Biochemical measurementsBlood was collected into a 4.5 ml tube containing 0.105 M sodium citrate for coagulation measurements and in a 4 ml tube containing 7.5% potassium selleck chemicals EDTA for hemocytometry (Becton Dickinson, Plymouth, UK). Citrated blood was centrifuged at 1500 g for 10 minutes, and plasma aliquots were stored at -80��C. Aliquots of ultrafiltrate samples were frozen at -80��C until use. The following assays were performed immediately after sampling: PTT (Innovin), aPTT (Actin FS) and antithrombin (Berichrom ATIII) on a Sysmex CA-1500 coagulation analyzer (all Siemens Healthcare Diagnostics, Deerfield, IL, USA), and platelet counts on a Sysmex XE-2100 hematology analyzer (Sysmex, Kobe, Japan).

Anti-Xa activity was determined in ultrafiltrates and citrated plasma to assess the anticoagulant activity of the LMWH nadroparin using the Coamatic Heparin kit (Chromogenix, Instrumentation Laboratory Company, Lexington, MA, USA). For determination of anti-Xa in ultrafiltrate, anti-Xa activity was determined after addition of an equal volume of normal plasma (Standard Human Plasma, Siemens Healthcare Diagnostics Deerfield, IL, USA) to the ultrafiltrate to provide for a suitable matrix and the presence of antithrombin. The sensitivity of our anti-Xa assay, (detection limit 0.01 U/ml) albeit negatively influenced by a factor 2 when measuring ultrafiltrate because of the need to add normal plasma, is sufficient to demonstrate relevant anti-Xa removal. Analytical precision, characterized by a coefficient of variation percentage of less than 2.

5 at the higher anti-Xa levels, is adequate to detect relevant accumulation in plasma if present.The ETP was measured as an overall indicator of hemostasis. The ETP monitors the thrombin-forming capacity of plasma, including the generation and inhibition of thrombin generation beyond the initiation of fibrin clot formation providing an overall assessment of hemostasis and potential extra-hemostatic effects of the generated thrombin [6]. The ETP is characterized by ‘lag time’ (ETPTlag (s)), ‘time to maximal activity’ (ETPTmax (s)), ‘maximal activity’ (ETPCmax (mA/min)) and the main parameter: ‘area under the curve’ (ETPAUC (mA)); the latter represents the total thrombin formation. ETP was determined on the BCS-XP (Siemens Healthcare Diagnostics, Deerfield, IL, USA) using the ETP-B protocol and reagents as provided and described by the manufacturer.

In this protocol, thrombin formation is triggered via the addition of Innovin up to a final concentration of 300 pM tissue factor, also providing for phospholipids. We have established a provisional reference range GSK-3 in our laboratory in 20 adults, representing +/- three standard deviations from the mean ETPTlag 14.4 to 22.1 s, ETOTmax 48.5 to 60.0 s, ETPCmax 115 to 148 mA/min, and ETPAUC 346 to 520 mA.