title: Thromboxane and cysteinyl-leukotriene formation are differentially activated in spontaneously clotting whole human blood in vitro.
abstract: 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.
authors.affiliation: Department of Pharmacology and Toxicology, Ruhr-University, Bochum, F.R.G.
authors.forename: T
authors.initials: T
authors.lastname: Simmet
authors.forename: I
authors.initials: I
authors.lastname: Weide
mesh_terms: 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*
chemical_list: D006629:Hirudins; D015289:Leukotrienes; D009842:Oligopeptides; D011994:Recombinant Proteins; D013929:Thromboxane B2; C020924:phenylalanyl-prolyl-arginine methyl chloride; D001094:Arachidonate 5-Lipoxygenase; D013917:Thrombin