Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate,and its mechanism

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Inhibition by heparin and dextran sulfate of stimulated rat pancreatic adenylate cyclase

Heparin inhibited the adenylate cyclase activity of semipurified rat pancreatic plasma membranes stimulated by hormones and by Gpp(NH)p but not by fluoride or when in the persistently active state. When observed, the inhibition was rapid and sustained. It was of a noncompetitive type and never exceeded 20% for secretin. The inhibition of Gpp(NH)p-stimulated activity was more pronounced (48% inhibition at a heparin concentration of 50 μg/ml). For the C-terminal octapeptide of pancreozymin (CCK-8)-stimulated adenylate cyclase, the inhibition amounted to 93% at 50 μg/ml. This inhibition was competitive at low heparin concentration and of a mixed type above 10 μg/ml. Besides, heparin inhibited (I₅₀ = 6 μg/ml) the binding of peptides of the CCK family to their specific receptors without affecting the apparent K_d value of binding. Taken together, these relatively specific effects of heparin gave evidence in favor of the existence of CCK spare receptors. Dextran sulfate was more potent than heparin as an inhibitor of adenylate cyclase activation while chondroitin-4-sulfate and chondroitin-6-sulfate were ineffective. Dansylated pancreatic plasma membranes exhibited characteristics of adenylate cyclase activation by CCK-8 which were similar to those found for untreated membranes exposed to heparin.     

Inhibition of factor VIII-associated platelet aggregation by heparin and dextran sulfate, and its mechanism.

Both ristocetin-induced aggregation in the presence of human factor VIII and bovine factor VIII-induced aggregation of washed normal human platelets were inhibited or reversed by the addition of heparin or dextran sulfate. These actions of dextran sulfate were stronger than those of heparin, and dependent on the sulfur content of dextran sulfate. In order to study the mechanism of actions of dextran sulfate and heparin, the affinity chromatographic experiment of factor VIII in human and bovine plasma, respectively, was carried out by using a dextran sulfate- and a heparin-Agarose column. Both human and bovine factor VIII have a strong affinity for dextran sulfate with high sulfur content and a weak affinity for heparin, but no affinity for dextran sulfate with low sulfur content. From these results, it is suggested that dextran sulfate or heparin binds directly the human and bovine factor VIII, which is an essential factor for the maintenance of the weak interplatelet bonds, and either inhibits or reverses the platelet aggregation.     

Inhibition of T4 polynucleotide kinase by the ATP analog, beta, gamma-imidoadenylyl 5'-triphosphate.

The inhibition of T4 polynucleotide kinase by beta,gamma-imidoadenylyl 5'-triphosphate has been investigated. It was found that the ATP analog was a competitive inhibitor with regard to ATP and a noncompetitive inhibitor with regard to DNA possessing a 5'-hydroxyl group. At pH 8.0, the Ki values were 3 and 11 mM, respectively. beta,gamma-imidoadenylyl 5'-triphosphate was not a substrate in the forward reaction, but would replace ADP and ATP in the reverse reaction. The reverse reaction was also used to make beta,gamma-imidoadenylyl 5'-tetraphosphate.     

Inhibition of fibroblast growth by polyanions; effects of dextran sulfate and lignin derivatives.

When macrophages prepared from rat bone marrow were cultured for more than one week, contaminating fibroblasts grew and formed large colonies which were observable with the naked eye after Giemsa staining. The addition of dextran sulfate or water-soluble lignin derivatives (acetyl or sulfonyl) to the culture medium almost completely inhibited the growth of fibroblasts at 10 micrograms/ml. These polyanions also inhibited the growth of chick embryo fibroblasts in a dose-dependent manner: at 5 micrograms/ml the inhibitory effect was 20-30% of the control and at 100 micrograms/ml it was 50-70%. In addition, these polyanions inhibited the mitosis of hepatocytes, although their inhibitory effects on the growth of hepatocytes were smaller than those observed on the growth of fibroblasts. On the other hand, these polyanions did not affect the growth of human urinary bladder carcinoma cells (HUB-4 and HUB-15). In conclusion, dextran sulfate and lignin derivatives may be useful in eliminating fibroblasts from certain cells.     

