Inherited AT-III deficiency: fast and slow inactivation of thrombin and factor Xa

A family with inherited AT-III deficiency is presented. This variant is unlike those formerly described, as the plasma concentration is high, the thrombin inhibition with heparin is slow, and factor Xa inhibition is decreased.     

Anticoagulant activities and effects on platelets of a heparin fragment with high affinity for antithrombin

A fragment of heparin containing 10–16 sugar units and retaining ability to bind to antithrombin III has been prepared by degrading standard heparin with nitrous acid. This fragment greatly potentiated the inhibition of factor X_a by antithrombin III but had virtually no effect on the inhibition of thrombin. Studies on heparin neutralization showed that the fragment was affected to a much lesser extent than standard heparin by heparin neutralizing components in plasma. The heparin-potentiated aggregation of platelets by low concentrations of ADP was measured for a number of heparin fractions and the fragment. The molecular weight of the heparin was found to be the most important factor determining the platelet aggregation activity, low molecular weight fractions including the fragment being much less active than high molecular weight ones.     

Cyclic AMP independent inhibition of platelet aggregation by prostaglandin E1 is mediated through factor Xa

Normal plasma exposed to an insolublized ω-NH2-hexyl-agarose derivative of PGE₁ inhibits ADP-induced platelet aggregation. To define the plasma macromolecule responsible, we tested plasma congenitally deficient in coagulation factors for this property. Only plasma deficient in factor X failed to inhibit platelet aggregation under these conditions. The inhibition could be restored by reconstituting the defective plasma with bovine or human factor X. Purified factor X or Xa exposed to insolubilized PGE₁ also inhibited ADP-induced platelet aggregation. Factor Xa could reverse the inhibition of altered factor X but the reverse was not true suggesting that factor Xa was the responsible component. This conclusion was further supported by the ability of heparin to reverse the inhibitory effect of altered factor Xa when the anti-coagulant was added simultaneously to platelets. The results were not due to the release of PGE₁ from the insoluble derivative. Factor Xa may have a heretofore unrecognized effect on modulating platelet functions.     

Abolition by dextran sulfate of the heparin-accelerated antithrombin III/thrombin reaction

Dextran sulfate did not inhibit the amidolytic activity of thrombin on Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide, but abolished inhibition of the enzyme with antithrombin III (AT III) in the presence of heparin.

Dextran sulfate did not bind to AT III and had less affinity than immobilized heparin for the protein. Dextran sulfate bound strongly to thrombin and had higher affinity than immobilized heparin for the enzyme.

These findings indicate that binding of dextran sulfate to a site other than the active site of thrombin to prevent the approach of AT III in the presence of heparin.     

Evaluation of the pharmacological properties and clinical results of the synthetic pentasaccharide (fondaparinux)

Fondaparinux (Arixtra) is the first of a new class of selective indirect antithrombin-dependent factor Xa inhibitors, which inhibits thrombin generation. Fondaparinux is a completely synthetic pentasaccharide. It is a single molecular entity with a well-defined pharmacological target. Fondaparinux has nearly complete bioavailability after subcutaneous injection. The pharmacokinetics of fondaparinux appears predictable and consistent. The peak plasma level is obtained about 2 h after the subcutaneous injection, indicating that a rapid onset of antithrombotic activity is obtained on initiation of treatment. The elimination half-life is about 17 h and it is dose-independent, which allows a convenient once-daily dosing regimen. Fondaparinux is eliminated exclusively by the kidneys. Thus, the estimation of the renal function especially in elderly patients is important for the treatment with fondaparinux, whereas it is contraindicated in patients with severe renal insufficiency. Phase II clinical studies have identified a subcutaneous dose of 2.5 mg once daily for prophylaxis of venous thromboembolism in patients undergoing major orthopaedic surgery. Four phase-III clinical trials using bilateral phlebography for the diagnosis of DVT, demonstrated a combined 50% relative risk reduction of asymptomatic venous thromboembolic events in orthopaedic surgery patients in comparison to the low-molecular-weight heparin (LMWH) enoxaparin. Hemorrhagic complications for fondaparinux were either comparable or higher than those for LMWH but the authors did not judge that the increased bleeding was clinically relevant. A dose ranging study led to the selection of the dose of 7.5 mg at a single daily subcutaneous injection as optimal for the treatment of VTE. In two phase III clinical trials, the dose of 7.5 mg/day is expected to be as efficacious and safe as heparin for the treatment of DVT or PE, respectively. Phase II studies show that the efficacy-to-safety ratio of fondaparinux in the treatment of unstable angina or as an adjunct to thrombolysis in acute myocardial infarction is promising. These results demonstrated that a single anti-Xa agent devoid of antithrombin activity is a potent antithrombotic drug. Fondaparinux has obtained FDA and European health authorities approval. Its use on a large scale will allow the evaluation of its efficacy and tolerance in the daily clinical practice. Chemical modifications of the original synthetic pentasaccharide increase the affinity to AT resulting in a more potent inhibition of FXa and longer half-life. Idraparinux is the first of these new oligosaccharides that we named "meta-pentasaccharides." After subcutaneous injection the half-life of idraparinux is about 80 h allowing a single injection per week. A dose-finding study has established the optimal dose given once a week to be compared with warfarin for the treatment of DVT.     

