Increase In Anticoagulant Activity Research Articles

Стан системи гемостазу за трансмісивної венеричної саркоми собак

Стан системи гемостазу за трансмісивної венеричної саркоми собак

The effect of increasing the sulfation level of chondroitin sulfate on anticoagulant specific activity and activation of the kinin system.

Oversulfated chondroitin sulfate (OSCS) was identified as a contaminant in certain heparin preparations as the cause of adverse reactions in patients. OSCS was found to possess both plasma anticoagulant activity and the ability to activate prekallikrein to kallikrein. Differentially sulfated chondroitin sulfates were prepared by synthetic modification of chondroitin sulfate and were compared to the activity of OSCS purified from contaminated heparin. Whilst chondroitin sulfate was found to have minimal anticoagulant activity, increasing sulfation levels produced an anticoagulant response which we directly show for the first time is mediated through heparin cofactor II. However, the tetra-sulfated preparations did not possess any higher anticoagulant activity than several tri-sulfated variants, and also had lower heparin cofactor II mediated activity. Activation of prekallikrein was concentration dependent for all samples, and broadly increased with the degree of sulfation, though the di-sulfated preparation was able to form more kallikrein than some of the tri-sulfated preparations. The ability of the samples to activate the kinin system, as measured by bradykinin, was observed to be through kallikrein generation. These results show that whilst an increase in sulfation of chondroitin sulfate did cause an increase in anticoagulant activity and activation of the kinin system, there may be subtler structural interactions other than sulfation at play given the different responses observed.

Photoswitchable Heparinase III for Enzymatic Preparation of Low Molecular Weight Heparin.

A photocontrollable biocatalyst was rationally designed by site-specifically linking a photoswitchable azobenzene derivative to the only cysteine residue in heparinase III mutant (K130C). Upon photoswitch, the enzymatic degradation of heparin could be artificially controlled to produce low molecular weight heparin with more uniform molecular weight and an increase in anticoagulant activity.

Antioxidant and anticoagulant activity of polyphenol and polysaccharides from fermented Sargassum sp.

The current investigation was carried out with an objective of determining the structural characteristic of polysaccharides extracted from fermented Sargassum sp. to be used as potent natural heparin substitute anticoagulant compound. Sargassum sp. fermented with marine lactic acid bacteria was initially subjected to ethanol precipitation for the recovery of bioactive compounds. Antioxidant activity was maximum in the soluble fraction whereas anticoagulant activity was observed to be high in the precipitate which correlated with the increased polyphenols and total sugars respectively. The precipitate was purified by anion exchange chromatography and the fractions collected were analyzed for total sugars and anticoagulant activity. There was 2.6–3.9-folds increase in anticoagulant activity in the final purified fractions, with a maximum activity in case of sample fermented with Enterococcus faecium (6.7±0.22IU/mg). Structural elucidation of potential anticoagulant polysaccharide by Fourier Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance (NMR) analysis indicated the presence of alginate rich in mannuronic acid.

Study on antithrombotic and antiplatelet activities of low molecular weight fucoidan from Laminaria japonica

The antithrombotic and antiplatelet effects of two fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica were compared in order to examine the influence of chemical character on their antithrombotic activity and the possible mechanism. Both LMW fucoidan fractions exhibited favorable antithrombotic activity in an Fecl3-induced arterial thrombosis. The antithrombotic activity of LMW fucoidan was related with decrease of TXB2 and whole blood viscosity and hematocrit. LMW fucoidan showed a correlation between anticoagulant, antiaggregant and antithrombotic effects in vivo. For LMW fucoidan, antithrombotic activity required high dose of 5–10 nmol kg−1, concomitantly with increase in anticoagulant activity and inhibition of platelet aggregation. Administration of LMW fucoidan significantly promoted the 6-keto-PGF1α content and decreased the TXB2 content, indicating its inhibition of tissue factor pathway and regulation of metabolism of arachidonic acid. By comparison, highly sulfated fucoidan LF2 with Mw 3900 seemed to be a more suitable choice for antithrombotic drug for its antithrombotic activity accompanied with specific inhibitory activity on platelet aggregation, low anticoagulant activity and low hemorrhagic risk in vivo.

Anticoagulant activity and platelet aggregation in blood plasma from rats after intranasal administration of adenosine triphosphate and its complex with heparin

Fourfold intranasal administration of ATP was followed by an increase in anticoagulant activity and decrease in platelet aggregation in blood plasma of healthy rats and, particularly, of animals with suppressed function of the anticoagulant system. These changes decrease the risk of the prethrombotic state. Complex of ATP and high-molecular-weight heparin decreased platelet aggregation only in the blood from healthy animals, but increased anticoagulant potential of plasma hemostasis in prethrombotic animals.

O-Acylated heparin derivatives with low anticoagulant activity decrease proliferation and increase α-smooth muscle actin expression in cultured arterial smooth muscle cells

Selectively O-acylated derivatives of various glycosaminoglycans were prepared and tested in vitro for their anticoagulant activity and their antiproliferative effect on rat and rabbit smooth muscle cells. When O-acylation (butyrylation or hexanoylation) had been performed on periodate-depolymerized heparin fragments having very low anticoagulant activity, the antiproliferative potency was markedly increased (IC 50 = 2 and 1 μg/ml respectively, versus 31 μg/ml for starting compound) without an increase in anticoagulant activity. The antiproliferative activity was related to the degree of acylation. The O-acylated derivatives of heparin fragments were also very active in reversing the de-differentiation of smooth muscle cell in culture, as estimated by the increase in the expression of α-smooth muscle actin and α-smooth muscle actin mRNA.

