Effect of heparin on the activation of the anticoagulation system by alpha-thrombin

Heparin-regulated alpha-thrombin ability to activate the response of the anticoagulation system has been studied by the perfusion of sinocarotid area of rabbits with DIP-alpha-thrombin-heparin complex. In a series of experiments the area was perfused with 1.8 micron DIP-alpha-thrombin and significant changes in anticoagulation parameters have been registered in systemic circulation. During perfusion of sinocarotid area by DIP-alpha-thrombin-heparin complex (2 microns) no activation of anticoagulation system was noted. DIP-alpha-thrombin-heparin perfusates contained no endogenic heparin, unlike DIP-alpha-thrombin perfusates. This confirms the absence of anticoagulation system response to DIP-alpha-thrombin. Control perfusion by heparin alone in equimolar concentrations revealed no changes in anticoagulation system. It is assumed that heparin, blocking cation subcentre of the recognition centre for high molecular compounds in the enzyme molecule, prevents the response of anticoagulation system, disturbing the enzyme ability to bind to specific receptors of the vascular walls.     

Heparin secretion by mast cells as an index of the status of the anticoagulant system

The status of the mast cell population was studied and compared after administration of trypsin or alpha-thrombin in similar molar concentrations. Morphometry disclosed a substantial shift of the mast cell population towards light, heparin-free cells within one minute after alpha-thrombin administration. The index of mast cell saturation with heparin dropped below 1. The maximal heparin secretion was observed at the 5th minute of experiment. The morphometric criteria of the mast cell population returned to basal level in 120 minutes. These data along with a significant increase in the level of complex heparin compounds and plasma thrombin time indicate heparin release as a result of the effector action of the anticoagulation system. No changes were observed in the activity of complex heparin compounds and in thrombin time after intravenous injection of trypsin. It is suggested that high heparin secretion by mast cells may serve as criterion of the active status of the anticoagulation system.     

Complex Compound of Heparin and Glutamic Acid: Synthesis and Effect on Hemostatic Indices in vitro and in vivo

A complex compound of high-molecular heparin and glutamic acid has been synthesized. This compound has anticoagulant, fibrinolytic, and antithrombotic properties in vitro. Single intravenous or chronic peroral introduction of the complex in normal animals imitates the activation of anticoagulation system. When the anticoagulation system in depressed, the complex restores or even activates the anticoagulant–fibrinolytic background of the serum it. The obtained data are discussed in physiological terms.     

Prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin administration

The possibility of prevention of intravascular blood coagulation in rats by DIP-alpha-thrombin devoid of proteolytic activity and capable of stimulating the reaction of anticoagulation system was studied. The injection of lethal thromboplastin dose was shown to produce a sharp increase in soluble fibrin blood content, total disappearance of fibrinolytic activity and intravascular blood coagulation. The animals died of thrombosis in 90% of cases. It was established that the injection of lethal thromboplastin dose 5 min after DIP-alpha-thrombin injection caused a 13% lethality from thrombosis. No reliable changes in fibrinolytic activity and soluble fibrin content were observed. A significant increase in thrombin and recalcification time was recorded. It is suggested that DIP-alpha-thrombin prevents intravascular blood coagulation induced by lethal thromboplastin dose due to mobilization of the reserve capacities of neuro-humoral anticoagulation system.     

Mathematical simulation of the blood coagulation system

The Blood coagulation system was converted to a mathematical model which was described using differential equations. The calculated output patterns of the mathematical model against various input stimulations were compared with results of in vitro assay. The simulated results of (1) Hemophilia A, (2) anticoagulation effect of antithrombin III and (3) anticoagulation effect of heparin corresponded to the results of in vitro assay and clinical reports. However, the simulated result of (4) anticoagulation effect of synthesized arginine derivative No. 805 (MD-805) did not correspond to the results of in vitro assay. Therefore, a new series simulation of MD-805 was done, supposing that MD-805 had an inhibitory activity not only on coagulation factor IIa but also VIIa. The new simulation pattern closely resembled the results of in vitro assay. From these facts, it was theoretically indicated that MD-805 also has an inhibitory activity on VIIa.     

