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. 1982 Aug 15;206(2):231–237. doi: 10.1042/bj2060231

Studies, with a luminogenic peptide substrate, on blood coagulation Factor X/Xa produced by mouse peritoneal macrophages

Ulf Lindahl 1,2, Svein O Kolset 1,2, Jarl Bøgwald 1,2, Bjarne Østerud 1,2, Rolf Seljelid 1,2
PMCID: PMC1158578  PMID: 7150243

Abstract

The formation and secretion of coagulation Factor X/Xa by mouse peritoneal macrophages was studied with a luminogenic peptide substrate (S-2613; t-butyloxycarbonylisoleucylglutamyl-γ-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 106 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-(α-acetonylbenzyl)-4-hydroxycoumarin; 12.5μg/ml of medium], but could be restored by adding vitamin K (0.1μg/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.

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Selected References

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