Magnesium Augments Platelet Activation and Coagulation in Citrated Blood Product

Abstract 3438

Massive transfusion is defined as infusion of 10 units of red blood cells in 24 hours during treatment of uncontrolled bleeding. Mortality rates are 28.3% higher for patients that undergo massive transfusion. Administered blood products are collected from donors using citrate inhibition for anti-coagulation. Citrate chelates calcium, thus preventing its interactions with clotting enzymes and eliminating coagulation activity. Severe trauma patients suffer compromised liver function slowing citrate clearance and further reducing calcium availability. Under this scenario, calcium is empirically replaced due to concerns regarding hemostasis, however magnesium, which is also chelated by citrate, is not empirically replaced. Our goal was to describe both platelet aggregation and coagulation under conditions of increasing magnesium concentration following citrate chelation to ascertain the true spectrum of magnesium influence upon hemostasis.

To analyze platelet activation/aggregation, de-identified whole blood (WB) was anti-coagulated with citrate (10.9mM) and corn trypsin inhibitor (CTI) (0.1mg/mL), or citrate and 50μL pPACK (final). Platelet-rich plasma (PRP) was prepared through centrifugation at 200g for 10 minutes. Subsequent spin at 2000g for 10 minutes yielded platelet-poor plasma (PPP). Remaining packed red blood cells were combined with PRP (platelets calcein-tagged to 10μM final) to simulate 40% hematocrit. Shear rate of 920/sec was induced for arterial flow conditions for 10 minutes at 37°C through a microchannel (350μm W × 70μm H × 5000μm L) pre-coated with a solution of 100μg/mL type-I collagen blocked with 0.5% BSA/1X PBS. Fluorescent signal intensity of calcein-tagged platelets was quantified through sequential image capture every 30 seconds using Fluxion BioFlux 1000. For coagulation analyses, citrate/CTI inhibited WB, PRP, and PPP was analyzed via thromboelastograph (TEG) following large volume pre-mixing with tissue factor (1:5000). TEG cups were preloaded with varying concentrations of calcium chloride (0M, 5.5mM, 10.9mM, 15mM), or calcium chloride (15mM) with magnesium sulfate (0M, 3mM, 3.5mM, 4.5mM) before blood or component addition. TEG data were collected for 90 minutes. Statistical analyses were performed with GraphPad Prism 5 software. Comparisons were drawn using linear analysis and area under the curve.

Data indicate that re-attainment of physiological magnesium concentration in citrated blood: (1) facilitates platelet activation/aggregation, and (2) augments coagulation parameters through accelerated clot time and increased clot strength. Rate of platelet aggregation was significantly increased at final magnesium concentrations of 1.5mM (p<0.01), and 3mM (p<0.005) when added to re-calcified WB. Further, addition of magnesium to PPP reduced time to initiation of clot formation (4.5mM), increased clot strength (1mM, 3mM), and increased rate of clot formation (1mM, 3mM). These data suggest supplemental magnesium improves hemostatic properties in citrated WB and PPP with or without re-calcification. The incidence and severity of hypocalcemia and hypomagnesimia in massive transfusion, and effects on hemostatic function remain to be established. Replacement of magnesium during massive transfusion may represent a therapeutic opportunity.