Inhibition of cyclic AMP dependent protein kinase by vanadyl sulfate

Vanadium salts influence the activities of a number of mammalian enzymes in vitro but the mechanisms by which low concentrations of vanadium ameliorate the effects of diabetes in vivo remain poorly understood. The hypothesis that vanadium compounds act by inhibiting protein tyrosine phosphatases has attracted most support. The studies described here further evaluate the possibility that vanadyl sulfate trihydrate (VS) can also inhibit 3′,5′-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA). Using conventional assay conditions, VS inhibited PKA only at high concentrations (IC₅₀>400 μM); however, PKA inhibition was seen at dramatically lower concentrations of VS (IC₅₀<10 μM) when sequestration of vanadyl ions was minimized. Vanadyl appears to be the effective PKA inhibitor because sodium orthovanadate did not inhibit PKA and inhibition by vanadyl was abolished by potential chelators such as ethylenediaminetetraacetic acid or glycyl peptides. PKA inhibition by vanadyl appears to be mixed rather than strictly competitive or uncompetitive and may replicate the inhibitory effects of high concentrations of Mg²⁺. The effect of vanadyl on PKA provides a possible explanation for the effects of vanadium salts on fat tissue lipolysis and perhaps on other aspects of energy metabolism that are controlled by cAMP-dependent mechanisms. Considering the high degree of conservation of the active sites of protein kinases, vanadyl may also influence other members of this large protein family. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Preparation and properties of several polysaccharide sulfates

Amylose, amylopectin, guaran, cellulose, and laminaran are sulfated to different degrees with very little degradation in dimethylformamide with triethylamine-sulfur trioxide complex at 0 °. A 1 → 6 linked dextran and alginate are unreactive.Periodate oxidation of the polysaccharide sulfates of degree of substitution of one suggests that half of the sulfuric ester groups may be on position C-6. Amylose, amylopectin, and guaran sulfates precipitate solutions of gelatin, egg albumin, and methylene blue but not human plasma. They form films but absorb large amounts of moisture and readily disperse in water. Unlike carrageenan their solutions do not gel with potassium or ammonium ions.Sulfuric ester groups are readily eliminated from amylopectin sulfate but not from laminaran sulfate.     

Stimulation of extramedullary hemopoiesis by dextran sulfate

The spleen of normal dogs (beagles) shows only slight hemopoietic activity, characterized by the presence of megakaryocytes in mitosis and small groups of erythroblasts scattered throughout the red pulp of the organ. Repeated intravenous injection of dextran sulfate, at a dose of 15 mg per kg body weight, produced markedly enhanced erythropoietic and megakaryocytopoietic activity in the splenic red pulp, without concomitant increase in splenic granulopoiesis. The probable existence of a micro-environment adequate for erythro- and megakaryocytopoietic differentiation of stem cells is discussed.     

Activation of calcium-ATPase of liver plasma membrane by dextran sulfates.

Dextran sulfate (DS) with average molecular weight (AMW) of 20,000 and sulfur content of 18%, which has a high lipemia clearing activity, enhanced Ca²⁺ binding to the plasma membrane of rat liver, and the DS itself bound the membrane, whereas there was little binding of DS and Ca²⁺. Various DSs slightly activated Na⁺-K⁺-ATPase, but not Mg²⁺-ATPase activity of the membrane. These results suggest that DSs, especially with high AMW of 20,000, bind the plasma membrane, resulting in enhancements of the Ca²⁺ binding to there and Ca²⁺-ATPase activity.     