Heparin with low affinity to antithrombin III inhibits the activation of prothrombin in normal plasma

Standard unfractionated heparin is known to have two actions on blood clotting. Unfractionated heparin enhances the rates at which antithrombin III inactivates activated clotting factors, and inhibits the activation of both Factor X and prothrombin by disrupting the calcium and phospholipid dependent assembly of the Factor X and prothrombin activator complexes. This latter inhibitory action of heparin occurs independently of antithrombin III. A heparin fraction with low affinity to antithrombin III was prepared from standard heparin by affinity chromatography on antithrombin-III-Sepharose and its properties compared with unfractionated heparin. The low affinity heparin fraction and the unfractionated heparin had equivalent inhibitory effects on prothrombin activation in antithrombin III depleted plasma. In normal plasma, the low affinity fraction inhibited the activation of prothrombin. Unlike the unfractionated heparin, however, the fraction of heparin with low affinity to antithrombin III did not enhance the inactivation of either Factor Xa or thrombin. This antithrombin III independent inhibition of the activation of prothrombin was also evident when activated platelets were used as the source of the procoagulant phospholipids. The antithrombin III independent effect of heparin is unlikely to be important therapeutically, however, if this property of heparin is shared by other naturally occurring glycosaminoglycans, it could be important in maintaining the fluidity of blood under physiological conditions.     

Mechanism of thrombin inhibition by heparin cofactor II and antithrombin in the presence of the ray (Raja radula) skin dermatan sulfate

Introduction

The kinetics of the thrombin inhibition by heparin cofactor II (HCII) and antithrombin (AT) have been studied as a function of the concentration of a dermatan sulfate (DS) from the skin of the ray Raja radula.

Materials and methods

The initial concentrations of inhibitor (I), HCII or AT, and thrombin (E) were set at equimolecular levels (3.10^{- 9} M). Analysis of the experimental data obtained for DS concentrations ranging from 10^{- 8} to 10^{- 4} M was performed according to a previously described model in which DS binds quickly to the inhibitor and forms a complex more reactive than the free inhibitor towards thrombin.

Results

The apparent rate constant of the thrombin inhibition, k_app, by either HCII or AT, increased in a concentration-dependent manner for DS concentrations up to 10^{- 5} M or 10^{- 6} M, respectively. At higher DS concentrations, k_app remained unchanged for thrombin inhibition by HCII whereas a decrease in k_app was observed for the thrombin-AT reaction. The dissociation constant of the polysaccharide-inhibitor complex, K_DSI, and the rate constant of the thrombin inhibition by this complex, k, were (7.81 ± 0.75).10^{- 7} M and (2.84 ± 0.42).10⁹ M^{- 1}.min^{- 1}, whereas they were (4.93 ± 0.31).10^{- 7} M and (2.47 ± 0.28).10⁸ M^{- 1}.min^{- 1}, when the inhibitor was either HCII or AT, respectively.

