Hemolytic disorders characterized by
complement-mediated
intravascular hemolysis, such as
autoimmune hemolytic anemia and
paroxysmal nocturnal hemoglobinuria, are often complicated by life-threatening thromboembolic complications. Severe hemolytic episodes result in the release of red blood cell (RBC)-derived proinflammatory and oxidatively reactive mediators (e.g., extracellular
hemoglobin,
heme, and
iron) into plasma. Here, we studied the role of these hemolytic mediators in coagulation activation by measuring
factor Xa (FXa) and
thrombin generation in the presence of RBC lysates. Our results show that hemolytic microvesicles (HMVs) formed during
hemolysis stimulate
thrombin generation through a mechanism involving FVIII and FIX, the so-called
intrinsic tenase complex.
Iron scavenging during
hemolysis using
deferoxamine decreased the ability of the HMVs to enhance
thrombin generation. Furthermore, the addition of
ferric chloride (FeCl3) to plasma propagated
thrombin generation in a FVIII- and FIX-dependent manner suggesting that
iron positively affects blood coagulation.
Phosphatidylserine (PS) blockade using lactadherin and
iron chelation using
deferoxamine reduced
intrinsic tenase activity in a purified system containing HMVs as source of
phospholipids confirming that both PS and
iron ions contribute to the procoagulant effect of the HMVs. Finally, the effects of FeCl3 and HMVs decreased in the presence of ascorbate and
glutathione indicating that oxidative stress plays a role in
hypercoagulability. Overall, our results provide evidence for the contribution of
iron ions derived from hemolytic RBCs to
thrombin generation. These findings add to our understanding of the pathogenesis of
thrombosis in hemolytic diseases.