A new activated factor X-based clotting method with improved specificity for procoagulant phospholipid.

Abstract

An improved activated factor X-based clotting method was used to investigate activity of procoagulant phospholipid (PPL) in blood samples collected into various anticoagulants and in plasmas with a range of abnormalities. The dilute activated factor X-activated clotting time (XACT) was carried out on a mixture of specimen with phospholipid-free porcine plasma. PPL from the test sample is then rate-limiting, controlling the clotting time so that the XACT is shortened from a maximum of approximately 120 s with citrated platelet-free plasma to approximately 30 s with freeze-thawed platelet-rich plasma. XACT results were only shortened slightly by fresh normal platelet-rich plasma, but were shortened significantly by platelets that had been activated by freeze-thawing. This improved method for PPL was not prolonged by deficiencies of known clotting factors and therapeutic levels of heparin, and it was surprisingly resistant to most lupus anticoagulants. However, it was extremely sensitive to PPL, detecting down to 50 ng/ml synthetic phospholipid added to phospholipid-free plasma. Excellent correlation was achieved between XACT shortening and microparticle count assessed by annexin V-binding and flow cytometry in normal plasma spiked with platelet microparticles. In citrated blood specimens, XACT shortened with time in a temperature-dependent manner. XACT results on blood samples anticoagulated with ethylenediamine tetra-acetate were more stable, and these would be preferable for assessing PPL expression ex vivo. XACT was significantly shorter in whole blood samples than in normal platelet-rich or platelet-poor plasmas, suggesting that PPL was normally expressed more by cells or aggregates larger than platelets or microparticles.