Conclusion

DS from ray skin catalyzes the thrombin inhibition by HCII or AT primarily by forming a DS-inhibitor complex more reactive than the free inhibitor towards the protease. The affinity of DS for HCII was approximately 2-fold higher whereas the catalyzed reaction rate constant was approximately 20-fold higher when compared to AT.     

Inhibition of human coagulation factor Xa by thrombin substrates

The thrombin specific chromogenic substrates, Phe-Pipecolyl-Arg-p-nitroanilide (S-2238) and benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) are inhibitors of factor Xa amidolytic and proteolytic activity. The inhibition is of the partial noncompetitive type in that no change in the Km for the hydrolyzed substrate was observed and some factor Xa activity remains even at infinite inhibitor concentration. High performance liquid chromatographic analysis indicated that the inhibitors were essentially free of either p-nitroaniline or free peptide contamination. Neither p-nitroaniline nor acid hydrolyzed inhibitors were capable of inhibiting factor Xa demonstrating that a minimal peptide structure is necessary for inhibition. Our results indicate that factor Xa contains a regulatory site which, when occupied by certain peptides, causes a decrease in the rate of catalysis.     

Heparin affinity of factor VIIa: implications on the physiological inhibition by antithrombin and clearance of recombinant factor VIIa

Factor VIIa (FVIIa), a trypsin-like serine protease, plays an essential role in haemostasis by initiating the coagulation in complex with its cofactor, tissue factor (TF). The TF pathway inhibitor is the main physiological inhibitor of FVIIa-TF complex, but FVIIa can also be inhibited by antithrombin, although little is known about this process.Functional analyses by second order kinetic determination and identification of FVIIa-antithrombin complex by electrophoresis, evaluating the effect of different cofactors: pentasaccharide, low molecular weight heparin (LMWH) and unfractionated heparin (UFH), confirmed that any activation of antithrombin significantly enhanced the inhibition of FVIIa. The analysis of the binding of FVIIa to heparin by surface plasmon resonance identified a high affinity interaction under physiologic conditions (K_D = 3.38 μM, with 0.15 M of ionic strength) strongly dependent on Ca²⁺ and ionic strength. This interaction was verified in cell models, indicating that FVIIa also binds to the surface of endothelial cells with similar requirements. Structural modeling suggests the presence of a potential exosite II in FVIIa. However, the binding of heparin did not display significant changes on both the intrinsic fluorescence and the associated functional consequences of FVIIa.These results indicate that FVIIa binds to exposed glycosaminglycans of the endothelium through an exosite II, structurally similar to that reported for thrombin and suggested for FIXa. This binding may favor its inhibition by antithrombin in the absence of TF, contributing to the physiological control of this protease. This process may also play an important role in the clearance of recombinant FVIIa administered to patients.     

Substitution of antithrombin III in shock and DIC: a randomized study

In 51 shock patients with DIC Antithrombin III (AT III) substitution, heparin or a combination of both substances respectively was administered. In the two groups which had been given AT III substitution the concentration of AT III rose considerably higher than the activity. There was a drop of the platelet count in both groups which had received heparin. C1 esterase inhibitor was diminished in the beginning but spontaneously increased in all groups. This increase was slowest in the group without substitution of AT III. The blood loss in cases of traumatic shock was considerably higher in the group which had received both substances. The consumption of AT III concentrates was slightly higher in the combined therapy group than in the AT III group. The duration of symptoms of DIC was considerably shorter in the two substituted groups than in the heparin group. It is concluded that additional administration of heparin does not improve the effect of AT III substitution in patients with DIC and that side effects such as thrombocytopenia and an increased blood loss are likely to develop when both substances are given simultaneously.     

