M.W.C. Hatton

Inhibition of thrombin by antithrombin III in the presence of certain glycosaminoglycans found in the mammalian aorta

Abstract Chondroitin-4-sulphate, chondroitin-6-sulphate, dermatan sulphate, heparan sulphate and hyaluronic acid were compared with heparin in their abilities to influence the inactivation of bovine thrombin by rabbit antithrombin III. The effect of the glycosaminoglycans on the enzymic activity of thrombin was examined using the substrate α-N-benzoyl arginine ethyl ester. Heparin, dermatan sulphate, heparan sulphate and, to a smaller extent, chondroitin-6-sulphate increased the esterase activity, whereas chondroitin-4-sulphate and hyaluronic acid had a negligible effect. Heparan sulphate and dermatan sulphate markedly accelerated the inactivation of thrombin by antithrombin III, but chondroitin-4-sulphate, chondroitin-6-sulphate and hyaluronic acid did not significantly affect the reaction. The glycosaminoglycans were adsorbed on Sepharose-lysine or polyacrylamide-ethylenediamine to test their ability to bind thrombin or antithrombin III under conditions where neither protein could directly bind to the conjugates. The binding of thrombin to immobilised heparan sulphate or dermatan sulphate compared well with that to heparin. On immobilised chondroitin-6-sulphate, thrombin was retarded and on chondroitin-4-sulphate no thrombin binding was observed. In contrast to thrombin, antithrombin III only bound to heparin: antithrombin III did not bind to dermatan sulphate, heparan sulphate or the other glycosaminoglycans. Antithrombin III inactivation of 125 I-thrombin adsorbed to either heparan sulphate or dermatan sulphate produced a thrombin-antithrombin III complex which was spontaneously released from the glycosaminoglycan. However, no complex was recovered from immobilised heparin unless the column was eluted by 1M NaCl. These results support the view that heparan sulphate and dermatan sulphate could play a role similar to that of heparin in the inactivation of thrombin by antithrombin III.

Cytokeratin 18 Is Expressed on the Hepatocyte Plasma Membrane Surface and Interacts with Thrombin-Antithrombin Complexes

During experiments to identify putative hepatic receptors for thrombin-antithrombin (TAT) complexes, a 45-kDa protein was identified by ligand blotting. Following gel purification, amino acid sequencing revealed the 45-kDa TAT-binding polypeptide to be cytokeratin 18 (CK18). The presence of CK18 on the surface of intact rat hepatoma cells was demonstrated by binding of125I-anti-CK18 antibodies. Anti-CK18 antibodies reduced the binding and internalization of 125I-TAT by rat hepatoma cells. Immunocytochemical analysis, to determine the location of CK18 in vivo, revealed a periportal gradient of CK18 staining; with hepatocytes around the portal triads demonstrating striking pericellular staining. In addition, anti-CK18 IgG associated with perfused livers to a significantly greater extent than preimmune IgG. Taken together, these data provide evidence that CK18 is found on the extracellular surface of hepatocytes and could play a role in TAT removal. Finally, these data, in conjunction with recent reports of CK8 (Hembrough, T. A., Li, L., and Gonias, S. L. (1996)J. Biol. Chem. 271, 25684–25691) and CK1 cell membrane surface expression (Schmaier, A. H. (1997) Thromb. Hemostasis 78, 101–107), indicate a novel role for these proteins as putative cellular receptors or cofactors to cellular receptors.

Inactivation of thrombin by antithrombin III on a heparinized biomaterial

Abstract Heparin covalently bonded to polyvinyl alcohol (PVA) is potentially useful as a nonthrombogenic coating in the preparation of small diameter vascular prostheses and blood sampling catheters. PVA-heparin is highly stable: the elution rate of 35 S-heparin from the polymer was determined to be negligible (approx. 2 × 10 −11 g/cm 2 min) when washed with either buffered saline (pH 7.4) or citrated human plasma. The inactivation of thrombin by antithrombin III was studied on PVA-heparin. Using small columns of PVA-heparin beads eluted by 0.14M NaCl buffered at pH 7.4 both thrombin and antithrombin III bound to the immobilized heparin. If thrombin was loaded before an excess of antithrombin III, significant inactivation of thrombin was observed; however, loading antithrombin III before thrombin did not measurably inactivate thrombin. The results suggest that the covalently-bound heparin effectively participates in the inactivation of thrombin through the formation of surface-bound heparin-thrombin, which then reacts with antithrombin III to yield a surface-bound thrombin-antithrombin III complex. The fate of this surface-bound complex has yet to be clarified.

