Review  综述

Haptoglobin Therapeutics and Compartmentalization of Cell-Free Hemoglobin Toxicity
触珠蛋白疗法与游离血红蛋白毒性的区室化作用

Within red blood cells (RBCs), Hb exists as a stable tetramer (64 kD), consistent with its high concentration in the erythrocytes. When diluted within the plasma and extracellular spaces following hemolysis, Hb exists in a dynamic equilibrium of tetramer and αβ-subunit heterodimers. The αβ-dimers are of a relatively small molecular size (32 kD), allowing for protein translocation across tissue barriers such as the renal glomerulus, endothelial barriers in the myocardium and coronary arteries, as well as the different barriers between the CSF and the brain (e.g., the pia mater and ependymal cell layer) [18., 19., 20.]. The mechanisms responsible for Hb decompartmentalization vary depending on the tissue sites (e.g., kidney, brain, and heart). Decompartmentalization from the blood plasma across the glomerular filtration barrier defines the main pathway for cell-free Hb clearance from blood in the absence of haptoglobin. Tissue localized biochemical reactions of Hb and cast formation in the kidney can induce renal injury in animals and humans [18,21,22]. The sequelae of tissue exposure to Hb is most evident by reduced renal function and tubular injury following chronic low-level hemolysis or as overt hemoglobinuria following severe acute hemolysis [23,24]. Hb is also capable of entering the vascular structures and the lymph fluid after hemolysis [25]. However, the mechanisms associated with Hb decompartmentalization from blood plasma or perivascular sites (e.g., the Virchow-Robin space in the brain) into vascular structures or tissue parenchyma are not adequately understood. These processes may occur by transcellular transport or by intercellular trafficking, analogous to experimentally defined processes of albumin transport [26]. In-depth mechanistic definitions of this phenomenon are required for cell-free Hb and its prevention by haptoglobin binding.
在红细胞（RBCs）内，血红蛋白（Hb）以稳定的四聚体形式（64 kD）存在，这与红细胞内高浓度状态相符。当溶血后进入血浆和细胞外空间被稀释时，血红蛋白处于四聚体与αβ亚基异源二聚体的动态平衡中。αβ二聚体分子量相对较小（32 kD），能够穿越肾小球、心肌和冠状动脉的内皮屏障，以及脑脊液与大脑之间的多层屏障（如软脑膜和室管膜细胞层）[18,19,20]。导致血红蛋白去区室化的机制因组织部位（如肾脏、大脑和心脏）而异。在缺乏结合珠蛋白的情况下，血红蛋白从血浆跨越肾小球滤过屏障的去区室化过程构成了游离血红蛋白从血液中清除的主要途径。血红蛋白在组织局部的生化反应及肾脏管型形成可导致动物和人类出现肾损伤[18,21,22]。 组织暴露于血红蛋白（Hb）的后果在慢性低水平溶血后表现为肾功能下降和肾小管损伤，或在严重急性溶血后表现为明显血红蛋白尿[23,24]。溶血后 Hb 还能进入血管结构和淋巴液[25]。然而，关于 Hb 从血浆或血管周围部位（如大脑中的 Virchow-Robin 间隙）解区室化进入血管结构或实质组织的机制尚未充分阐明。这些过程可能通过跨细胞运输或细胞间运输发生，类似于实验定义的清蛋白运输过程[26]。针对游离 Hb 及其被触珠蛋白结合所阻止的现象，需要深入阐明其机制定义。

From a biochemical perspective, Hb-toxicity is determined by heme-iron oxidation and by the reaction of Hb with NO. Following the decompartmentalization of extracellular Hb from the vascular space into the subendothelial space and into smooth muscle layers of arteries, Hb reacts with NO, leading to reduced bioavailability of the vasodilator and, subsequently, systemic, pulmonary, and microvascular vasoconstriction [19,27]. NO depletion by extracellular Hb is a widely accepted hypothesis to explain the acute hypertensive response that occurs during hemolysis and is one mechanism for oxidation to ferric (Fe³⁺) Hb in tissue. NO depletion occurs via two well established reactions, NO dioxygenation (oxy-Hb) and iron nitrosylation (deoxy-Hb), [28]:
从生化角度来看，血红蛋白毒性取决于血红素铁的氧化及其与一氧化氮的反应。当血管外血红蛋白从血管腔室解离进入内皮下层和动脉平滑肌层后，会与一氧化氮发生反应，导致这种血管舒张剂的生物利用度降低，继而引发全身性、肺循环及微血管收缩[19,27]。细胞外血红蛋白消耗一氧化氮这一被广泛接受的假说，可解释溶血过程中出现的急性高血压反应，也是组织中血红蛋白氧化为三价铁（Fe ³⁺ ）的机制之一。一氧化氮消耗通过两种明确反应实现：一氧化氮双加氧反应（氧合血红蛋白）和铁亚硝基化反应（脱氧血红蛋白）[28]：

