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Purified antithrombin III has been reported to have bound glucocerebroside, the major glycolipid of plasma. We have separated whole plasma by ultracentrifugation into lipoprotein-rich and lipid-deficient fractions and demonstrated that glucocerebroside and antithrombin III clearly separate into different fractions. Antithrombin III does not have glucocerebroside associated with it.
alpha2-Macroglobulin level, trypsin protein esterase and progressive antithrombin activities were measured in normal and nephrotic sera and plasma. Trypsin protein esterase activity was proportional to the alpha2-macroglobulin concentration in serum and plasma from both normal and nephrotic patients. The results were different, however, with progressive antithrombin activity: in normal plasma, antithrombin III is the main thrombin inhibitor, then alpha2-macroglobulin and alpha1-antitrypsin, whereas in nephrotic syndrome patients, alpha2-macroglobulin is the main thrombin inhibitor.
The alpha-2-protease inhibitor, of 65,000 daltons molecular weight, described by several authors in horse plasma and also present as a contaminant in alpha-1-isoinhibitor isolates previously described by us (Pellegrini & von Fellenberg (1980) Biochim. biophys. Acta 616, 351-361) has now been isolated to purity and identified as antithrombin III. The inhibitor is composed of a single polypeptide chain as judged by SDS polyacrylamide gel electrophoresis. The inhibitor was effective only against trypsin and thrombin. Serological cross-reaction existed between the inhibitor and the antiserum to human antithrombin III. An antiserum to our isolate, however, did not react with human antithrombin III. This confirms the results reported by Kurachi et al. (1976, Biochemistry 15, 368-372).
Antithrombin III activity and plasma fibronectin levels were determined in patients with preeclampsia. In several cases low antithrombin III and high plasma fibronectin concentrations could be related to proteinuria. Altered plasma fibronectin and antithrombin III concentrations might contribute to a hypercoaguable state in patients with preeclampsia.
Heparin cofactor II (HC II) has been purified from human plasma by a modification of the method described by Tollefsen et al. (J. Biol. Chem., 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 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.
It has been reported that patients with preeclampsia have decreased levels of antithrombin III heparin cofactor and that the degree of depression parallels the severity of disease. To determine the time course of development and resolution of this deficiency, and to gain insight into the cause of this deficiency, plasma antithrombin III antigen and heparin cofactor activity levels were measured serially in 11 women with mild preeclampsia. Although the ATIII antigen level remained stable during the antepartum period, the level of heparin cofactor was noted to fall progressively during the antepartum period. Both ATIII antigen and heparin cofactor increased promptly postpartum. The discrepancy between the levels of ATIII antigen and heparin cofactor activity suggests that in both mild and severe preeclampsia there is utilization of antithrombin III to form inactive protease inhibitor complexes.
The clearance of thrombin-antithrombin (TAT) complexes from blood by the liver is through a receptor mediated pathway. We have used the established human hepatoma cell line, Hep G2, to determine if these hepatocytes have the capacity to bind this enzyme-inhibitor complex. The TAT complex was bound to the cells in a time and temperature dependent manner, reaching an apparent steady state at 90 minutes at both 4 and 37 degrees C. Binding at 4 degrees C was 5-7-fold less extensive than at 37 degrees C. The bound TAT was structurally similar to the added ligand. This interaction was specific, as it was inhibited by nonlabeled TAT but not by 50-fold molar excesses of unrelated proteins or by the individual constituents, thrombin or antithrombin. Factor Xa- antithrombin complex inhibited the binding reactions slightly. Specific binding isotherms at 37 degrees C were subjected to Scatchard plots. The apparent dissociation constant was 247 +/- 74 nM, and the number of TAT molecules bound per cell was 5.19 +/- 0.89 X 10(5). Bound TAT complexes did not undergo degradation at 4 or 37 degrees C for up to 2.5 hr, as greater than 85% of the bound ligand was acid precipitable during the time course of binding. Internalization of the TAT complex was compared with transferrin, a molecule known to be internalized by Hep G2 cells, by resistance of the cell-bound ligands to degradation by trypsin or pronase. In contrast to transferrin, most of the TAT complexes remained cell-surface associated for at least 2 hr at both 4 degrees and 37 degrees C, indicating that TAT was not substantially internalized by the Hep G2 cells.
