Thrombin inhibitory activity of some polyphenolic compounds.

Thrombin, also known as an active plasma coagulation factor II, belongs to the family of serine proteases and plays a crucial role in blood coagulation process. The process of thrombin generation is the central event of the hemostatic process and regulates blood coagulant activity. For this reason, thrombin inhibition is key to successful novel antithrombotic pharmacotherapy. The aim of our present study was to examine the effects of the well-known polyphenolic compounds on the activity of thrombin, by characterization of its interaction with selected polyphenols using different biochemical methods and biosensor BIAcore analyses. Only six compounds, cyanidin, quercetin, silybin, cyanin, (+)-catechin and (-)-epicatechin, of all examined in this study polyphenols caused the inhibition of thrombin amidolytic activity. But only three of the six compounds (cyanidin, quercetin and silybin) changed thrombin proteolytic activity. BIAcore analyses demonstrated that cyanidin and quercetin caused a strong response in the interaction with immobilized thrombin, while cyanin and (-)-epicatechin induced a low response. Lineweaver-Burk curves show that used polyphenol aglycones act as competitive thrombin inhibitors. Our results suggest that polyphenolic compounds might be potential structural bases and source to find and project nature-based, safe, orally bioavailable direct thrombin inhibitors.

Evolution of the contact phase of vertebrate blood coagulation.

BACKGROUND: Previous reports have noted that factor (F) XI and FXII and prekallikrein (the contact phase proteases) are absent in fish. OBJECTIVES: A broad survey of recently completed genomes was undertaken to find where during the course of vertebrate evolution these coagulation factors appeared. METHODS: BLAST searches were conducted for the various factors on genomes of lamprey, puffer fish, zebra fish, frog, chicken, platypus, and opossum. RESULTS: It was confirmed that FXII is absent from fish; it is present in frog, platypus, and opossum, but is absent in chicken, an apparent example of gene loss. A single gene corresponding to the evolutionary predecessor of FXI and prekallikrein occurs in frog, chicken, and platypus. The opossum (a marsupial) has both prekallikrein and FXI, completing the full complement of these genes that occurs in eutherian mammals. CONCLUSIONS: The step-by-step accrual of genes for these factors by a series of timely gene duplications has been confirmed by phylogenetic analysis and other considerations.

The effects of nitronium ion on nitration, carbonylation and coagulation of human fibrinogen.

The effect of nitronium ion on nitration, carbonylation and coagulation of human fibrinogen (Fg) in vitro was investigated. We observed that nitration of tyrosine, induced by NO2BF4 (0.01 mmol/l), was increased. No changes in carbonylation by NO2BF4 (0.01 mmol/l) were noticed. Mentioned alterations were associated with amplified coagulation of Fg. Higher concentrations of NO2BF4 (1 and 0.1 mmol/l) triggered growth of nitration and carbonylation of Fg, but led to inhibition of polymerization. Slight nitration may be responsible for increase, whereas sizable nitration and oxidation may lead to inhibition of Fg coagulation.

Resveratrol, a natural phenolic compound may reduce carbonylation proteins induced by peroxynitrite in blood platelets.

Resveratrol (3,4',5-trihydroxystilbene) has a very broad range of biological properties, including antiplatelet and antioxidative activity. We investigated in vitro the effect of resveratrol on carbonylation of proteins (indicators of oxidative stress) in blood platelets treated with peroxynitrite (ONOO(-)), a strong biological oxidant and inflammatory mediator. We observed that carbonylation of proteins induced by ONOO(-) (0.1 mmol/l), in the presence of resveratrol (0.25-0.1 mmol/l) is reduced. Resveratrol may scavenge ONOO(-), and may be useful in the prevention of ONOO(-)-related diseases, such as inflammatory and cardiovascular diseases.

Regulation of cytosolic free calcium concentration by extracellular nucleotides in human hepatocytes.

The effects of extracellular ATP and other nucleotides on the cytosolic free Ca2+ concentration ([Ca2+]i) have been studied in single primary human hepatocytes and in human Hep G2 and HuH-7 hepatoma cells. ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and UTP caused a concentration-dependent biphasic increase in [Ca2+]i with an initial peak followed by a small sustained plateau in most cells. In some cells, however, repetitive Ca2+ transients were observed. The rank order of potency was ATP >/= UTP > ATPgammaS, and complete cross-desensitization of the Ca2+ responses occurred between ATP and UTP. The initial transient peak in [Ca2+]i was resistant to extracellular Ca2+ depletion, which demonstrates mobilization of internal Ca2+ by inositol 1,4,5-trisphosphate whose formation was enhanced by ATP and UTP. In contrast, the sustained plateau phase required influx of external Ca2+. Ca2+ influx occurs most likely through a capacitative Ca2+ entry mechanism, which was shown to exist in these cells by experiments performed with thapsigargin. On the molecular level, specific mRNA coding for the human P2Y1, P2Y2, P2Y4, and P2Y6 receptors could be detected by RT-PCR in Hep G2 and HuH-7 cells. However, ADP and UDP, which are agonists for P2Y1 and P2Y6 receptors, respectively, caused no changes in [Ca2+]i, demonstrating that these receptors are not expressed at a functional level. Likewise, alpha,beta-methylene-ATP, beta,gamma-methylene-ATP, AMP, and adenosine were inactive in elevating [Ca2+]i, suggesting that the ATP-induced increase in [Ca2+]i was not caused by activation of P2X or P1 receptors. Thus, on the basis of the pharmacological profile of the nucleotide-induced Ca2+-responses, extracellular ATP and UTP increase [Ca2+]i by activating P2Y2 and possibly P2Y4 receptors coupled to the Ca2+-phosphatidylinositol signaling cascade in human hepatocytes. This suggests that extracellular nucleotides from various sources may contribute to the regulation of human liver cell functions.

Emergency subtotal colectomy for obstructing carcinoma of the left colon.

Eight patients with obstructing carcinoma of the left colon underwent emergency subtotal colectomy and ileosigmoidostomy or ileoproctostomy. Based on the results obtained by us and those reported in the literature, it is our opinion that emergency subtotal colectomy with primary ileocolonic anastomosis should be considered as the procedure of choice for patients with obstructing carcinoma of the left colon.