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.

Brain-derived microparticles induce systemic coagulation in a murine model of traumatic brain injury

“…To overcome this technical challenge, we generated BDMPs from the brains of adult C57BL/6J mice by freeze-thawing injury. 37 The BDMPs thus produced resemble those detected in the plasma of FPI mice in size and levels of PS and GFAP expression (supplemental Figure 7). When viewed under TEM, these BDMPs were irregular spheres of 50 to 500 nm in diameter, with a clearly defined and relatively intact membrane, but without apparent granules (Figure 2A).…”

“…The first was a PS-dependent assay that tested the ability of BDMPs to promote clot formation in the presence of coagulation factor Xa. 37 For each reaction, 25 mL of phosphate-buffered saline (PBS) containing a known number of BDMPs was mixed with 25 mL of phospholipid-deficient porcine plasma (IMVS, Adelaide, Australia) and 100 mL of bovine factor Xa (0.02 U/mL in a buffer containing 15 mM CaCl 2 , 100 mM NaCl, 20 mM N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid, and 0.001% polybrene [pH 7.0]). Clot formation was monitored at 37°C on a CoaScreener coagulation analyzer (American Labor Corp., Durham, NC).…”

Cardiolipin-mediated procoagulant activity of mitochondria contributes to traumatic brain injury–associated coagulopathy in mice

“…Blood samples were collected at multiple points after mice were subjected to FPI (1.9 6 0.1 atm) to measure the dynamic state of coagulation (supplemental Methods, available on the Blood Web site) 12,14,15 and the production of BDMPs and anti-brain antibodies. To test the procoagulant activity of CL and mtMPs without the confounding influence of brain trauma, uninjured C57BL/6J mice (12-16 weeks and 22-25 g; The Jackson Laboratory, Bar Harbor, ME) were injected through the tail vein with bovine heart CL (Avanti Polar Lipids, Alabaster, AL), mouse mtMPs, or phosphate-buffered saline (PBS; pH 7.4).…”

Association of the Plasma Platelet-Derived Microparticles to Platelet Count Ratio with Hospital Mortality and Disseminated Intravascular Coagulopathy in Critically Ill Patients

“…Moreover, Joop and coworkers used FCM and suggested that the PDMP level is simply a reflection of the platelet count 16) . Therefore, according to the findings of several studies, the PDMP/Plts ratio should be a constant value, regardless of the presence of thrombopenia [40][41][42] . In our study using ELISA, the thrombopenia group has significantly elevated PDMP values, and there was a weak but significant negative correlation between the max PDMP value and the nadir platelet count.…”