Procoagulant Activity In Stored Units Of Red Blood Cells

It has been observed that the procoagulant activity of stored units of red blood cells (RBC) increases over time, which is, in part, related to the expression/exposure of tissue factor (TF) on blood cells and microparticles (Keating et al, Transfusion 2011;51:1086). However, in many instances there is a discrepancy between the levels of TF measured in stored units of RBC and changes in procoagulant activity observed over the storage time. We hypothesized that, in addition to TF, an exposure of an acidic phospholipid, phosphatidylserine (PS), on blood cells (Whelihan et al, Blood 2012;120:3837), could be the cause of an elevated procoagulant activity.

Four healthy donors were recruited. RBC units were prepared and stored at 4°C for 30 days. On selected days, aliquots were removed, reconstituted with autologous plasma and recalcified, and were tested in the presence of corn trypsin inhibitor in the thromboelastography assay for procoagulant activity. For all 4 donors, the clotting time decreased from ∼3000-4000 s on day 1 to ∼1000-1600 s on day 30, with the most dramatic changes occurring between days 1 and 10. Addition of an inhibitory anti-TF antibody slightly prolonged clotting times, suggesting the presence of endogenous TF in RBC units. The concentration of TF, quantitated in an activity-based assay, did not change significantly over time for 3 of 4 donors and was within the range of 40-130 fM. For the fourth donor, TF concentration reached a maximum of 310 fM on day 10 and then decreased steadily to 60 fM on day 30.

In an attempt to identify other components in RBC units responsible for the procoagulant activity, the effect of lactadherin, which binds to the phosphatidylserine exposed on cell membranes, was investigated. An addition of 100 nM lactadherin substantially prolonged the clotting time of the reconstituted RBC. Thus, in days 3-5 the clotting time in the presence of lactadherin was by 2.4-3.4-fold longer than in the absence of it. With increasing age of the RBC units, the effect of lactadherin diminished and at day 30, a prolongation of the clotting time did not exceed 1.3-1.8-fold. The addition of anti-TF monoclonal antibody in the presence of lactadherin further enhanced the clotting time of the reconstituted RBC.

In conclusion, our data indicate that in stored units of RBC, the procoagulant activity is dependent upon the presence of active monocyte TF and phosphatidylserine exposed on blood cells and platelets. However, the failure of lactadherin in combination with the anti-TF antibody to completely inhibit the procoagulant activity in RBC units suggests that other components of blood (most likely platelets) support this activity as well.