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.

以补体介导的血管内溶血为特征的溶血性疾病，如自身免疫性溶血性贫血和阵发性夜间血红蛋白尿，常常并发危及生命的血栓栓塞并发症。严重的溶血事件导致红细胞 (RBC) 衍生的促炎和氧化反应介质（例如，细胞外血红蛋白、血红素和铁）释放到血浆中。在这里，我们通过测量 Xa 因子 (FXa) 和在 RBC 裂解物存在下的凝血酶生成来研究这些溶血介质在凝血激活中的作用。我们的研究结果表明，溶血过程中形成的溶血微泡 (HMV) 通过涉及 FVIII 和 FIX 的机制刺激凝血酶的产生，即所谓的内在 Tenase 复合物。在溶血过程中使用去铁胺清除铁会降低 HMV 增强凝血酶生成的能力。此外，添加氯化铁（FeCl₃ ) 以 FVIII 和 FIX 依赖性方式传播到血浆中的凝血酶生成，表明铁对血液凝固有积极影响。在含有 HMV 作为磷脂来源的纯化系统中，使用乳粘素的磷脂酰丝氨酸 (PS) 阻断和使用去铁胺的铁螯合降低了内在张力酶活性，这证实了 PS 和铁离子都有助于 HMV 的促凝作用。最后，在抗坏血酸和谷胱甘肽存在的情况下，FeCl ₃和 HMV 的作用降低，表明氧化应激在高凝状态中起作用。总体而言，我们的结果提供了来自溶血性红细胞的铁离子对凝血酶生成的贡献的证据。这些发现增加了我们对溶血性疾病血栓形成发病机制的理解。