Isotopic studies of therapeutic anticoagulation with a coagulating enzyme.

The kinetics of the depletion of plasma fibrinogen were studied in seven patients who received fibrinogen-(131)I 1 hr before an intravenous injection of the coagulating enzyme (CE) derived from the venom of the pit viper, Agkistrodon rhodostoma. Disappearance of the clottable radioactivity labeled fibrinogen from the circulation conformed to an exponential decay with an average half-life of 0.85 hr. The mean clearance rate for protein-bound radioactivity, composed of fibrinogen and it's split products, was 12% of the intravascular pool per hour. The breakdown products of fibrin produced by CE inhibited polymerization of fibrin in vitro. Studies in five patients performed between the 3rd and 10th day following the administration of CE revealed that the absolute catabolic rates of fibrinogen were subnormal initially, but gradually increased as the fibrinogen concentration returned to normal. In rabbits, after the administration of CE, regeneration of the fibrinogen pool was markedly prolonged. This delayed regeneration time was not influenced by an excess of antivenene, but rapid regeneration to pretreatment values of plasma fibrinogen was immediately initiated by stimulating fibrinogen synthesis with subcutaneous turpentine.

Platelet interaction with polymerizing fibrin.

Interaction of washed pig, rabbit, or human platelets with fibrinogen was studied during its transition to fibrin using photometric, isotopic, and electron microscopic techniques. Untreated fibrinogen and fully polymerized fibrin had no detectable effect on platelets. Fibrinogen, incubated with low concentrations of reptilase or thrombin, formed intermediate products which readily became associated with platelets and caused their aggregation. Neutralization of the thrombin did not prevent this interaction. In the absence of fibrinogen, reptilase did not affect platelets. The interaction of polymerizing fibrin with platelets was accompanied by small losses of platelet constituents (serotonin, adenine nucleotides, platelet factor 4, and lactic dehydrogenase). This loss did not appear to be the result of the platelet release reaction. Inhibitors of the release reaction or of adenosine diphosphate (ADP)-induced aggregation did not prevent the interaction of platelets with polymerizing fibrin. Apyrase or prostaglandin E(1) (PGE(1)) reduced the extent of platelet aggregation by polymerizing fibrin, but the amount of protein associated with platelets was slightly increased. The interaction of polymerizing fibrin with platelets was completely inhibited by ethylenediaminetetraacetate (EDTA) or ethylene glycol bis (beta-aminoethyl ether) N, N,N',N'-tetraacetic acid (EGTA).Fibers formed in solutions of polymerizing fibrin were larger in the presence than in the absence of washed platelets, suggesting that platelets affect fibrin polymerization. The adherence of platelets to polymerizing fibrin may be responsible for the establishment of links between platelets and fibrin in hemostatic plugs and thrombi.

Resolution of experimental pulmonary emboli with heparin and streptokinase in different dosage regimens.

Thrombolytic agents may be useful in acute pulmonary embolism, but their optimal dosage remains uncertain. We have examined the relative efficacy of heparin and different doses of streptokinase, either alone or in combination, in acute experimental pulmonary embolism. A standardized massive embolus of autologous blood clot incorporating canine [(125)I]-fibrinogen was given to 40 dogs; the degree of resolution after 24 h was quantitated by measuring the radioactivity in the lungs and was compared with detailed postmortem observations. The amount of residual embolus was 49% in control animals, 28% after heparin (200 U/kg loading dose and 800 U/kg/24 h maintenance dose), and 6% after high dose streptokinase (250,000 U loading dose and 100,000 U/h maintenance dose); it was 31% after low dose streptokinase (25,000 U loading dose and 10,000 U/h maintenance dose), 7% after low dose streptokinase with heparin, 14% after very low dose streptokinase (5,000 U/h without a loading dose) with heparin, and 9% after short course streptokinase (250,000 U loading dose and no maintenance dose) with heparin. The combination of heparin and low doses or brief courses of streptokinase appeared to be synergistic and produced as much resolution as did standard high dose streptokinase alone. The enhanced resolution of pulmonary emboli in heparin-treated animals may have been due to the prevention by heparin of further deposition of fibrin on the embolus. It appears that dosage regimens of thrombolytic therapy other than those in current use may be worthy of clinical examination.

The relevance of the structure of lysine bound to Sepharose for the affinity of rabbit plasminogen;.

