thromboxane-b2 and phenylalanyl-prolyl-arginine-methyl-chloride

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.

Topics: Adult; Amino Acid Sequence; Arachidonate 5-Lipoxygenase; Blood Coagulation; Blood Platelets; Enzyme Activation; Hirudins; Humans; In Vitro Techniques; Leukotrienes; Male; Molecular Sequence Data; Neutrophils; Oligopeptides; Recombinant Proteins; Thrombin; Thromboxane B2