The inhibition by heparin of the intrinsic pathway activation of factor X in the absence of antithrombin-III

Heparin was found to inhibit the activation of purified factor X by purified factors IXa and VIII (or VIIIa) in the presence of calcium and phospholipid but in the absence of antithrombin-III. In contrast, heparin did not inhibit the activation of factor X by either the extrinsic pathway or by the factor X activator isolated from Russell's Viper Venom (RVV-X). The heparin inhibition of the intrinsic pathway activation of factor X could be totally reversed by polybrene. These results suggest that even in the absence of antithrombin-III, heparin reversibly interferes with the interactions of one or more of factors IXa, VIII (or VIIIa) and X with calcium and phospholipid that result in the activation of factor X.     

Assay of unfractionated and LMW heparin with chromogenic substrates: Twin methods with factor Xa and thrombin

Utilizing two newly synthesized chromogenic substrates (CS), two different assay methods for heparin in plasma have been developed. The assay with bovine factor Xa and the highly reactive “Substrate FXa-1” (CH₃OCO-D-CHA-Gly-Arg-pNA-AcOH) measures both unfractionated (UF) heparin and low molecular weight (LMW) heparin within a single standard curve in the 0.05–1.5 U/ml plasma range. The very similar (and less expensive) assay with bovine thrombin and “Substrate Th-1” (2AcOH-H-D-CHG-Ala-Arg-pNA), measures OF heparin, but not LMW heparin. The standard curves are highly reproducible (CV 3.5–4.7%). For clinical work, a linear standard curve is obtained with three standards and lin-log plot. The “within run” SD was 0.007–0.026 U/ml. Mean recovery of 0.5 U/ml heparin added to 10 pathological plasma samples ranged 0.46–0.53 U/ml (SD 0.034–0.040). Activities of three of heparin and three LMW heparin preparations are reported.     

Purification and biological property of heparin cofactor II: Activation of heparin cofactor II and antithrombin III by dextran sulfate and various glycosaminoglycans

Heparin cofactor II (HC II) has been purified from human plasma by a mocification of the method described by Tollefsen $\text{et al}$.(J. Biol. Chem., $\text{257}$, 2162, 1982) and abilities of dextran sulfate and various glycosaminoglycans to activate the antithrombin activities of HC II and antithrombin III (AT III) were studied. By the purification method described here, highly purified HC II with the same specific activity as reported by Tollefsen $\text{et al}$. was obtained with a higher yield and in a shorter purification time. Heparin, dextran sulfate and chondroitin polysulfates 1 and 5 activated both HC II and AT III, while dermatan sulfate activated only HC II. Dextran sulfate was almost as active as heparin in the activation of HC II and AT III, indicating that in the interactions of heparin with HC II and AT III, sulfate groups of heparin are more important than carboxyl groups. When mixed with thrombin in the presence of dermatan sulfate, normal human plasma showed antithrombin activity which was not due to AT III but to HC II only. HC II did not inhibit factor Xa or plasmin in the presence of any glycosaminoglycans or dextran sulfate, suggesting that HC II would be a specific inhibitor of thrombin.     

Relative influence of different disulphate disaccharide clusters on the HCII-mediated inhibition of thrombin by dermatan sulphates of different origins

Besides the major monosulphated disaccharide sequences (IdoA-GalNAc4SO₃), dermatan sulphates (DS) contain the oversulphated sequences (IdoA2SO₃-GalNAc4SO₃) and (IdoA-GalNAc4,6SO₃), the concentration of which is correlated with the HCII-mediated inhibition of thrombin by DS. The effect of the chemical removal of the sulphate groups on the HCII-mediated activity was studied. The base-catalyzed sulphate group displacement from IdoA2SO₃ residues, leading to formation of the epoxyde aGulA, is a 1st order reaction. When the content of sequences (IdoA-GalNAc4,6SO₃) is higher than that of sequences (IdoA2SO₃-GalNAc4SO₃), removal of the sulphate groups from Ido2SO₃ reduces the HCII activity less than when the latter sequences prevail. (IdoA-GalNAc4,6SO₃) cooperates with (IdoA2SO₃-GalNAc4SO₃) in the activation of the HCII. Also the removal of 6-SO₃ groups from GalNAc4,6SO₃, in absence of IdoA2SO₃-GalNAc4SO₃, considerably reduces the activity. A low molecular mass natural fraction rich in IdoA2SO₃ as well as glucuronic acid, having higher electrophoretic mobility than the higher molecular mass DS which contains less glucuronic acid, is remarkably active.     

