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 m M 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 m M 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 m M 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 m M might be a new therapeutic for sepsis-PDIC.

Effects of cyclooxygenase inhibition on canine coronary artery blood flow and thrombosis

“…Further studies with these compounds were conducted on arterial thrombosis in the presence of lipopolysaccharide (LPS)-induced systemic inflammation. LPS pretreatment was employed to increase the expression of endothelial-derived COX-2 and establish a simulated environment of an inflammatory state (36,47) and its influence on arterial thrombus formation in response to vessel wall injury.…”

The anticoagulant capacity of plasmatic unfractionated heparin decreases at 23°C

“…Folding of this molecule might create some polynegative niches that interact with prekallikrein or factor XII [6][7][8][9]. This contact activation might be favoured at 238C [3]. The procoagulant changes occur especially with UFH and not with LMWH [10].…”

“…Room temperature favours intrinsic haemostasis activation by the polynegative molecule SiO 2 [2]. The aim of the present study was therefore to quantify the anticoagulant capacity of heparin and low-molecular-weight heparin (LMWH) at 37 and at 238C, using a new specific thrombin generation assay [3].…”

Thrombolysis for indwelling catheter related thrombosis and superior vena cava obstruction in cystic fibrosis: a case series

“…No systematic review addressing the rate of candidaemia with thrombosis related to TIVAD has been published to date; however, in vitro studies have shown that zymosan A from Candida spp. does induce thrombin formation [11].…”