No abstract

No abstract

Complement-mediated autoimmune hemolytic anemia (CM-AIHA) is characterized by destruction of red blood cells (RBCs) by autoantibodies that activate the classical complement pathway. These antibodies also reduce transfusion efficacy via lysis of donor RBCs. Since C1-inhibitor (C1-INH) is an endogenous regulator of the classical complement pathway, we hypothesized that peritransfusional C1-INH in patients with severe CM-AIHA reduces complement activation and hemolysis, and thus enhance RBC transfusion efficacy. We conducted a prospective, single center, phase 2 open-label trial (EudraCT2012-003710-13). Patients with confirmed CM-AIHA and indication for the transfusion of two RBC units were eligible for inclusion. Four intravenous C1-INH doses (6000, 3000, 2000 and 1000 U) were given with 12 h intervals around RBC transfusion. Serial blood samples were analyzed for hemolytic activity, RBC opsonization, complement activation and inflammation markers. Ten patients were included in the study. C1-INH administration increased plasma C1-INH antigen and -activity, peaking at 48 h after the first dose, accompanied by a significant reduction of RBC C3d deposition. Hemoglobin levels increased briefly after transfusion but returned to baseline within 48 h. Overall, markers of hemolysis, inflammation and complement activation remained unchanged. Five grade 3 and one grade 4 adverse event occurred, but were considered unrelated to the study medication. In conclusion, peritransfusional C1-INH temporarily reduced complement activation. However, C1-INH failed to halt hemolytic activity in severe transfusion-dependent CM-AIHA. We cannot exclude that posttransfusional hemolytic activity would have been even higher without C1-INH. The potential of complement inhibition on transfusion efficacy in severe CM-AIHA remains to be determined.

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.

Background C1-inhibitor (C1-inh) therapeutics can reduce neutrophil activity in various inflammatory conditions. This ‘novel’ anti-inflammatory effect of C1-inh is attributed to the tetrasaccharide sialyl LewisX (SLeX) present on its N-glycans. Via SLeX, C1-inh is suggested to interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain plasma glycoprotein α1-antichymotrypsin (ACT) as a co-purified protein impurity. Objective This article investigates the contribution of ACT to the effects observed with C1-inh. Materials and Methods We have separated C1-inh and ACT from a therapeutic C1-inh preparation and investigated the influence of these proteins on SLeX–selectin interactions in a specific in vitro model, which makes use of rolling of SLeX-coated beads on immobilized E-selectin. Results We find that ACT and not C1-inh, shows a clear sialic acid-dependent interference in SLeX–selectin interactions, at concentrations present in C1-inh therapeutics. Furthermore, we do not find any evidence of SLeX on C1-inh using either Western blotting with anti-SLeX antibodies (CSLEX1 and KM93) or by mass spectrometric analysis of N-glycans. C1-inh reacts weakly to antibody HECA-452, which detects a broad range of selectin ligands, but ACT gives a much stronger signal, suggesting the presence of a selectin ligand on ACT. Conclusion The ‘novel’ anti-inflammatory effects of C1-inh are unlikely due to SLeX on C1-inh and can in fact be due to SLeX-like glycans on ACT, present in C1-inh products. In view of our results, it is important to assess the role of ACT in vivo and revisit past studies performed with commercial C1-inh.

No abstract

Immune haemolytic anaemia (IHA) is characterized by an increased breakdown of red blood cells (RBCs) due to allo-or auto-antibodies directed to RBC antigens with or without complement activation. Based on the nature of the antibodies, IHA can be divided in three main categories: autoimmune, drug-induced and alloimmune-mediated IHA. There is growing evidence that the innate immune system plays an important role in the pathogenesis of IHA. Complement-mediated haemolysis with the subsequent release of cell-free haemoglobin and cellfree haem resulting in the generation of reactive oxygen species and cytotoxicity as well as the production of anaphylatoxins induce a systemic inflammatory response, which contributes to morbidity and mortality in IHA. The natural plasma scavengers of cell-free haemoglobin and cell-free haem, haptoglobin and hemopexin, respectively, are depleted in cases of chronic or severe IHA. The inducible enzyme haem oxygenase 1 (HO-1) is an efficient cellular scavenger degrading haem into anti-inflammatory products to partially limit haemmediated oxidative damage in cases of saturated scavenging capacity. Complement-targeted therapy and the therapeutic replenishment of haptoglobin and hemopexin as well as the induction of HO-1 expression might be suitable targets in the treatment of IHA.

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 pro-inflammatory 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 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.