Elimination of asialofetuin and asialoorosomucoid by the intact rat quantitative aspects of the hepatic clearance mechanism

The capacity of the liver to eliminate asialofetuin and asialoorosomucoid was investigated in intact rats. From plasma radioactivity curve measurements and assays on tissue homogenates the liver is shown to be able to dispose of an average of 19.8 microgram of asialofetuin/min per 100 g body weight. No other major route is identified for the disappearance of asialofetuin from the plasma, although trace amounts of the protein were detectable in the urine. From analyses of the plasma radioactivity curves the elimination process for asialoorosomucoid appears to be comparatively complex because of the existence of extrahepatic disposal routes. Quantification of labelled asialoorosomucoid in liver homogenates indicates, however, that the hepatic clearance rate for asialoorosomucoid is similar to that for asialofetuin. Urinary excretion significantly contributes to the disappearance of asialoorosomucoid from the plasma but the hepatic and renal routes do not account for all the protein lost from this compartment. At plasma concentrations above the maximal eliminative capacity of the liver, the hepatic clearance of asialofetuin obeys zero-order kinetics and is remarkably constant. Elimination of a quantity of asialoglycoprotein which exceeds the calculated total number of binding sites in the liver does not reduce the efficiency of the pathway, and studies of [3H]leucine incorporation indicate that the lectin, unlike the bound asialoglycoprotein, is not destroyed in the elimination process. Cytochalasin B (80 microgram/100 g body wt.) had no measureable effect on the hepatic clearance of asialofetuin. Administration of colchicine (10 mg/100 g body wt.) resulted in transitory accumulations of asialoorosomucoid in the liver, presumably due to interference with the intracellular transport of the endocytised protein.

The proteolytic nature of commercial samples of galactose oxidase. Purification of the enzyme by a simple affinity method.

Several commercially available samples of galactose oxidase (D-galactose: oxygen 6-oxidoreductase, EC 1.1.3.9) were found to contain high proteolytic activity on proteins such as fibrinogen, transferrin, albumin and casein. A simple, efficient method was devised for the purification of galactose oxidase which relies on the affinity of the enzyme for agarose (Sepharose 6B). The purified galactose oxidase was recovered in high yield free from proteolytic activity. The enzymic affinity for Sepharose and Sephadex was investigated to clarify the absorption mechanism.

Tritiation of Commercial Heparins by Reaction with NaB3H4: Chemical Analysis and Biological Properties of the Product

Abstract A simple method to tritiate commercial samples of heparin is described. Heparin is reacted with NaB3H4 at pH 8.0 for 3 h at room temperature, after which the 3H-labeled mucopolysaccharide is isolated by gel filtration. Using NaB3H4 of low specific radioactivity (227–555 mCi/mmol), [3H]heparins containing 3.7–8.0 × 105 dpm/mg are made whereas with NaB3H4 of higher specific activity (5–10 Ci/mmol), preparations of 1.2–1.8 × 107 dpm/mg are obtained. From analysis of [3H]heparin using several techniques (periodate oxidation; mild acid hydrolysis accompanied by ion-exchange chromatography and high-voltage electrophoresis), the labeling procedure largely relies on a small proportion (approximately 4–6%) of heparin molecules possessing a terminal monosaccharide which, on reaction with NaB3H4, is reduced to yield an alditol. Consequently, 3H is incorporated on C1 of this modified terminus. The product, [3H]heparin as the Na salt, is very stable under normal conditions of treatment and storage. The behavior of [3H]heparin when chromatographed on Sepharose-antithrombin III and on Sepharose-thrombin compares closely with that of unlabeled heparin. However, gel filtration on Sephadex G-200 reveals that [3H]heparin ( V e V 0 = 2.19 ) chromatographs more slowly than native heparin ( V e V 0 = 2.08 ), a feature which may reflect the true nature of the constituent molecules present in the heparin sample.