Inhibition of alpha-fetoprotein synthesis in hepatocytes of adult mice by dextran sulfate in vivo and in vitro

Embryospecific serum protein alpha-fetoprotein (AFP) is known to be synthesized in the adult liver only during regeneration and development of hepatocellular carcinomas. It was shown that collagenase digestion of hepatic tissue followed by monolayer cell cultivation was a powerful inducer of AFP synthesis, more potent than the liver regeneration in vivo. The treatment of hepatocytes in culture with 50-100 micrograms/ml of dextran sulphate caused a remarkable inhibition of cell proliferation, formation of cord-like multicellular structures and reduction of AFP synthesis. Mouse liver regeneration after CCL4 poisoning was accompanied by a 1000-fold increase in blood AFP levels. Blood AFP levels and the content of AFP-positive cells in the liver tissue were maximum on the 3rd-4th day after poisoning. Injections of 50 micrograms of dextran sulphate per g body weight 3-5 h after poisoning and 24 and 48 h later caused nearly tenfold reduction in AFP blood level and a decrease in the content of AFP-positive cells in the liver on the 3rd day of regeneration.     

Inhibition of dextran synthesis by glucoamylase and endodextranase

In a model experiment, glucoamylase was shown to inhibit α-D-glucan synthesis as catalyzed by potato phosphorylase. Both glucoamylase and endodextranase inhibited dextran synthesis with dextransucrases of Leuconostoc mesenteroides. The inhibition could be ascribed to competition between glucoamylase and dextransucrase for the glucosyl groups at the non-reducing end of dextran. The inhibition caused by endodextranase may result from rapid and random hydrolysis of acceptor dextrans. Moreover, significantly low units of glucoamylase, as compared with endodextranase, effectively inhibited dextran synthesis. These results thus present evidence that bio-synthesis of dextran occurs by the addition of glucosyl groups at the non-reducing end of the growing dextran. The measurement of initial velocity suggested that the ping-pong Bi-Bi mechanism proposed for the levansucrase of Bacillus subtilis is also applicable to dextransucrase.     

RNA with polynucleotide kinase properties, isolated by in vitro selection

Data on isolation from a large pool of RNAs of a fragment characterized by high-affinity ATP binding are reviewed. This ATP-binding domain flanking the regions of randomly sequenced nucleotide residues was used for preparation of an RNA pool, from which ribozymes displaying a polynucleotide kinase activity were isolated. The isolated ribozymes catalyzed the transfer of gamma-thiophosphate from ATP-gamma S to the 5' hydroxyl or to internal 2'-hydroxyls of their own chains. ATP was also used as a donor of phosphate; however, in this case the reaction rate was 55-300 times lower. Similarly to a true enzyme, one of these ribozymes shortened by 40 nucleotide residues at the 5' end repeatedly catalyzed the transfer of thiophosphate or phosphate to the 5' hydroxyl of an exogenous oligonucleotide.     

Diffusion of sucrose and dextran through agar gel membranes

Mass transfer limitations severely impede the performance of bioreactions involving large molecules by gel-entrapped microorganisms. This paper describes a quantitative investigation of such diffusional limitations in agar gel membranes. Sucrose and commercial dextran fractions with (weight-average) molecular weights ranging from 10,000 to 2,000,000 Da were used as standard diffusants. For all tested solutes but sucrose, the values of the agar/water partition coefficients highlighted steric hindrance at the entrance of the membrane pores. The effective diffusivity of sucrose in agar was similar to that in water. All dextran fractions, however, displayed restricted diffusion in the agar membranes. Their effective diffusivities were a decreasing function of the agar content of the gel membrane (0.5, 1.0, or 1.5% w/v). The effective diffusivity in a given membrane decreased as the molecular weight of the diffusing molecule increased. T500 (ucbar|M_w = 470,000 Da) and ucbar|M_w = 1,950,000 Da) fractions were unable to diffuse through 1.0 or 1.5% agar membranes. The diffusion data did not agree with the classical (Renkin) model for a hard sphere diffusing through a cylindrical pore. These results are discussed in terms of gel and diffusant characteristics.