Isolation and properties of high molecular weight heparin

A heparin fraction comprising the highest molecular weight components of beef-lung heparin preparations was isolated by chromatography on Biogel P-100. This fraction, all of which is highly active in accelerating the inhibition of thrombin by antithrombin III, includes material with considerably greater activities than previously reported. All of the high-molecular weight fraction binds to antithrombin-Sepharose. The fraction has a molecular weight of 3.5 × 10⁴ and binds to antithrombin III in a 1:1 molar ratio. The dissociation constant of the complex is 1.38 × 10^?8 M ± 0.26 × 10^?8.     

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.     

Measurement of the heparin enhanced-antithrombin III/thrombin reaction rate in the presence of synthetic substrate

An assay approach, designed to allow accurate measurement of the rate of thrombin inhibition by antithrombin III in the presence of heparin, is described. In essence the approach takes advantage of the fact that synthetic substrates, e.g. N-α-p-tosyl-L-glycyl-L-prolyl-L-arginine-p-nitroanilide, bind to the active site of thrombin, which slows the rate of reaction with antithrombin III. The rate of thrombin inhibition can be monitored continuously by change in absorbance at 400 nm or measured by end point determination. In either case, reproducible rate measurements can be made under a variety of experimental conditions not easily examined with conventional assay approaches.     

Antithrombin III modulates the effect of thrombin on the metabolism of glycosaminoglycans in cultured endothelial cells

We previously reported that a treatment of cultures of endothelial cells from bovine aorta with thrombin resulted in a less accumulation of glycosaminoglycans (GAG) in the cell layer. In the present study, we found that thrombin-induced decrease in the accumulation of [³⁵s]sulfate-labeled GAG (³⁵S-GAG) such as heparan sulfate was prevented by antithrombin III (AT III) but not by heparin cofactor II (HC II). However, AT III did not show a significant effect on the ³⁵S-GAG accumulation individually. Pretreatment of the cell layer with neither AT III not HC II showed any preventive effect. When GAG in the cell layer was labeled with both [³⁵s]sulfate and [³⁵h] glucosamine, neither thrombin nor a combination of thrombin with AT III changed the ratio of the radioactivity of ³⁵S to that of ³H. Although thrombin stimulated the release of ³⁵S-GAG from the cell layer, AT III completely prevented the stimulatory effect. In conclusion, it was suggested that AT III may inhibit the thrombin action on GAG metabolism of endothelial cells to prevent thrombosis in vivo.     

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.     

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.     

The catalytic activity and physical properties of bovine thrombin in the presence of dimethyl sulfoxide

Dimethyl sulfoxide produces an opposite effect on the esterase and amidase activities of bovine thrombin. The esterase activity is increased by two fold but the amidase activity is decreased to 9% of the initial activity in 20% dimethyl sulfoxide. The stimulation of the esterase activity is due to the change in V_max rather than K_m for the substrate p-Tosyl-L-Arginine methyl ester. The inhibition of the esterase activity of thrombin by NaCl is not affected due to the addition of dimethyl sulfoxide. K_i for NaCl, 0.03 M, is the same for both in the absence and in the presence of 10% dimethyl sulfoxide. The catalytic activity of thrombin is inhibited by heparin. This effect is significantly decreased by dimethyl sulfoxide . The dissociation constant of heparin-thrombin complex, measured in the absence and in the presence of 10% dimethyl sulfoxide are 4 nM and 28 nM respectively. Thermal stability of thrombin, determined by monitoring catalytic activity, is increased in the presence of dimethyl sulfoxide. The enhancement of the fluorescence intensity of thrombin in the presence of dimethyl sulfoxide reflects the contribution of more exposed tryptophanyl residues. The alteration of the conformation of the enzyme structure due to the perturbation of the aqueous medium by dimethyl sulfoxide, has been attributed to these observed effects.     

