Cirrhosis was diagnosed on the basis of

Cirrhosis was diagnosed on the basis of Selleckchem PD0325901 clinical, laboratory, and ultrasound evidence. Criteria for exclusion were the use of drugs known to interfere with blood coagulation, ongoing bacterial

infections, hepatocellular carcinoma, and extrahepatic malignancy. The severity of the disease was estimated according to Child-Turcotte-Pugh score.11 One-hundred-five healthy individuals matched with the patient population for age and sex, were enrolled in this study as controls. In addition to the above controls, a population of 37 individuals (4 with and 33 without a previous history of thrombosis) known to be carriers of the factor V Leiden mutation was included for comparative purposes. Blood for laboratory analyses was drawn by clean venipuncture and collected in vacuum tubes (Becton Dickinson, Meylan, France) containing 109 mM trisodium citrate as anticoagulant in the proportion of 1/9 parts of anticoagulant/blood. Blood was centrifuged within 30 minutes from collection at 2880g for 15 minutes at room temperature. Platelet-free plasma was then harvested, quick frozen in liquid nitrogen and stored at −70°C until tested. This is a commercially available chromogenic assay (HemosIL Thrombopath;

Instrumentation Laboratory, Orangeburg, NY) designed to globally evaluate the functionality of the protein C anticoagulant system.12 It is based on the ability of endogenous activated protein C generated after activation of protein C by a snake venom extract (Protac) to reduce the tissue factor–induced thrombin generation. The amount of thrombin is evaluated by recording changes in optical CX-4945 ic50 density (OD) at 405 nm in the presence (A) or absence (B) of Protac after adding a thrombin-specific chromogenic 上海皓元 substrate. The assay kit contains all the reagents that are needed to run the test. All reagents, except for the diluent are lyophilized and were reconstituted with distilled water before use according to

the manufacturer’s specification. Briefly, 10 μL of the plasma sample and 40 μL of the Thrombopath diluent were incubated with either the Thrombopath activator A or the Thrombopath activator B (45 μL) for 120 seconds, before the Thrombopath thromboplastin reagent (50 μL) was added. After 90 seconds of incubation, 50 μL of the Thrombopath substrate was added, and change in OD at 405 nm was recorded for 45 seconds. Results are expressed as the Protac-induced coagulation inhibition percentage (PICI%) calculated by the following equation: PICI% = ([B − A]/B) × 100, where B and A are the OD for plasma tested in the absence (B) or presence (A) of Protac. The smaller the PICI% value, the greater the procoagulant imbalance. Testing with Thrombopath was performed by a fully automated coagulation analyzer (ACL 10000; Instrumentation Laboratory, Bedford, MA) according to the manufacturer’s specification. To minimize methodological variability, equal numbers of plasmas from patients and controls were included on each test occasion.

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