The interactions between antithrombin III,thrombin and surface immobilized heparin

The interactions between antithrombin III (ATIII), thrombin, and surface immobilized heparin were investigated. Carboxylated polystyrene modified with covalently immobilized albumin-heparin conjugate contain sites which can bind ATIII from buffer and plasma solutions. Approximately 65% of the ATIII molecules present at the heparinized surface either adsorbed from a buffer or plasma solution, exchanged with ATIII in buffer solution. The exchange between surface bound ATIII and ATIII in solution was repeated several times on the same heparinized surface. The number of binding sites that could bind and release ATIII was much higher when the heparinized surface was exposed to an ATIII containing buffer solution than to a plasma solution. The reduction in binding sites available for ATIII using plasma solutions as compared to buffer solutions could be explained by the competition of other plasma proteins with ATIII for the heparinized surface. It was observed that heparin binding proteins were able to compete with ATIII for binding to the immobilized heparin. Furthermore adsorption of proteins on the heparinized surface significantly reduced the availability of binding sites for ATIII. Exposure of thrombin to the heparinized surface resulted in thrombin activity at the surface. The thrombin activity on the heparinized surfaces was lower on surfaces with a higher ATIII concentration. The activity of surface bound thrombin was not affected by the presence of other plasma proteins. Enzymatically active thrombin molecules present at the heparinized surface were completely inactivated when the surface was exposed to a solution containing ATIII. The inactivation rate of surface bound thrombin by ATIII was higher than the rate of the uncatalyzed inactivation of thrombin in solution. Part of the Thrombin-Antithrombin III (TAT) complexes (10-20%) that were formed upon inactivation of thrombin remained bound to the heparinized surface. In general it was concluded that only the surface immobilized heparin molecules that can bind ATIII in a reversible way determine the anticoagulant properties of the surface. The mechanism of inactivation of a protease on a heparinized surface depends either on the catalytic effect of heparin on the inactivation rate of proteases by ATIII or on an increased uncatalytic inactivation due to increased concentrations of ATIII near the surface as compared to the concentration of ATIII in the bulk phase.     

Antithrombotic activity of Russian Antithrombin III preparation on the model of induced venous thrombosis

Antithrombotic activity of Russian preparation Antithrombin III was studied on rat model of induced venous thrombosis. Optimal doses of antithrombin and heparin preventing thrombus growth were determined. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 7, pp. 75–77, July, 2006     

Effect of antithrombin III on local hemostasis in the kidneys during experimental nephritis

Administration of antithrombin III-enriched plasma to rabbits with acute Masugi nephritis inhibited prothrombinase formation and increased the release of component C₃ from the kidneys. This treatment had a cytoprotective effect and was probably followed by dissociation of antigen—antibody complexes. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 8, pp. 210–213, August, 2004     

Adsorption of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: II. Competition with the other plasma proteins

In the preceding paper, we described results concerning the adsorption of purified thrombin and antithrombin III on two insoluble anticoagulant polystyrene derivatives. We now report similar results obtained in a plasma system. In each case, the purified protein was mixed with fresh platelet poor plasma in order to maintain the same concentrations of all the other plasma proteins. The thrombin molecule was modified by alkyl phosphorylation of the active serine site prior to mixing with plasma. The adsorption of antithrombin was found to be reduced 8 to 9 times when the protein solution was substituted by diluted plasma. In contrast the thrombin adsorption only depends on the substituents bound on the polymeric chain. These results are supported by those of the study of the competition between purified antithrombin and albumin.     

Affinity of purified thrombin or antithrombin III for two insoluble anticoagulant polystyrene derivatives: I. In vitro adsorption studies

In previous papers, we described insoluble polystyrene derivatives which exhibit a heparin-like antithrombic activity in plasma. In order to ascertain the heparin-like mechanism of this activity we have studied the interactions of thrombin and antithrombin III with two polymers of this series: sulphonated polystyrene and sulphonate-glutamic acid sulphonamide polystyrene. The adsorption was measured using purified enzyme and enzyme inhibitor and polymer beads whose average diameter was about 25 μm. The maxima of adsorption approximately correspond to a monolayer of protein. The results are discussed with respect to the most common isotherms used in chemisorption and the affinities of the enzyme and its inhibitor for both materials are evaluated: k_T- 10⁷(M/I)⁻¹, k_AT- 3.10⁵(M/I)⁻¹.     

Clinical and genetic aspects of antithrombin III deficiency

We have reviewed the clinical data on 120 individuals (from four personally studied families and 14 families from the literature) with inherited symptomatic antithrombin III deficiency in order to obtain information useful for genetic counseling and short- and long-term prophylaxis in heterozygotes of the mutant gene.     

Disturbance of interaction between thrombin and the vessel wall and its inactivation by antithrombin III in an experimental nephrotic syndrome

Laboratory of Pathology and Pharmacology of Hemostasis, All-Union Hematologic Scientific Center, Ministry of Health of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR A. l. Vorob'ev.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 110, No. 8, pp. 133–136, August, 1990.     

Effect of Antithrombin III on Local Hemostasis in the Kidneys during Experimental Nephritis

Administration of antithrombin III-enriched plasma to rabbits with acute Masugi nephritis inhibited prothrombinase formation and increased the release of component C3 from the kidneys. This treatment had a cytoprotective effect and was probably followed by dissociation of antigen--antibody complexes.     