The features of the structure of lysine, linked to Sepharose by the alpha-amino group, which are important for affinity chromatography of rabbit plasminogem were studied. Nine lysine and lysine-like conjugates, including epsilon-aminohexanoic acid DL-norleucine, DL-alpha-aminoadipic acid, DL-alpha-epsilon-diaminopimelic acid, cadaverine L-ornithine, L-arginine and D-lysine, were prepared; Using labelled rabbit plasminogen added to plasma, the ability of each conjugate to absorb plasminogen and separate the allomeric forms, type I and type II, during epsilon-aminohexanoic acid gradient elution was compared to Sepharose-L-lysine. Plasminogen had no affinity for Sepharose-epsilon-aminohexanoic acid, and was only weakly attracted by Sepharose-norleucine, Sepharose-cadaverine and others. Sepharose-ornithine held a greater attraction to the protein but the strongest binding was obtained with Sepharose-arginine. The affinity of plasminogen type I was always less than type II for the Sepharose-lysine analogues and the recovery of type II greater than type I from Sepharose-ornithine and Sepharose-arginine. Plasminogen affinity was in the order of Sepharose-arginine greater than Sepharose-lysine greater than Sepharose-ornithine. However, because of the present difficulty in recovering plasminogen from Sepharose-arginine the use of Sepharose-lysine in the affinity chromatography of rabbit plasminogen remains unchallenged. It is concluded that binding of rabbit plasminogen to conjugates of lysine and its analogues is determined by the presence of both a free carboxyl and a free amino group and that the distance between these groups is critical.

The effect of heparin on the affinity chromatography of plasminogen. Demonstration of heparin-plasminogen interaction.

Evidence is presented that heparin binds rabbit plasminogen types I and II under affinity chromatographic conditions using the single stage technique earlier described (Hatton, M.W.C. and Regoeczi, E. (1974) Biochim. Biophys. Acta 359, 55-65). Thus, the affinity of types I and II for Sepharose-lysine is markedly increased in the presence of heparin and elution by epsilon-aminohexanoic acid requires a steeper gradient to recover the plasminogen types. Furthermore by adding sufficient epsilon-aminohexanoic acid to non-heparinised plasma to suppress plasminogen affinity, the presence of heparin is shown to encourage binding of plasminogen (type II more so than type I) to the gel. However, the heparin effect is quickly reversed by washing the column with 0.5 M NaCl prior to elution by epsilon-aminohexanoic acid. No evidence of a stable plasminogen-heparin complex has been found from gel filtration studies and any interaction between plasminogen and heparin probably only takes place when heparin is bound to an affinity site. Studies with 35-S-labelled heparin have shown the mucopolysaccharide to bind to the free amino group of Sepharose-lysine and Sepharose-cadaverine and to be displaced by 0.5 M NaCl elution but not by 0.1 M epsilon-aminohexanoic acid. The plasminogen types produced from heparinised plasma are free from heparin and closely resemble preparations from non-heparinised plasma when compared by polyacrylamide gel electrophoresis, Sephadex gel filtration and arginine esterase activity after urokinase activation.

The affinity of human, rabbit and bovine thrombins for sepharose-lysine and other conjugates.

Human, rabbit and bovine thrombins are shown to possess marked affinities for Sepharose-lysine. Using either Xa-activated crude prothrombins (human, rabbit) or a commercial thrombin sample (bovine), the enzyme was isolated in a single chromatographic step by the affinity medium and preparations of high specific activity were obtained. The relevance of bound-lysine for the affinity of the thrombins was studied using other Sepharose conjugates with structures related to Sepharose-lysine. Using freshly activated prothrombins it was found that human and rabbit thrombin uptake required a conjugate with a spacer chain containing a minimum of four carbon atoms in length which supported a terminal amino group. As the thrombin activity aged, affinity for the terminal amino group decreased but the hydrophobic spacer chain became essential for enzyme binding. The active centre of thrombin was not involved in binding to Sepharose-lysine.

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.

Hepatic uptake and degradation of trace doses of asialofetuin and asialoorosomucoid in the intact rat.

