. PATIENTS: We studied 1,008 patients (43.6% of the overall intention-to-treat population, n = 2,314) with a predicted mortality rate of 30-60% at study entry as defined by the Simplified Acute Physiology Score II. INTERVENTIONS: Patients were randomized in a 1:1 fashion to receive either high-dose antithrombin III (30,000 IU intravenously over the period of 4 days) or placebo. MEASUREMENTS AND MAIN RESULTS: In a Kaplan-Meier analysis of patients with a predicted mortality of 30-60%, the survival time when followed up for 90 days after admission was increased in the high-dose antithrombin III group compared with placebo (p = .04). If heparin was avoided during the 4-day treatment phase with high-dose antithrombin III (n = 140) or placebo (n = 162), the treatment effect appeared to be even more pronounced: 28-day mortality rate, 35.7% vs. 44.4% (risk ratio, 0.804; 95% confidence interval, 0.607-1.064); 56-day mortality rate, 39.9% vs. 52.2% (risk ratio, 0.764; 95% confidence interval, 0.593-0.984); 90-day mortality rate, 42.8% vs. 55.1% (risk ratio, 0.776; 95% confidence interval, 0.614-0.986). Like in the overall population, the percentage with any bleeding was increased in patients receiving high-dose antithrombin III compared with placebo. Survival rates were in favor of high-dose antithrombin III in patients both with and without bleeding complications. CONCLUSIONS: Treatment with high-dose antithrombin III may increase survival time up to 90 days in patients with severe sepsis and high risk of death. This benefit may even be stronger when concomitant heparin is avoided.

APACHE↗

PubMed1989

Antithrombin Sheffield: amino acid substitution at the reactive site (Arg393 to His) causing thrombosis.

A Sheffield family with a predisposition towards thrombosis has been shown to have a functional abnormality of antithrombin. The abnormality was detected as reduced heparin cofactor activity, with normal antigenic levels of antithrombin. Crossed immunoelectrophoresis performed in the absence and presence of heparin was normal. The antithrombin was isolated by heparin Sepharose affinity chromatography. It had normal mobility on SDS polyacrylamide gel electrophoresis. However, the second order rate constant of inhibition of thrombin was about half that of normal, and this was compatible with a heterozygous abnormality involving the reactive site. The antithrombin was further purified by chromatography on thrombin-Sepharose (to remove the normal component), reduced, S-carboxymethylated and fragmented with cyanogen bromide. A pool containing the reactive site region was digested with trypsin and the molecular size of peptides generated determined by fast atom bombardment mass spectrometry. The two peptides adjacent to the Arg393-Ser394 bond of mass 2290 and 700 were almost absent from the mass spectrum, but an additional peptide of mass 2952 was present. Subdigestion with V8 protease reduced the mass of this peptide to 1748. These peptides generated by trypsin and V8 protease were almost identical to those obtained when another variant, antithrombin Glasgow, was treated in the same way (Erdjument et al, 1988). It is concluded that the molecular abnormality of antithrombin Sheffield is identical to that of antithrombin Glasgow, Arg393 to His.

Adult↗

PubMed1990

Hypercoagulability during L-asparaginase treatment: the effect of antithrombin III supplementation in vivo.

To evaluate the occurrence of hypercoagulability during treatment with L-asparaginase (L-ase), thrombin-antithrombin complex (TAT) and D-dimer levels in plasma were serially measured in 15 consecutive adult patients with acute lymphoblastic leukaemia or lymphoblastic lymphoma who had recently completed a chemotherapy cycle with cytosine arabinoside and methotrexate. The first eight patients (group A) received i.v. L-ase alone (20,000 U/m2 on alternate days over 10 d); the last seven patients (group B) received, in addition to L-ase, bolus injection of antithrombin concentrate (2000 U) on alternate days for a total of six administrations, beginning with the second L-ase infusion. Increased levels of TAT (P less than 0.05) and D-dimer (P less than 0.01) were observed prior to L-ase, possibly related to inflammation and cytolysis secondary to previous chemotherapy. In patients treated with L-ase alone, further elevation of TAT (P less than 0.05) and persistence of increased D-dimer were observed, associated with marked reduction of the anticoagulant activities of protein C, protein S and antithrombin III. At variance, in patients receiving antithrombin III supplementation there was no increase of TAT and a normalization of D-dimer levels occurred during L-ase treatment. In these patients, mean plasma antithrombin III activity was maintained at levels higher than 70% of normal throughout the treatment. The rate of decline of fibrinogen, factor IX, protein C and protein S was unaffected by antithrombin III supplementation, indicating that hypercoagulability has little if any relevance for the reduction of coagulation factors and inhibitors induced by L-ase treatment. The usefulness of antithrombin III concentrates in preventing thromboembolic complications in patients submitted to L-ase treatment remains to be determined.

Antithrombin III↗

PubMed1990

Antithrombotic potencies of heparins in relation to their antifactor Xa and antithrombin activities: an experimental study in two models of thrombosis in the rabbit.