• NO dioxygenation: HbFe²⁺O₂ + NO → Hb(Fe³⁺OONO•) → HbFe³⁺ + NO₃−
  一氧化氮双加氧反应：HbFe ²⁺ O ₂ + NO → Hb(Fe ³⁺ OONO•) → HbFe ³⁺ + NO ₃ −
• Iron nitrosylation: HbFe²⁺ + NO → Hb(NO)
  铁亚硝基化反应：HbFe ²⁺ + NO → Hb(NO)

Oxidation reactions that lead to higher oxidation states of Hb (i.e., HbFe⁴⁺) and redox cycling are dependent on the presence of supra-physiological concentrations of oxidants (e.g., lipid peroxides, R-OOH and hydrogen peroxide, H₂O₂) and differ based on the initial oxidation state of the heme-iron in Hb. These peroxidation reactions generate oxo-ferryl Hb (1) (HbFe⁴⁺=O) and transient radical species (2) (∙HbFe⁴⁺=O).
导致血红蛋白（Hb）氧化态升高（即 HbFe ⁴⁺ ）和氧化还原循环的氧化反应依赖于超生理浓度氧化剂（如脂质过氧化物 R-OOH 和过氧化氢 H ₂ O ₂ ）的存在，其反应进程会因血红素铁初始氧化态的不同而有所差异。这类过氧化反应会生成氧合正铁血红蛋白（1）（HbFe ⁴⁺ =O）及瞬态自由基（2）（∙HbFe ⁴⁺ =O）。

A third mechanism by which Hb toxicity occurs is through release of heme from HbFe³⁺ [28]. This allows for the transfer of the lipophilic metallo-porphyrin to cell membrane components, soluble plasma proteins, cell surface receptors, and lipids, particularly low-density lipoprotein (LDL) and high-density lipoprotein (HDL) [32., 33., 34.]. The ultimate binding protein for free heme is hemopexin (Hpx), which irreversibly binds heme in a hexacoordinated complex, blocking its oxidative reactivity and delivering it to liver parenchymal cells for degradation [35]. Several proteins with lower heme-binding affinity exist as intermediate heme-carriers in plasma, such as albumin and alpha-1-microglobulin (A1M) [36., 37., 38.], while the small 27-kDa A1M is also a relevant tissue-binding protein for heme [36]. These proteins may modulate heme-distribution between plasma and different tissue compartments and could contribute toward organ-specific toxicity patterns when Hpx is depleted, such as in patients with SCD [39].
血红蛋白毒性的第三种作用机制是通过 HbFe ³⁺ 释放血红素[28]。这使得这种亲脂性金属卟啉能够转移至细胞膜组分、可溶性血浆蛋白、细胞表面受体以及脂质（特别是低密度脂蛋白(LDL)和高密度脂蛋白(HDL)）[32., 33., 34.]。游离血红素的最终结合蛋白是血凝素(Hpx)，其通过六配位复合物不可逆地结合血红素，阻断其氧化活性并将其递送至肝实质细胞进行降解[35]。血浆中存在几种血红素结合亲和力较低的中间载体蛋白，如白蛋白和α-1-微球蛋白(A1M)[36., 37., 38.]，而分子量仅 27-kDa 的 A1M 同时也是重要的组织血红素结合蛋白[36]。当 Hpx 耗竭时（如镰状细胞病(SCD)患者），这些蛋白可能调节血红素在血浆与不同组织区室间的分布，并可能导致器官特异性毒性模式[39]。

One of the most identifiable end-products of heme release are oxidized LDLs and HDLs, oxLDL and oxHDL [16,40., 41., 42.]. Lipid oxidation is considered to be a critical event in the pathogenesis of endothelial dysfunction, progressive vasculopathy, and atherosclerosis in animals and humans [43,44]. Free heme has also been defined as an activator of innate immunity pathways in endothelial cells and leukocytes [45., 46., 47., 48., 49., 50.].
血红素释放最易识别的终产物之一是氧化型低密度脂蛋白(LDL)和高密度脂蛋白(HDL)，即 oxLDL 与 oxHDL[16,40-42]。脂质氧化被认为是导致动物和人类内皮功能障碍、进行性血管病变及动脉粥样硬化发病机制中的关键事件[43,44]。游离血红素还被证实是内皮细胞和白细胞中天然免疫通路的激活剂[45-50]。