The influence of increasing concentrations of heparin of different molecular mass (Mr) has been compared in potentiation of the rate of heparin cofactor II:thrombin interaction and of antithrombin III:thrombin interaction. Unfractionated and fractionated heparin showed a concentration dependent ascending and descending limb of stimulation of the rate for both inhibitors. Unfractionated heparin and fractions of 16.5 KDa or less showed a peak acceleration of the rate of interaction of thrombin with both inhibitors at 0.3 X 10(-6) M heparin although the observed maximum rate at this peak decreased with fall in Mr. For both inhibitors two high Mr fractions showed peak stimulation at a lower heparin concentration (0.3 X 10(-7) M) and approximately two-fold greater increase in rate than that observed with unfractionated heparin. Potentiation of heparin cofactor II inhibitory activity differed from that of antithrombin III in that it was reversed by lower ionic strength and was not reversed by a heparin pentasaccharide with high affinity for antithrombin III. It is proposed that differences in the profiles of stimulation by high Mr fractions to those of lower Mr are related to higher binding affinities for the inhibitor permitting maximal binding of heparin before the descending part of the slope due to saturation of thrombin (according to the template hypothesis).
The abnormal Antithrombin-III (AT-III) Toyama has no ability to interact with heparin in the mast cells or heparin-like glycosaminoglycans on the surface of vascular endothelial cells, so that it has low heparin cofactor activity. In the present study, we have measured the binding capacity of 125I-labeled AT-III to cultured porcine aortic endothelial cells and estimated the inactivation of thrombin by AT-III in the presence of endothelial cells. The abnormal AT-III Toyama had low binding capacity to endothelial cells and had lower heparin cofactor antithrombin activity in the presence of endothelial cells than that of normal AT-III. Our results suggested that heparin-like glycosaminoglycans on endothelial cells play as important a role as heparin does in the regulation of antithrombin activity.
According to the reaction conditions selected, chemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5 nitrobenzyl) sulfonium bromide (HNBSB) generated products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl (HNB) incorporated/mole of antithrombin III) but with high or low affinity for heparin. These products were subjected to digestion by cyanogen bromide and shown to be modified equivalently in fragment II containing Trp 189 and Trp 225 and fragment III containing Trp 49. The molar level of incorporation of HNB into these fragments was similar in the high and low affinity forms. Both high and low affinity forms showed loss of heparin cofactor activity. A recovery of heparin cofactor activity towards coagulation factor Xa was observed upon prolonged storage of low affinity forms at -70 degrees C. It is considered that the loss of high affinity for heparin upon modification of antithrombin III arises from change or stabilization of conformation associated with tryptophan modification and is not a singular property of modification of Trp 49.
The mechanism of antithrombin action of a fucan sulfate (C-II), which was isolated from the brown seaweed Ecklonia kurome, was examined by clotting method using a thrombin-fibrinogen system and by amidolytic method using a chromogenic substrate in the presence and the absence of antithrombin III (AT III) or heparin cofactor II (HC II). C-II significantly inhibited the clotting of fibrinogen by thrombin even in the absence of the protease inhibitors, and the amidolytic activity of the protein only in the presence of HC II. C-II was not adsorbed on an AT III-agarose column and its anticoagulant activity in AT III-depleted plasma was the same as that in normal one. Examination of interaction of C-II with fibrinogen by gel filtration chromatography demonstrated that C-II bound to the protein. These results indicated that the antithrombin activity of C-II was mediated by HC II and not by AT III, and that the polysaccharide bound to fibrinogen, thereby blocking thrombin action, and also that its direct thrombin inhibition was very weak.