Structural Requirements of Human Tissue Factor Pathway Inhibitor (TFPI) and Heparin for TFPI-Heparin Interaction

Heparin affinity chromatography of synthetic peptide fragments mimicking tissue factor pathway inhibitor (TFPI) indicated that the minimal heparin binding sequence consists of 12 amino acid residues located at the C-terminal tail. Within this minimal sequence, Arg-257 and Arg-259 appeared to contribute most significantly to interaction with heparin. Affinity chromatography of TFPI using immobilized heparin derivatives regiospecifically desulfated at O-6 of the glucosamine residue, N-2 of the glucosamine residue, and/or O-2 of the iduronic acid residue indicated that all the sulfate groups in heparin appeared to be required for TFPI-heparin interaction. Among them, however, the 6-O-sulfate groups appeared to make the largest contribution to the interaction, while the 2-O-sulfate groups contributed the least . In vitro experiments on the inhibition of factor Xa by TFPI enhanced with native and chemically modified heparins afforded similar results.     

Activity of antithrombin III and effect of heparin on coagulation in shock

In 16 patients admitted for DIC and shock,series of coagulation tests were carried out prior to and during heparin therapy. Plasma heparin concentrations measured were corresponding to expected levels. In some cases however,considerably higher or lower levels were found. This could be partly explained by release of PF 4 from platelets and by a decreased turnover of heparin because of a malfunction of the kidneys or of the liver. A relation between the effect of heparin on coagulation and the activity of antithrombin III could be established. A diminution of this activity resulted in a diminished or even missing effect of heparin. In some instances,the PTTand the thrombin clotting time were considerably prolonged when FDP were present or when procoagulant factors were diminished. These tests therefore,did not reflect true heparin concentrations in shock. For this reason,regular assays of antithrombin III and of heparin are proposed.     

Effects of activated factor X inhibitor,JTV-803, in a pig model of hemodialysis

JTV-803 (4-[(2-amidino-1,2,3,4-tetrahydroisoquinolin-7-yloxy)methyl]-1-(4-pyridinyl)piperidine-4-carboxylic acid monomethanesulfonate trihydrate), a specific inhibitor of factor Xa, was evaluated in a pig hemodialysis model with ligation of renal arteries. In this model, JTV-803 administered into the dialysis circuit showed an anticoagulant effect (prolongation of dialysis time) at doses of 0.3 mg/kg+0.3, 1.0 and 3.0 mg/kg/h. The prolongation of dialysis time in the 0.3 mg/kg+1.0 mg/kg/h JTV-803 group was comparable to that in the 15 U/kg+7.5 U/kg/h heparin group. The plasma concentrations of JTV-803 following 0.3 mg/kg+1.0 mg/kg/h infusion of the drug into the dialysis circuit was 500 ng/ml or less, and prothrombin time was 1.5-fold or less than the pretreatment value. JTV-803 was removed by passing the blood through a dialyzer, resulting in a clearance rate of 53.4–81.8%. After the end of dialysis, plasma concentrations of JTV-803 decreased rapidly with time. These results suggest that human factor Xa inhibitor JTV-803 may have good potential as an antithrombotic agent during hemodialysis, with lower likelihood of bleeding after the end of dialysis.     

Demonstration of a direct anti-factor Xa activity in certain heparin-related glycosaminoglycans

Heparan sulphate/heparin subfractions with high plasma anti-Xa activity have an unusual uronate composition, i.e. high proportions of both glucuronate and sulphated iduronate. These preparations inhibit the amidase activity of factor Xa in an uncompetitive mode and the prothrombin-activation catalyzed by Xa, both in the absence of antithrombin III. Subfractions of low affinity for antithrombin III are equally potent against Xa. The anti-X activity is destroyed by a 3-h periodate oxidation.