Antithrombin III inhibition of the reaction to thrombin excitation of the anticoagulation system

It has been established that intravenous administration of alpha-thrombin-antithrombin III preparations (1 mkM) has practically no effect on anticoagulation parameters (thrombin time, additive fibrinolytic activity, nonenzymatic fibrinolysis and nonenzymatic fibrinolytic activity). Administration of 1 mkM of alpha-thrombin caused a statistically significant increase of all the parameters. The experiments on perfusion of the humorally isolated sinocarotid area of the rabbit with alpha-thrombin-antithrombin III preparations (1.25 mkM) showed no changes peculiar to the induction of anticoagulation response with thrombin. It is concluded that antithrombin III blocks the ability of thrombin to activate anticoagulation system function.     

The antiheparin thrombocyte factor 4 and its interaction with heparin

A rat platelet factor has a high antiheparin activity. It also decreases nonenzymatic fibrinolytic activity of normal rat plasma and antithrombin III-heparin complex. The platelet factor 4 formed inactive complexes with heparin in molar ratios of 1:1 and 2:1. Intravenous injection of the platelet factor 4 before injection of albino rats with tissue thromboplastin prevented the reaction of anticoagulation system inactivated the synthesis of endogenous thrombin. This effect is accompanied by high hypercoagulation and depression of nonenzymatic fibrinolysis in blood.     

Heparin clearance during activation of the anticoagulation system in animals

Retarded heparin-35S clearance from the blood stream during activation of the blood anticoagulation system was observed. Under these conditions the half-life of heparin-35S increased from 1.6 h in control animals to 2.35 h in the animals after intravenous injection of a threshold dose of thrombin causing activation of the anticoagulation system. Retarded heparin-35S clearance from the blood stream during activation of the blood anticoagulation system is accompanied by increase of its absorption in the liver and in the auricular atrii.     

Formation of thrombin and its inactivation by antithrombin III following repeated intravenous injections of tissue thromboplastin in animals

A breach in the inactivation of thrombin activity by antithrombin III following numerous repeated intravenous injections of tissue thromboplastin to albino rats was established. Seven injections of tissue thromboplastin to animals (at 30-40 min-interval) caused functional exhaustion of anticoagulation system and increased thrombin blood circulation level.     

Myxobacterial slime and proteolytic activity

An extracellular protein-polysaccharide-lipide (PPL) complex from exponentially growing cultures of Myxococcus virescens was purified by phosphate precipitation and gel chromatography. The high molecular weight slime polymer appeared homogenous upon isoelectric focusing. The PPL complex exhibited proteolytic activity against gelatin and the activity was only partly reduced by heat treatment. The function of the slime polymer as protein denatured was studied. The complex formed micelles similar to anionic detergents and it inhibited the precipitation and coagulation of proteins by trichloroacetic acid. Lysozyme was totally inactivated when treated with the PPL complex. By gel chromatography binding studies, the PPl complex was found to bind lysozyme in the ratio of 1 to 5.8 (w/w). After separation of added protein from the complex the anticoagulation effect on the protein remained. The biological function of the PPL complex was demonstrated with hemoglobin. When all susceptible peptide bonds in PPL-treated hemoglobin were hydrolyzed by trypsin only 20% in the urea-denatured protein were attacked. The combined role of slime and proteolytic activity is discussed.Abbreviations Used PPL protein-polysaccharide-lipide - TCA trichloroacetic acid - BSA bovine serum albumin - Tris tris-(hydroxymethyl)aminomethane - CMC critical micelle concentration - DNFB 2,4-dinitrofluorobenzene - DNP N-dinitrophenyl - SDS sodium dodecylsulphate - H.U. Hultin units     

Antidiabetic and Anticoagulant Properties of Heparin–Glutamic Acid Complex

Natural heparin complexes proved to activate the anticoagulation system. The obtained experimental data convincingly confirm that glutamic acid alone, and particularly in a complex with heparin, has a considerable preventive potential and efficiently protects experimental animals with induced diabetes mellitus.     