Hydrogels by irradiation of a synthetic heparinoid polyelectrolyte

Gamma irradiation of aqueous solutions of a synthetic heparinoid polyelectrolyte results in the formation of hydrogels, varying in water content and mechanical strength. The equilibrium water content and the mechanical strength of the hydrogels are dependent on the initial polyelectrolyte concentration, the molecular weight of the polyelectrolyte, the percentage of double bonds in the polyelectrolyte and the radiation dose. The polyelectrolyte hydrogels do not deplete Antithrombin III from blood and there is no activation of factor XII according to an in vitro kallikrein generation test. However, in a very sensitive test for factor XII activation (contact promoted shortening of the thrombotest) a slight activation of this factor was observed.     

Haemostasis and antithrombin III in the full-term newborn foal

Thirty-three Standardbred foals received a physical examination and had blood drawn for anti-thrombin III (AT III), immunoglobulin G (IgG), and haemostasis evaluation between 24 and 72 h of age. Based on physical examination, a normal haemogram within reference intervals and serum IgG concentration >600 mg/dl, 19 foals remained in the study. AT III values for protein concentration (mg/dl) and activity (mg/dl and percentage thrombin inhibition) were determined by rocket immunoelectrophoresis and chromogenic substrate assay, respectively, and compared to the reference intervals for adults. In 19 healthy, full-term foals, the mean plasma AT III activity for percentage thrombin inhibition (75.9%), mean amount of active AT III (19.2 mg/dl) and the mean plasma AT III concentration (28.7 mg/dl) were significantly (P<0.05) lower than the reference interval of adult values. The mean active AT III concentration for both foals (19.2 mg/dl) and adults (24.6 mg/dl) was significantly (P<0.05) less than their AT III concentration, 28.7 and 44.3 mg/dl, respectively. Fibrinogen degradation products (FDPs) were increased in 100% of the foals, with 12 of 19 (63%) having FDPs >10 <40 g/dl). Platelet count, prothrombin time, activated partial thromboplastin time and fibrinogen did not differ significantly from those of adult values.These findings support the view that haemostasis in the full term foal is characterised as a hypercoagulable state by the significant decrease in plasma AT III activity and concentration and increase in split products of fibrin and fibrinogen.     

Instability of succinyl ester linkages in O2'-monosuccinyl cyclic AMP-protein conjugates at neutral pH

Chromatographic and immunological evidence is presented regarding the hydrolysis of the ester linkage of O2'-monosuccinyl cyclic AMP in neutral solutions. Such hydrolysis occurs whether the nucleotide derivative is present in free form in solution or conjugated through its succinyl carboxyl group via an amide bond to proteins. The latter process apparently occurs when succinyl cyclic AMP is conjugated to human serum albumin for use as an immunogen in the production of anti-cyclic AMP antibodies and when the derivative is coupled to the enzyme glucose-6-phosphate dehydrogenase (E.C. 1.1.1.49). The enzyme conjugate has been used in developing a homogeneous enzyme immunoassay for cyclic AMP. Inhibition of the catalytic activity of enzyme-cyclic AMP conjugates by anti-cyclic AMP antibody decreases with time, apparently due to the loss of cyclic AMP from enzyme-cyclic AMP conjugates stored in neutral solutions. In addition, the ability of free cyclic AMP to completely reverse the inhibition process decreases with time because of the presence of antibodies in the anti-cyclic AMP sera that apparently inhibit enzyme activity because of their binding specificity for the residual succinate-protein determinant sites of the enzyme conjugates. Lyophilization of the conjugates immediately after preparation helps to overcome the problem; however, in vivo hydrolysis of immunogens prepared with the succinyl cyclic AMP derivative may always occur. The consequence of this hydrolysis reaction and the subsequent formation of anti-succinyl-protein antibodies will be discussed with regard to existing RIAs for cyclic AMP and a new homogeneous enzyme immunoassay for the nucleotide.     

A patient defines the interstitial 1q deletion syndrome characterized by antithrombin III deficiency

A patient with microbrachycephaly, high forehead, long philtrum, thin upper lip, downturned corners of the mouth, low set ears with overlapping helix, fifth‐finger clinodactyly, small hands and feet, bilateral transverse palmar crease, low total finger ridge count, hypotonia, severe growth and psychomotor delay, mild hypoplasia of corpus callosum, and Arnold‐Chiari type 1 malformation is reported. The karyotype showed 46, XY, del(1)(q23q31.2). Coagulation factor V (F5, 1q23) and coagulation factor XIII (F13B, 1q31‐q32.1) levels were normal. As expected, antithrombin III (AT3, 1q23‐q25.1) serum level and activity were half of normal. We performed a review of the literature on proximal and intermediate deletion 1q syndrome, and we hypothesize the existence of only one 1q interstitial deletion syndrome, clinically characterized by ATIII deficiency. © 2001 Wiley‐Liss, Inc.     

Decreased production or increased turnover of antithrombin III in severe acquired coagulopathy?

Summary During the course of severe coagulopathy in an infant suffering from septicaemia and shock, antithrombin III levels were determined repeatedly before and during substitution therapy with human antithrombin. By mathematical analysis of these data, using a biexponential function, the plasma elimination half-life of the antithrombin III was estimated to be 7.5–10.5 h. Compared with known plasma half-lives of radioactively labelled antithrombin III in adults the increase was five-to ten-fold. This indicates that the significantly decreased levels of antithrombin III in this case of coagulopathy were at least partly due to an accelerated consumption of antithrombin III. The estimation of the plasma elimination half-life of antithrombin III helps to differentiate decreased production from increased consumption in cases of severe coagulopathy. Thus, a more precise diagnosis of disseminated intravascular coagulation can be made whilst taking advantage of substitution therapy and avoiding the hazards of radioactive tracer proteins.