Our purpose was to determine the relative contribution of the antifactor Xa and antithrombin activities of heparin to its antithrombotic potency. The antithrombotic activities of unfractionated heparin (UH), two low molecular weight heparins (LMWH, CY 216 and CY 222) with increasing anti-factor Xa/antithrombin ratio and a synthetic pentasaccharide (PS) with high affinity to antithrombin III and no antithrombin activity were evaluated. In the Wessler-thromboplastin model, the most potent antithrombotic agent, on a weight basis, was UH followed by CY 216, CY 222 and the PS which was 40 times less potent than UH. On an antithrombin unit basis, the antithrombotic potencies of UH, CY 216 and of CY 222 were equivalent. Thus, in this model, the antithrombotic effect results from the catalytic action of UH or LMWH on thrombin inhibition. In the Wessler-serum model, on a weight basis, the antithrombotic effectiveness of UH was unchanged, those of CY 216 and CY 222 were doubled, and that of the PS was increased 10 times. On an anti-factor Xa unit basis, CY 216 was as effective as UH, and PS as effective as CY 222. On an antithrombin unit basis, CY 216 and CY 222 were equivalent and more potent than UH. Thus, in this model, the antifactor Xa activity of heparin becomes important for its antithrombotic property. After a single subcutaneous injection of 1000 antifactor Xa U/kg, the antithrombotic effects of UH were maintained for more than 14 h in the two models. After injection of the same dose of CY 216 significant antithrombotic effects were observed only for 9 h, in the Wessler-thromboplastin model but for 18 h in the Wessler-serum model. At that time, no detectable antithrombin activity was measurable in the plasma while 0.11 units of antifactor Xa activity/ml was detected. Thus, the relative contribution of the anti-factor Xa and antithrombin activities to the antithrombotic effect of a LMWH differs according to the nature of the thrombogenic stimulus.

Animals↗

PubMed1986

S protein modulates the heparin-catalyzed inhibition of thrombin by antithrombin III. Evidence for a direct interaction of S protein with heparin.

The interference of S protein with the heparin-catalyzed inhibition of thrombin by antithrombin III was studied in a purified system and in plasma. The effect of S protein to counteract heparin activity was documented by kinetic analysis of the initial phase of the inhibition reaction. Addition of S protein induced a concentration-dependent reduction of the inhibition rate, reflected in a decrease of the apparent pseudo-first-order rate constant by a factor of 5-8 in the presence of a twofold molar excess of S protein over antithrombin III. A non-competitive interaction of S protein with the thrombin--antithrombin-III--heparin inhibition reaction with Ki = 0.6 microM was found. While the association constant of thrombin--antithrombin III in the presence of 0.05 U/ml heparin amounted to 2.5 X 10(8) M-1, an approximately 200-fold decrease of this value was observed in the presence of S protein. The fast formation of the covalent complex between thrombin and antithrombin III in the presence of heparin was impaired as a result of the presence of S protein, as was shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In the absence of heparin the inhibition of thrombin by antithrombin III alone was not influenced by S protein. The heparin-counteracting activity of S protein was found to be mainly expressed in the range of 0.01-0.1 U/ml heparin, thereby shifting the point of 50% inhibition of thrombin from 0.003 U/ml to 0.1 U/ml heparin with a second-order rate constant of k2 = 1.4 X 10(6) M-1. A direct interaction of S protein with heparin was demonstrated by crossed immunoelectrophoresis with purified proteins as well as in plasma and serum. The analysis of plasma and serum by crossed immunoelectrophoresis against rabbit anti-(human S protein) serum revealed an additional cathodal peak in the serum sample, resulting from the interaction of S protein with serum components. These findings not only indicate a direct interaction of S protein with heparin in the onset of the inhibition of thrombin by antithrombin-III--heparin, but also a contribution of S protein during enzyme-inhibitor complex formation.

Antithrombin III↗

PubMed1998

Inactivation of antithrombin III in synovial fluid from patients with rheumatoid arthritis.

OBJECTIVE: To investigate the thrombin inhibitory capacity of antithrombin III in the inflamed human joint. METHODS: Thrombin inhibitory capacity was measured, using a kinetic spectophotometric method, in matched plasma and synovial fluid samples of patients with rheumatoid arthritis (n = 22) and osteoarthritis (n = 16), together with normal control plasma samples (n = 13). In the same samples, the concentration of antithrombin III was also determined by the method of radial immunodiffusion. The combination of these measurements allowed the calculation of the specific thrombin inhibitory capacity of these samples. RESULTS: An increased concentration of antithrombin III in rheumatoid compared with osteoarthritic synovial fluid was noted (p < 0.05). However, there was a significant depression in the specific activity of antithrombin III in rheumatoid synovial fluid when compared with matched plasma samples (p < 0.001) or with osteoarthritic synovial fluid (p < 0.05). CONCLUSION: In rheumatoid synovial fluid the thrombin inhibitory capacity of antithrombin III is disproportionately depressed relative to the concentration of antithrombin III, indicating the inactivation of antithrombin III in the rheumatoid joint.

Aged↗

PubMed1973

Antithrombin-3 deficiency in a Dutch family.

A Dutch family (family A) with inherited antithrombin-III deficiency and an increased incidence of venous thrombosis was investigated. Antithrombin-III levels were measured by means of a coagulation assay in plasma and by single radial immunodiffusion in plasma and serum.THREE GROUPS COULD BE DISTINGUISHED: group I comprised the relations-in-law of family A, group II the members of family A with a plasma antithrombin-III level higher than 90% of normal, when determined by the immunoassay, and group III the members of family A with an antithrombin-III level of less than 60%. To group III belonged all eight adults with an abnormal tendency to thrombosis, and furthermore nine children, all having a parent with abnormally low antithrombin-III levels. Mean plasma and serum antithrombin-III levels were significantly decreased in group III. However, the results of the coagulation assay showed some overlap between groups II and III. In addition, the immunoassay appeared to be much less laborious than the coagulation assay. Therefore, the former assay is recommended in any search for similar families. The results of our family investigation confirm the findings of Egeberg (1965) that inherited antithrombin-III deficiency, giving rise to plasma levels between 50 and 60% of normal, causes thrombophilia and that the pattern of inheritance is autosomal dominant.

Adult↗