Glycosylation of human protein C affects its secretion, processing, functional activities, and activation by thrombin

Human protein C (HPC) is an antithrombotic serine protease that circulates in the plasma as several glycoforms. To examine the role of glycosylation in the function of this protein, we singly eliminated each of the four potential N-linked glycosylation sites by site-directed mutagenesis of Asn to Gln at amino acid positions 97, 248, and 313 (HPC derivatives Q097, Q248, and Q313) or at the unusual consensus sequence Asn-X-Cys at 329 (HPC derivative Q329). The cDNAs for wild type and each derivative were inserted into expression vectors and expressed both transiently and stably in human 293 and hamster AV12-664 cells. We demonstrate that N-linked glycosylation at position 97 in the light chain of HPC is critical for efficient secretion and affects the degree of core glycosylation at Asn-329. Glycosylation at position 248 affects the intracellular processing of the internal Lys-Arg (KR) KR cleavage site, and partial glycosylation at the sequence Asn-329-X-Cys is responsible for the natural alpha-glycoform. Altering the glycosylation pattern of the protein had no significant effect on the level of fully gamma-carboxylated HPC secreted from the 293 cell line. However, elimination of glycosylation sites in the heavy chain resulted in a 2- to 3-fold increase in anticoagulant activity. Utilizing synthetic substrate, both the Km and kcat were affected, depending on the specific glycosylation site eliminated. However, there were no significant differences in the inhibition kinetics by alpha-1-antitrypsin (association rate constants of 10-11 M-1s-1 and t1/2 of 27-29 min at 40 microM alpha-1-antitrypsin) or t1/2 in human plasma (17-18 min). A comparison of the rate of activation of each derivative by thrombin alone or in complex with thrombomodulin revealed that Q313 was activated approximately 2.5-fold faster than wt HPC, independent of calcium concentration. This increase in rate was due to an enhanced affinity of thrombin-thrombomodulin for Q313, as indicated by a 3-fold reduction in Km. Overall, our studies demonstrate that glycosylation at different sites in HPC affects distinct properties of this complex protein. Furthermore, we demonstrate the ability to improve the catalytic efficiency of this enzyme through carbohydrate modifications.

Anticoagulant Properties of Purified X-Like Fragments of Human Fibrinogen Produced by Degradation with Leukocyte Elastase

The anticoagulant properties of fibrinogen digestion products change with stage of digestion. On digestion with leukocyte elastase, in the presence of calcium ions, the anticoagulant potency of fibrinogen digests first increases, then decreases sharply, and in late stages increases again. This is different from plasmin digestion where only an increase in anticoagulant activity is seen followed by a slow decrease. From SDS-gel electrophoresis it appears that both the early rise and the decrease in anticoagulant activity are associated with the stage of elastase-produced X-like fragments. This is confirmed with pure fragments: X-like fragments (purified from elastase digests of fibrinogen of different stages by ammonium sulphate precipitation and ion-exchange chromatography) give an increase and decrease in anticlotting activity which correlates very well with that of the potency of the digest from which they are purified. As expected, and in contrast with (late) plasmic X-fragments, late elastase X-like fragments have a low anticoagulant potency. The molecular basis for the gain and loss in anticoagulant activity going from early to late X-like fragments is obscure. Immunological tests, calcium-binding experiments and affinity chromatography on immobilized thrombin-activated NDSK suggest that the changes in anticoagulant activity are not due to a proteolytic change in the carboxyl-terminal part of the gamma-chain in the D moiety of the molecule. Our data suggest a correlation with the stage of digestion of the A alpha-chain in the X-like fragments.

Anticoagulant activity of pectic acid derivatives

Pectic acid propionate is reduced with gaseous diborane to yield a series of polysaccharides having different carboxyl group content. Sulfation of the reduced polymers with various molar ratios of sulfur trioxide- N,N-dimethylformamide complex yields a series of polysaccharides having different carboxyl and O-sulfate group contents. Treatment of the reduced polymers with ethylenimine prior to sulfation yields a second series of polysaccharides having various levels of carboxyl and N- and O-sulfate groups. All of the sulfated polysaccharide preparations show activity in delaying the clotting time of recalcified human plasma. The anticoagulant activity of the reduced and sulfated polysaccharides increases with increasing degree of substitution of sulfate groups and with decreasing amounts of carboxyl groups. Activities of 44 and 43 units/mg are observed for preparations reduced to 7.7 and 4.9% carboxyl content and having degrees of sulfate substitution of 1.55 and 1.46, respectively. An increase in anticoagulant activity with decreasing carboxyl group content is also observed for preparations containing both N- and O-sulfate groups. The amount of nitrogen present, however, representing N-sulfate groups has little, if any, effect on the anticoagulant activity.

Leukocyte anticoagulant with myelogenous leukemia

This is a case of subacute myelogenous leukemia in a man who had a bleeding diathesis which was attributed to a leukocyte anticoagulant. The presence of the anticoagulant was first suggested by a significant prolongation of the silicone clotting time and was neutralized in vitro by phospholipid. An assay for the anticoagulant activity was developed and the results in this patient compared with assays in other subjects with chronic and acute leukemia. The assay clearly indicated a “twentyfold” increase in activity. The anticoagulant persisted after therapy even when the number of circulating leukocytes was reduced to 20,000 per cu mm. A “blastic” relapse of the leukemia was associated with a striking increase in anticoagulant activity. The marked leukopenia which developed following therapy with cystosine arabinoside resulted in the disappearance of the anticoagulant activity. To our knowledge this is the first such case to be documented. The studies suggest that leukocyte anticoagulants should be investigated in patients with leukemia who exhibit bleeding in the absence of thrombocytopenia or specific clotting factor deficiencies.