Thromboxane and cysteinyl-leukotriene formation are differentially activated in spontaneously clotting whole human blood in vitro.
We have previously demonstrated that clotting of whole human blood in vitro not only triggers the production of thromboxane (TX) B2 but is also accompanied by formation of 5-lipoxygenase-derived cysteinyl-leukotrienes (LT). In order to further characterize the mechanisms leading to activation of the cysteinyl-LT production, we have now investigated the effects of thrombin on cysteinyl-LT as well as TXB2 formation in whole human blood. Addition of exogenous human alpha-thrombin (0.1 - 3.0 U/ml) to whole human blood incubated in vitro led to a concentration- and time-dependently increased release of TXB2 into the serum samples. The serum contents of cysteinyl-LT were, however, not significantly affected. Inactivation of endogenously generated thrombin by inhibitors such as recombinant hirudin (HBW 023, 0.43 - 1.43 microM) or the peptidyl chloromethyl ketone, D-Phe-Pro-Arg-CH2Cl (1.0 - 100 microM) concentration- and time-dependently inhibited the release of TXB2 into the serum or plasma samples. In contrast, however, serum contents of cysteinyl-LT remained unchanged. The identity of immunoreactive material was confirmed by thin-layer chromatography of immunoreactive TXB2 and by reversed phase HPLC of immunoreactive cysteinyl-LT. As expected, washed human platelets stimulated with alpha-thrombin were identified as the major source of TXB2 generation but purified monocytes were also found to release some TXB2 upon alpha-thrombin stimulation. Release of TXB2 by isolated human polymorphonuclear leukocytes (PMN) was negligible in the presence of this stimulus. None of the cells which are known to possess 5-lipoxygenase activity such as PMN or monocytes did release neither cysteinyl-LT nor LTB4 upon stimulation with human alpha-thrombin up to 10 U/ml. These data demonstrate that TXB2 production and cysteinyl-LT formation are differentially activated in spontaneously clotting whole human blood in vitro, the former being dependent on endogenously generated thrombin the latter being dependent on a stimulus yet to be identified.
Simmet
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Department of Pharmacology and Toxicology, Ruhr-University, Bochum, F.R.G.
Weide
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D000328:Adult; D000595:Amino Acid Sequence; D001094:Arachidonate 5-Lipoxygenase / Q000378:metabolism*; D001777:Blood Coagulation / Q000187:drug effects / Q000502:physiology*; D001792:Blood Platelets / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology; D006629:Hirudins / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D015289:Leukotrienes / Q000096:biosynthesis*; D008297:Male; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013929:Thromboxane B2 / Q000096:biosynthesis*
D006629:Hirudins; D015289:Leukotrienes; D009842:Oligopeptides; D011994:Recombinant Proteins; D013929:Thromboxane B2; C020924:phenylalanyl-prolyl-arginine methyl chloride; D001094:Arachidonate 5-Lipoxygenase; D013917:Thrombin