Biophysical characteristics of anionic density-fractionated mucosal heparins in relation to potencies in anticoagulant and thrombin-inhibition assays

The apparent molecular weights determined by high pressure liquid chromatography (HPLC), specific rotations, anticoagulant potencies and thrombin-inhibition potencies of a series of heparin fractions of different anionic density were compared. The fractions were prepared by sequential extraction of the heparin-quaternary ammonium complexes from butanol with aqueous solutions of successively increasing NaCl concentration. Surprisingly, their molecular weights determined by HPLC varied with anionic density, whereas molecular weights by gel chromatography on agarose did not. The specific rotation of the fractions which contained only heparins was constant at 52°, but those fractions which contained dermatan sulfate showed lower rotations. The anticoagulant potencies of the heparin-containing fractions, measured by the activated partial thromboplastin time (APTT) assay in human plasma, were very similar to previous measurements by the United States Pharmacopeia (USP) method in sheep plasma and, when plotted against the square of the anionic density (Z), showed initial linear portions with a sharp decrease in slope for the higher-charged fractions. In contrast the thrombin-inhibition potency measured in an amidolytic assay system using purified antithrombin and thrombin was linear over the entire range of Z². The ratio of the thrombin inhibition to anticoagulant potency varied systematically from 0.55 for the heparin fraction with the lowest anionic density to 2.0 for the fraction with the highest anionic density.     

Isolation and characterization of a hereditary abnormal antithrombin III 'Antithrombin III Toyama'

A hereditary abnormal antithrombin III (AT-III) 'Antithrombin III Toyama' was purified from the plasma of a patient with recurrent thrombophlebitis by a procedure involving barium chloride and ammonium sulfate fractionations, affinity chromatography on anti-AT-III-Sepharose gel, and DEAE-Sephadex chromatography. Purified abnormal AT-III was shown to be the same as normal one in the molecular size, having the same molecular weight, amino-terminal sequence and carboxy-terminal amino acid. Abnormal AT-III gave the same UV spectrum as normal AT-III and both proteins were immunologically identical. Abnormal AT-III, however, showed the different electrophoretic mobility on agarose gel electrophoresis and immunoelectrophoresis. Abnormal AT-III was more electronegative than normal one, before and after a neuraminidase digestion of both proteins. These results suggest that in antithrombin III Toyama an amino acid residue at the heparin-binding site has been replaced by less basic or more acidic one which has no ability to interact with heparin, resulting in a loss of heparin cofactor activity of this protein.     

Effect of thrombin and endotoxin on the in vivo metabolism of antithrombin III (AT III) in dogs

Effect of thrombin and endotoxin on the metabolism of I-125-labelled canine AT III was studied in mongrel dogs. Under control condition, mean total amount of intravascular AT III with standard deviation was 23.4 +/- 2.4 mg/kg, plasma half life of i.v. injected I-125-AT III was 1.7 +/- 0.2 days, and the fractional catabolic flux (j3x) was 16.3 +/- 1.6 mg/kg/day. The total amount of intra- and extra-vascular AT III was 36.0 +/- 0.34 mg/kg. Neither a 3 hour infusion of a small dose (30 units/kg/hr) of thrombin nor i.v. injection of a large amount of thrombin (5,000-15,000 units/day) with heparin significantly affected AT III metabolism except for a transient decrease in AT III concentration in the latter case, although decrease in plasma fibrinogen concentration and platelet count was observed in both cases. Two injections with 200 micrograms/kg of endotoxin resulted in an evident acceleration of AT III metabolism with significant decrease in the plasma AT III, fibrinogen concentrations and platelet count. More marked changes in AT III metabolism were induced by a single infusion with 1 mg/kg of endotoxin. Changes in hemostatic system coincided with those observed in DIC.     

The effect of operation and subcutaneous heparin on plasma levels of antithrombin-iii

Plasma antithrombin III (AT-III) levels were measured in 61 patients undergoing general surgery. The patients were randomly allocated to a test or a control group. The test group received 5000iu of subcutaneous heparin two hours before operation and then twelve hourly for seven days. The control group received subcutaneous saline. Plasma levels of AT-III and of heparin were estimated before operation, at two, four and six hours after heparin injection on the day of operation and thereafter before the morning injection on the first, third and sixth postoperative days. Antithrombin III decreased progressively during and after operation in both groups. The decrease was greater in the test group at six hours after the first injection. These findings support the hypothesis that AT-III is consumed during coagulation and its utilization is increased in the presence of heparin.     