Homogeneous sulfation of bagasse cellulose in an ionic liquid and anticoagulation activity

Homogeneous sulfation of bagasse cellulose (BC) with chlorosulfonic acid-dimethylformamide was accomplished in an ionic liquid 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl). The BCS products from the sulfation had degrees of substitution (DS) in the range of 0.52-2.95 and a simultaneous substitution pattern at C-6, C-2 and C-3 positions. The sulfated BCS attained significant anticoagulation activity, causing a dose-dependent prolongation of coagulation time and inhibition of FIIa and FXa activities in human plasma. The anticoagulation activity of BCS showed a positive correlation with DS, and some of the activity indexes exceeded those of heparin.     

Apparent decrease of "inhibition" in the dilution curve system with the increase of anticoagulation. A paradox that is against the presence of inhibitors in coumarin treated patients.

The apparent "inhibition" found in coumarin plasma by means of the dilution curve system seems to decrease with the increase of anticoagulation. Three groups of anticoagulated patients with a P/N prothrombin time ratio of less than 1.8, between 1.8 and 2.3 and over 2.3, showed an "inhibition" of 1.6, 1.1 and 0.9 conventional units, respectively. An inverse correlation was found to exist between Thrombotest clotting times, P/N ratio, antigen/activity ratios on one side and inhibition units on the other side. These findings do not confirm the presence of "inhibitors" in coumarin treated patients.     

Thyroid hormonal axis regulates protein C anticoagulation pathway in rats

Effects of the hormones of the hypothalamic-pituitary-thyroid axis on some basic parameters of the activity of protein C anticoagulation pathway in rats are studied. Thyrotropin-releasing hormone (0.06 mg/kg body mass), thyrotropin (1 IU/kg), triiodothyronine (T3) (0.08 mg/kg), thyroxine (T4) (0.08 mg/kg), administered subcutaneously for three consecutive days on four different groups of rats increased significantly activated protein C, free protein S and protein S activity, and reduced the soluble endothelial protein C receptor. Protein C antigen and total protein S were significantly elevated only by thyrotropin-releasing hormone and thyroid-stimulating hormone, but they were not affected by T3 and T4 treatment. The data indicate the hypothalamic-pituitary-thyroid axis is involved in the regulation of the protein C anticoagulation pathway in rats by activation of this system, suggesting a tendency of hypocoagulability.     

Antidiabetic properties of high molecular weight heparin-adenosine triphosphate complex

Repeated intramuscular administration of the heparin-adenosine triphosphate (ATP) complex or ATP increased plasma anticoagulant and fibrinolytic activities and depressed the anticoagulation system in rats at the age of 10–11 months. Diabetogenic dose of alloxan induced no diabetes mellitus in such animals.     

Protective antithrombotic effects of proline-containing peptides under the influence of stress on the animal organism

We discovered that simple proline-containing peptides Gly-Pro, Pro-Gly, Pro-Gly-Pro, and semax had an antistress protective effect on the organism appearing as anticoagulation system activation. Repeated intranasal injection of each of these peptides to rats prior to acute immobilization stress prevented a hypercoagulation response to prolonged stress lasting 60 min. At the same time there was increase of anti-thrombotic, anticoagulant, and fibrin depolymerization activity and recovery of enzymatic fibrinolytic activity. Dipeptides were found to have the greatest antistress effect. Our results showed that semax had a protective effect against enhanced blood coagulability resulting from repeated immobilization stress.     