Asialoorosomucoid and asialofetuin were prepared by using sialidase, which was removed chromatographically before the proteins were labelled with radioactive iodine. After intravenous administration of a small amount oa asialoglycoprotein (3--4 microgram/100 g body wt.) protein-bound and non-protein radioactivities in plasmas and livers of rats were determined at intervals over a period of 30 min. Transfer of either tracer protein from plasma to liver was almost complete in 5 min. Proteolysis of asialofetuin was evident very shortly thereafter, but degradation of asialoorosomucoid commenced after a significant delay and was initially slow relative to that of asialofetuin. Studies in vitro with crude hepatic lysosomal enzyme preparations indicated that asialoorosomucoid was less readily digested than asialofetuin, and that desialylation of orosomucoid or fetuin did not noticeably increase the susceptibility of these proteins to protease action. Proteolysis of asialofetuin was also demonstrable in liver homogenates in conditions under which albumin and asialotransferrin were stable. A generalized mathematical model was devised to represent the uptake and degradation of asialoglycoproteins by the liver. The theoretical assumptions that gave the best fits with experiment are outlined and discussed.

The physicochemical and chemical properties of alpha 1-acid glycoproteins from mammalian and avian plasmas.

We have isolated from the plasma of man, rabbit, pig, rat and chicken alpha 1-acid glycoproteins that show a single band on polyacrylamide gel electrophoresis and a single arc 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. Ultra centrifugal 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.

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man.

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.

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.

Synthesis of antithrombin III and alpha-1-antitrypsin by the perfused rat liver.

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.

Metabolic stability of the fucose in rat transferrin.

The metabolic behaviour of the chitobiose core fucose that is a natural constituent of a large proportion of rat transferrin molecules was studied in rats comparatively to that of the polypeptide portion of the glycoprotein by using appropriate labels ([3H]fucose and 125I) and affinity chromatographic techniques (lentil-Sepharose). No evidence was obtained to suggest that this residue is cleaved from the glycan in significant amounts before removal of the entire glycoprotein for catabolism. Similarly, [14C]fucose linked to GlcNAc residues in the antennae of human asialotransferrin was being eliminated in pigeons at the same rate as the polypeptide itself. It is concluded that in spite of transferrin's exposure to the cellular milieu, the fate of its fucose is distinctly different from that of the same in plasma membrane glycoproteins.

A simple bioassay for monocyte-derived hepatocyte stimulating factor: increased synthesis of alpha 2-macroglobulin and reduced synthesis of albumin by cultured rat hepatocytes.

Cytokines released from monocytes upon stimulation by lipopolysaccharide cause a number of cells to undergo proliferative and synthetic changes. At least one of these cytokines affects hepatocytes in vivo causing increased synthesis of a series of acute-phase proteins. We have established an in vitro micro-assay for hepatocyte stimulating factor (HSF) using primary cultures of normal rat hepatocytes. Measurement of increased synthesis of alpha 2-macroglobulin and decreased synthesis of albumin caused by exogenously added factor constitute a sensitive parameter for quantifying HSF. For comparing various cytokines preparations, we have defined a unit of HSF activity in terms of a stimulation index. We have used this assay to follow some preliminary attempts to isolate the factors responsible for stimulation of synthesis of acute-phase reactant by the liver.

Alpha-1-acute phase globulin in the blood of tunicamycin-injected rats. Isolation of the non-glycosylated form, its inhibitory properties and synthesis in liver slices.

Plasma of rats injected with tunicamycin (2 micrograms/g body wt) shows on crossed immunoelectrophoresis the presence of an additional, slowly migrating component of alpha 1-acute-phase globulin (alpha 1-AP-globulin). The native and modified forms of alpha 1-AP-globulin were jointly isolated on the column of immobilized antibodies and then separated by chromatofocusing on polybuffer exchanger. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate demonstrated that tunicamycin-induced form of rat alpha 1-AP-globulin has Mr of 50,000 and is devoid of carbohydrates as inferred from the lack of staining with Schiff reagent. However, during incubation with papain in vitro it is only slightly less effective than mature glycosylated alpha 1-AP-globulin (Mr 68 000) in inhibiting hydrolysis of CBZ-Lys-ONp. Incubation of liver slices from control and tunicamycin-injected rats with 14C-leucine demonstrated that tunicamycin reduces synthesis and release to the medium of alpha 1-AP-globulin and some other plasma proteins, but the proportion of aglyco-alpha 1-AP-globulin is higher than in plasma.