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.     

In vitro studies of a new synthetic thrombin inhibitor

(2R, 4R)4-methyl-1-[N alpha-(3-methyl-1,2,3,4-tetrahydro-8-quinoline- sulfonyl)-L-arginyl]-2-piperidine carboxylic acid monohydrate (MCI-9038) was found to be a potent synthetic inhibitor of thrombin. In concentrations as low as 1 microM, the thrombin time, prothrombin time, and partial thromboplastin time were more than doubled. The venom (Bothrops atrox) time was similarly prolonged. The drug also inhibited the thrombin-induced activation of factors VIII and XIII. While MCI-9038 in concentrations of 10(-4) M had no effect on platelet aggregation induced by collagen, ADP, epinephrine, and arachidonate, nanomolar concentrations inhibited thrombin-induced platelet aggregation and the release of platelet ADP. The drug also significantly inhibited the adhesion of thrombin-treated platelets to cultured bovine aortic endothelial cells. We conclude that MCI-9038 is an extremely potent inhibitor of the effects of thrombin on platelets and clotting factors.     

Heparin-like glycosaminoglycan is a receptor for Antithrombin III-dependent but not for thrombin-dependent prostacyclin production in human endothelial cells

Antithrombin III (ATIII) induced a marked increase in prostacyclin (PGI₂) release from cultured human umbilical vein endothelial cells (HUVEC) after incubation for more than 2 hr and the induction continued for 8 hr, while thrombin induced the increase within 10 min. ATIII-dependent production of PGI₂ was abolished by addition of heparin, but pretreatment of HUVEC with polyclonal antibody against thrombomodulin could not prevent the PGI₂ productions by ATIII and thrombin. ATIII-dependent PGI₂ production was significantly inhibited by pretreatment of HUVEC with β- -xylosides or heparitinase, though neither pretreatment affected thrombin-induced PGI₂ production. After treatment of HUVEC with 1 μg/ml cycloheximide, ATIII-dependent PGI₂ production was completely abolished. These results indicate that the mechanism of the induction of PGI₂ production by ATIII involves heparin-like glycosaminoglycans on HUVEC and the stimulation of synthesis of a protein related to PGI₂ production. The ATIII-induced PGI₂ production is very different from that induced by thrombin.     

Isolation and partial characterization of two distinct types of antithrombin III from rabbit

Heparin-Sepharose chromatography of rabbit plasma or serum yields two fractions of free antithrombin III (AT). The first of these elutes at a lower salt concentration, represents about 90% of the AT in plasma, and has an approximately 2000 dalton higher molecular weight by SDS polyacrylamide electrophoresis. An antibody to the lower affinity species reacts with the second form. The higher affinity AT is not formed from the lower affinity type during blood coagulation as demonstrated by approximately equal levels in plasma and serum, and lack of conversion of ¹²⁵I-labelled lower affinity AT to the higher affinity form during blood coagulation. Heparin cofactor and progressive AT activities of the two forms are essentially identical when assayed by chromogenic substrates. The forms are separable by crossed immunoelectrophoresis.     

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.     

Antithrombin III Toyama: a hereditary abnormal antithrombin III of a patient with recurrent thrombophlebitis

A familial abnormal antithrombin III (AT-III) is reported. The characteristic of the abnormality in this family is low heparin cofactor activity with normal progressive antithrombin activity and normal or rather increased level of AT-III antigen. The patient is a 23-year-old female who had suffered from recurrent thrombophlebitis involving her lower extremities. Her plasma AT-III antigen concentration was 54 mg/dl and progressive antithrombin and factor Xa inhibitory activities were of normal level. However, the heparin cofactor activity of her plasma was as low as 26% of normal control. On crossed immunoelectrophoresis (CIE) containing heparin in the first dimension agarose, patient's AT-III showed no increase in electrophoretic mobility compared to that in the absence of heparin, suggesting that the patient's AT-III has no affinity for heparin. From CIE pattern in the presence of heparin, the patient was found to be a homozygote, and parents and one of her younger sisters were heterozygotes. Thus, the mode of inheritance is proposed to be autosomal dominant.