Studies, with a luminogenic peptide substrate, on blood coagulation factor X/Xa produced by mouse peritoneal macrophages.
The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-gamma-piperidylglycylarginylisoluminol). Amidolysis was quantified by measuring the light emitted during oxidation of isoluminol, released by Factor Xa. A lower detection limit of about 0.5ng of Factor Xa was established; the assay was linear with enzyme concentration up to at least 100ng/ml. Factor X was determined after treatment with the Factor X-activating component of Russell's-viper (Vipera russelli) venom. Macrophages, cultured in the absence of serum, released Factor X/Xa into the culture medium. The concentration of coagulation enzyme in the medium increased in an essentially linear fashion over a period of at least 3 days, at a rate corresponding to 6-8ng produced/24h per 10(6) cells. The ratio of Factor Xa/X+Xa varied from about 60 to 100%, showing that activation of Factor X to Xa is not prerequisite to release of the enzyme from the cells. Factor Xa activity was suppressed in the presence of warfarin [3-(alpha-acetonylbenzyl)-4-hydroxycoumarin; 12.5mug/ml of medium], but could be restored by adding vitamin K (0.1mug/ml) along with the warfarin. Cultures to which Sepharose beads containing covalently bound anti-(Factor X) antibodies had been added showed decreased amounts of free Factor X/Xa in the culture medium. The missing activity could be demonstrated by incubating the recovered conjugate with the substrate peptide S-2613. Factor Xa produced by the macrophages was efficiently inactivated by heparin in the presence of antithrombin, heparin with high affinity for antithrombin being more effective than the corresponding low-affinity species.
D000818:Animals; D000991:Antithrombins / Q000494:pharmacology; D001202:Ascitic Fluid / Q000166:cytology / Q000378:metabolism; D002478:Cells, Cultured; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D006493:Heparin / Q000494:pharmacology; D008165:Luminol / Q000031:analogs & derivatives / Q000378:metabolism*; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D009842:Oligopeptides / Q000378:metabolism*; D011724:Pyridazines / Q000378:metabolism*; D014812:Vitamin K / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology
D000991:Antithrombins; D009842:Oligopeptides; D011724:Pyridazines; D014812:Vitamin K; D008165:Luminol; D014859:Warfarin; C036212:S 2613; D005170:Factor X; D006493:Heparin; D015951:Factor Xa