Intimal alterations in rabbit aortas during the first 6 months of alloxan-induced diabetes.

Diabetes mellitus is a major risk factor for atherosclerosis. Since endothelial alteration is probably associated with the development of atherosclerosis, we questioned whether morphological evidence of endothelial injury could be observed during the first 6 months of diabetes induced by a single intravenous injection of alloxan in normally fed rabbits compared with age-matched controls. Diabetes (plasma glucose greater than 16 mM) was established by 5 days after alloxan injection. Endothelial alterations consistent with injury, including adhesion of white blood cells, platelets, and fibrin-like material to the endothelial surface, were seen in diabetic rabbit aortas by 2 weeks. These alterations became more severe during the next 6 months. Increased endothelial replication in diabetic vessels was shown by the uptake of tritium-labeled thymidine at 2 weeks and at 3 and 6 months. Hyperplasia of intimal smooth muscle cells progressed during 3 months after treatment. About one third of the diabetic rabbits also showed an elevated plasma cholesterol level, which correlated with increased intimal proliferation but not with endothelial injury or replication. The onset of alloxan-induced diabetes in rabbits is associated with nondenuding endothelial injury and subsequent intimal hypertrophy, changes that are consistent with atherogenesis.

Antithrombin III-beta associates more readily than antithrombin III-alpha with uninjured and de-endothelialized aortic wall in vitro and in vivo.

The properties of two isoforms, alpha and beta, of rabbit antithrombin III (ATIII) were compared in the presence of undamaged or de-endothelialized rabbit aortic wall. Similar quantities of ATIII-alpha and ATIII-beta bound to and rapidly saturated the endothelium in vitro, but the rate of transendothelial passage of ATIII-beta exceeded that of ATIII-alpha by 22%. Furthermore, ATIII-beta was adsorbed approximately twice as rapidly as ATIII-alpha by the subendothelium of the de-endothelialized aorta. Binding of both isoforms was decreased (ATIII-beta more than ATIII-alpha) by pretreating the subendothelial surface with heparitinase. Also, subendothelium-bound ATIII-beta was desorbed more readily than bound ATIII-alpha by thrombin. In vivo, the rate of uptake of iodine-131-labeled ATIII-beta from the circulation by the aortic wall and the major organs was 30-50% faster than that of iodine-125-labeled ATIII-alpha. In contrast, the uptake of 131I-ATIII-beta by the de-endothelialized aorta in vivo was three times faster than that of 125I-ATIII-alpha. By these criteria, ATIII-beta is the more active of the two isoforms. We surmise that plasma and, consequently, vessel wall levels of ATIII-beta may be vital for controlling thrombogenic events caused by injury to the vascular wall.

Galactose-specific elimination of human asialotransferrin by the bone marrow in the rabbit

Abstract Rabbit bone marrow accretes and degrades human asialotransferrin in vivo through a mechanism that recognizes the exposed galactose groups in this glycoprotein. After the liver, rabbit bone marrow is the second mammalian tissue now being identified as possessing a galactose-specific pathway for the elimination of a plasma protein. However, comparative studies with desialylated glycoproteins containing bi-, tri-, and tetraantennary glycans (asialofetuin, asialoorosomucoid, chicken α 1 -acid glycoprotein, and rabbit transferrin) indicate that the bone marrow recognizes fewer glycan structures than does the liver. Optimal uptake and degradation of an asialoglycoprotein by the bone marrow requires the presence of biantennary glycans.

On the physicochemical and chemical properties of α1-acid glycoproteins from mammalian and avian plasmas

Abstract We have isolated from the plasma of man, rabbit, pig, rat and chicken α1-acid glycoproteins that show a single band on polyacrylamide gel electrophoresis and a single are in immunodiffusion. Amino acid and carbohydrate compositions are presented. The human protein is very similar to preparations described by earlier workers and, although significant differences in amino acid composition exist among them, the proteins from the other species are assumed to be analogous to it. Ultracentrifugal studies, despite showing single, fairly symmetrical peaks at high speed, produced evidence for the physical heterogeneity of each protein in agreement with some earlier reports. Although many models consisting of mixtures of components were tested, none was found that fitted adequately all observations on any one of the glycoproteins.