Microvesicles in stored red blood cells activate the contact pathway resulting in both FXIIa- and kallikrein-mediated activation of FIX.
These pathways are potential targets to prevent thrombotic or inflammatory complications of red cell transfusion.
Storage lesion-induced red cell-derived microvesicles (RBC-MVs) propagate coagulation by supporting the assembly of the prothrombinase complex. It has also been reported that RBC-MVs initiate coagulation via the intrinsic pathway. To elucidate the mechanism(s) of RBC-MV-induced coagulation activation, the ability of storage lesion-induced RBC-MVs to activate each zymogen of the intrinsic pathway was assessed in a buffer system. Simultaneously, the thrombin generation (TG) assay was used to assess their ability to initiate coagulation in plasma. RBC-MVs directly activated factor XII (FXII) or prekallikrein, but not FXI or FIX. RBC-MVs initiated TG in normal pooled plasma and in FXII-deficient or FXI-deficient, but not in FIX-deficient plasma, suggesting an alternate pathway that bypasses both FXII and FXI. Interestingly, RBC-MVs generated FIXa in a prekallikrein-dependent manner. Similarly, purified kallikrein activated FIX in buffer and initiated TG in normal pooled plasma, as well as FXII-deficient or FXI-deficient, but not FIX-deficient plasma. Dual inhibition of FXIIa by corn trypsin inhibitor and kallikrein by soybean trypsin inhibitor was required to abolish RBC-MV-induced TG in normal pooled plasma, whereas kallikrein inhibition alone was sufficient to abolish TG in FXII-deficient or FXI-deficient plasma. Heating RBC-MVs at 60°C for 15 minutes or pre-treatment with trypsin abolished TG, suggesting the presence of microvesicle-associated protein(s) that is(are) required for contact activation. In summary, RBC-MVs activate both FXII and prekallikrein, leading to FIX activation by two independent pathways, namely the classical FXIIa-FXI-FIX pathway and via direct kallikrein activation of FIX. These data suggest novel mechanisms by which RBC transfusion mediates inflammatory and/or thrombotic outcomes.