Antidote-mediated control of an anticoagulant aptamer in vivo

Patient safety and treatment outcome could be improved if physicians could rapidly control the activity of therapeutic agents in their patients. Antidote control is the safest way to regulate drug activity, because unlike rapidly clearing drugs, control of the drug activity is independent of underlying patient physiology and co-morbidities. Until recently, however, there was no general method to discover antidote-controlled drugs. Here we demonstrate that the activity and side effects of a specific class of drugs, called aptamers, can be controlled by matched antidotes in vivo. The drug, an anticoagulant aptamer, systemically induces anticoagulation in pigs and inhibits thrombosis in murine models. The antidote rapidly reverses anticoagulation engendered by the drug, and prevents drug-induced bleeding in surgically challenged animals. These results demonstrate that rationally designed drug-antidote pairs can be generated to provide control over drug activities in animals.     

Preparation of sulfated-chitins under homogeneous conditions

Sulfated-chitins of varying degrees of sulfation were prepared by the reaction of chitin with sulfur trioxide–pyridine complex under homogeneous conditions in 5% LiCl/DMAc solvent system. Sulfation at 8 °C or room temperature was regio-selective for the C6–OH position with the degree of sulfation (D.S.) ranging from 0.53 to 1.00 depending on the reaction time. When the reaction temperature was elevated, sulfation at the C3–OH position also occurred. The extent of sulfation at the C3 position was a function of the concentration of sulfating reagent, reaction time and temperature. The structure of sulfated-chitins was established by ¹H, ¹³C NMR and 2D HMQC. The degree of sulfation at the C6 position was estimated by ¹H NMR while that of the C3 position was by elemental analyses. The anticoagulant activity of the prepared sulfated-chitins correlated closely with D.S. The higher the D.S. yielded, the better the anticoagulant activity. In particular, a continuous sequence of 36S units was critical for obtaining high anticoagulation activity.     

Synthesis and anticoagulant activity of the quaternary ammonium chitosan sulfates

Quaternary ammonium chitosan sulfates with diverse degrees of substitution (DS) ascribed to sulfate groups between 0.52 and 1.55 were synthesized by reacting quaternary ammonium chitosan with an uncommon sulfating agent (N(SO₃Na)₃) that was prepared from sodium bisulfite (NaHSO₃) through reaction with sodium nitrite (NaNO₂) in the aqueous system homogeneous. The structures of the derivatives were characterized by FTIR, ¹H NMR and ¹³C NMR. The factors affecting DS of quaternary ammonium chitosan sulfates which included the molar ratio of NaNO₂ to quaternary ammonium chitosan, sulfated temperature, sulfated time and pH of sulfated reaction solution were investigated in detail. Its anticoagulation activity in vitro was determined by an activated partial thromboplastin time (APTT) assay, a thrombin time (TT) assay and a prothrombin time (PT) assay. Results of anticoagulation assays showed quaternary ammonium chitosan sulfates significantly prolonged APTT and TT, but not PT, and demonstrated that the introduction of sulfate groups into the quaternary ammonium chitosan structure improved its anticoagulant activity obviously. The study showed its anticoagulant properties strongly depended on its DS, concentration and molecular weight.     

Factor X antigen--factor X activity discrepancy in coumarin treated patients. No relationship with dilution curve

The plasma of 12 patients on coumarin medication was investigated with regard to factor X antigen, factor X activity, factor X antigen--factor X activity difference (delta) and dilution curve. The plasmas were divided into 3 groups according to the level of anticoagulation (under anticoagulated, normally anticoagulated, over anticoagulated). The average factor X antigen and the average factor X activity were 56.7, 53.0, 34.6 and 25.5, 20.1, 11.1%, respectively for the three groups of patients. The antigen-activity differences (delta) were 21.2, 22.5 and 23.5%, respectively. The degree of inhibition in conventional units was 2.6, 1.7 and 0.4 units for the three groups of patients, respectively. The decrease of "inhibition" with the increase of anticoagulation is not in agreement with the presence of similar levels of pre-factors or delta antigen-activity in the plasma of the three groups of patients. These data are against the presence of inhibitors in coumarin treated patients.