We investigated heparin cofactor II (HC II) levels and their relationship to other haemostatic factors in the elderly in comparison with antithrombin III (AT III). We measured plasma HC II activity levels in 166 subjects aged from 61 to 99 years using a chromogenic method. HC II levels (94.4 +/- 18.5%) in the healthy elderly subjects were significantly (p less than 0.001) lower than in 40 healthy adult controls under 60 years of age (mean age: 51.5 years; 111.6 +/- 21.2%). HC II levels in the elderly subjects decreased further with age (r = 0.308, p less than 0.001) and the extent of the decrease was more marked than that for AT III (r = 0.179, p less than 0.05). There was no significant sex difference in HC II levels in the elderly. HC II levels correlated significantly with AT III levels and with acute phase reactants including sialic acid, fibrinogen, and PAI-1. HC II levels also correlated with factor VII, plasminogen, alpha 2-plasmin inhibitor, serum lipid, pseudocholinesterase, and albumin levels. These correlations were also found for AT III except active PAI-1 and tPA-PAI-1 complexes, but the correlations with acute phase reactants were stronger for HC II than AT III. We divided 154 elderly subjects into 4 groups by their pseudocholinesterase and albumin levels to estimate the effect of nutritional status on antithrombin activity in the elderly. HC II levels were normal in the elderly subjects with a good nutritional state (103 +/- 18%), but were significantly decreased in those with malnutrition (85 +/- 15%, p less than 0.001). AT III levels also showed the same tendency. These results indicate a decrease in the reserve capacity to inhibit thrombin generation at sites of atherosclerosis in response to trigger events. The deficiency of two major antithrombin factors in the elderly may indicate a tendency to thrombosis, especially in individuals with malnutrition. When considering the clinical significance of HC II, several other parameters, including age, nutritional status, hepatic synthetic ability, and the presence or absence of acute phase reaction should also be assessed.
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.
Commercially obtained (Diosynth) heparin was covalently bonded to poly (vinyl alcohol) (PVA) hydrogels and to polyethylene oxide (PEO) hydrogels activated by tresyl chloride. We found that as tresyl chloride activation of PVA increased, the specific activity of the bound heparin toward thrombin and antithrombin decreased by nearly a factor of 10 and that commercial heparin bound to PEO had nearly ten-fold greater activity than when bound to PVA at comparable concentrations. These findings suggest that the long 'leash' provided by PEO hydrogels may give the heparin more access to the thrombin-antithrombin pair than the tight bond to PVA, and that crowding of heparin units on a surface limits access of the thrombin-antithrombin pair.
T-lymphocytes contain a cytoplasmic granule associated homo-dimeric protease designated granzyme A. Upon T-cell target cell interaction, the granules undergo exocytosis and granzyme A, and other granule constituents, are released. Here we show that granzyme A secreted into plasma is immediately inactivated by antithrombin III. The rate of complex formation is enhanced 400-fold in the presence of heparin. Two different complexes are generated: granzyme A-antithrombin III and granzyme A-(antithrombin III)2, respectively, indicating that both active centers of granzyme A are functional. Thus, the proteolytic activity of lymphocyte protease granzyme A, whose physiologically relevant function is unknown, is well regulated in plasma.
Derivatives of human thrombin and antithrombin III with fluorescent labels covalently attached to their carbohydrate moieties were prepared by reaction of periodate-oxidized proteins with amino derivatives of dansyl, fluorescein and pyrene. The labeled derivatives retained full biological activity, including their ability to form stable enzyme-inhibitor complexes, a reaction whose rate could be monitored by the increase in fluorescence polarization. When the dansyl-labeled derivatives were heated, they exhibited sigmoidal increases in polarization with midpoints near 50 degrees C for thrombin and 60 degrees C for antithrombin III. By contrast, a complex between antithrombin III and dansyl-thrombin showed no change in polarization up to 70 degrees C, suggesting that the individual components are more stable in the complex. These studies show that fluorescent labels attached to carbohydrate moieties of glycoproteins provide convenient probes for monitoring conformational changes and protein-protein interactions with minimum interference by the probe.
Livers isolated from control or turpentine-injected rats were perfused for 3 h with human red cells suspended in Krebs-Henseleit solution containing bovine serum albumin, dextran, glucose, heparin, cortisol, insulin, a mixture of 20 amino acids and [3H]leucine. Changes in the concentrations of antithrombin III and alpha-1-antitrypsin were evaluated by rocket immunoelectrophoresis using specific antisera, and incorporation of the 3H radioactivity into the total protein, albumin, antitrhombin III and alpha-1-antitrypsin in the perfusate was measured. The results indicate that both antithrombin III and alpha-1-antitrypsin are synthesized in the liver. Local inflammation induced in the liver donors moderately stimulated the synthesis of alpha-1-antitrypsin but it affected only marginally that of antithrombin III.
The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.