Thrombin Generation by Exposure of Blood to Endotoxin: A Simple Model to Study Disseminated Intravascular Coagulation

Abstract

Pathologic disseminated intravascular coagulation (PDIC) is a serious complication in sepsis. In an in-vitro system consisting of incubation of fresh citrated blood with lipopolysaccharides (LPS) or glucans and subsequent plasma recalcification plasmatic thrombin was quantified. Five hundred microliters of freshly drawn citrated blood of healthy donors were incubated with up to 800 ng/mL LPS (Escherichia coli) or up to 80 μg/mL Zymosan A (ZyA; Candida albicans) for 30 minutes at room temperature (RT). The samples were centrifuged, and 30 μL plasma were recalcified with 1 volume or less of CaCl₂ (25 μmoles Ca²⁺/mL plasma). After 0 to 12 minutes (37°C), 20 μL 2.5 M arginine, pH 8.6, were added. Thirty microliters 0.9 mM HD-CHG-Ala-Arg-pNA in 2.3 M arginine were added, and the absorbance increase at 405 nm was determined. Fifty microliters plasma were also incubated with 5 μL 250 mM CaCl₂ for 5, 10, or 15 minutes (37°C). Fifty microliters 2.5 M arginine stops coagulation, and 50 μL 0.77 m M HD-CHG-Ala-Arg-pNA in 2.3 M arginine starts the thrombin detection. The standard was 1 IU/mL thrombin in 7% human albumin instead of plasma. Arginine was also added in the endotoxin exposure time (EET) or in the plasma coagulation reaction time (CRT). Tissue factor (TF)-antigen and soluble CD14 were determined. LPS at blood concentrations greater than 10 ng/mL or ZyA at greater than 1 μg/mL severalfold enhance thrombin generation, when the respective plasmas are recalcified. After 30 minutes EET at RT, the thrombin activity at 12 minutes CRT generated by the addition of 200 ng/mL LPS or 20 μg/mL ZyA is approximately 200 mIU/mL compared to approximately 20 mIU/mL without addition of endotoxin, or compared to about 7 mIU/mL thrombin at 0 minutes CRT. Arginine added to blood or to plasma inhibits thrombin generation; the inhibitory concentration 50% (IC 50) is approximately 15 mM plasma concentration. Endotoxin incubation of blood increases neither TF nor sCD14. This assay allows the study of the hemostasis alteration in PDIC, particularly in PDIC by sepsis. The thrombin generated by blood plus endotoxin incubation and plasma recalcification suggests that the contact phase of coagulation; e.g., triggered by cell components of (phospholipase-) lysed cells such as monocyte or endothelium DNA or phospholipid-vesicles (microparticles), is of primary pathologic importance in sepsis-PDIC. Arginine at plasma concentrations of 10 to 50 mM might be a new therapeutic for sepsis-PDIC.