Hb and heme are known to trigger adaptive cellular responses that can be protective in certain tissues and during certain pathophysiological conditions (e.g., intraplaque hemorrhage with macrophage polarization toward increased Hb metabolism) [56., 57., 58., 59.]. Heme and other metal-porphyrins can selectively bind to several transcription factors, receptors, and enzymes and thereby alter gene transcription and cellular metabolism. The best defined interaction is the binding of heme to the transcriptional repressor Bach-1, which regulates heme-oxygenase 1 (HMOX) and other antioxidant and iron-metabolism genes essential for the adaptive response to enhanced intracellular heme levels [60,61]. Heme also activates liver-X-receptor (LXR)-coordinated gene expression in macrophages, which may support an anti-inflammatory macrophage phenotype (Mhem) [58,62]. In addition, under certain conditions heme is suggested to be a damage-associated molecular pattern and an agonist of the endotoxin receptor toll like receptor 4 (Tlr4) [47,49,50,63] and an inhibitor of the proteasome [64].
血红蛋白（Hb）和血红素已知能触发适应性细胞反应，这些反应在特定组织及某些病理生理条件下（如斑块内出血伴随巨噬细胞向增强 Hb 代谢方向极化）具有保护作用[56-59]。血红素及其他金属卟啉可选择性地与多种转录因子、受体和酶结合，从而改变基因转录及细胞代谢。其中最明确的相互作用是血红素与转录抑制因子 Bach-1 的结合，该因子调控血红素加氧酶 1（HMOX）及其他抗氧化和铁代谢基因，这些基因对细胞内血红素水平升高的适应性反应至关重要[60,61]。血红素还能激活巨噬细胞中肝脏 X 受体（LXR）协调的基因表达，这可能支持抗炎型巨噬细胞表型（Mhem）的形成[58,62]。此外，在某些条件下，血红素被认为是一种损伤相关分子模式，可作为内毒素受体 Toll 样受体 4（Tlr4）的激动剂[47,49,50,63]及蛋白酶体的抑制剂[64]。

Most critical to homeostasis of Hb, heme, and iron, and for the prevention of Hb-driven toxicity, are the three high-affinity binding and transport proteins that are abundant in plasma. These include haptoglobin, Hpx, and transferrin (ApoTf) [65,66]. Depending on the nature of the hemolytic condition, supplementation with these proteins as therapeutic agents may prove effective at controlling the acute and chronic disease-modifying effects of Hb and its degradation products [67., 68., 69., 70.]. Figure 1 illustrates the progression of RBC damage and intravascular hemolysis that results in Hb and heme release and transfer to haptoglobin and Hpx, respectively. Erythrophagocytosis of damaged or senescent RBCs by macrophages and the subsequent export of iron through ferroportin [71] leads to transferrin saturation and, eventually, non-transferrin bound iron and labile plasma iron [72,73]. From this graphical representation of hemolysis, we focus on the critical role of haptoglobin in the prevention of cell-free Hb exposure in the kidney, cardiovascular system, lung, and brain. Herein, we suggest and describe a mechanism of compartmentalization that underpins the potential therapeutic efficacy of haptoglobin as critical modifier of the downstream toxicological effects of intravascular and tissue hemolysis.
对于维持血红蛋白（Hb）、血红素和铁的稳态以及预防 Hb 介导的毒性作用最为关键的，是血浆中含量丰富的三种高亲和力结合转运蛋白：结合珠蛋白（haptoglobin）、血色素结合蛋白（Hpx）和转铁蛋白（ApoTf）[65,66]。根据溶血性疾病的性质，补充这些蛋白作为治疗剂可能有效控制 Hb 及其降解产物引发的急慢性疾病修饰效应[67-70]。图 1 展示了红细胞损伤与血管内溶血导致 Hb 和血红素释放，并分别转移至结合珠蛋白与血色素结合蛋白的过程。巨噬细胞对损伤或衰老红细胞的吞噬作用，以及随后通过膜铁转运蛋白输出的铁[71]，将导致转铁蛋白饱和并最终形成非转铁蛋白结合铁和不稳定血浆铁[72,73]。基于该溶血过程的图示，我们重点探讨结合珠蛋白在防止肾脏、心血管系统、肺部和大脑接触游离 Hb 方面的关键作用。 本文提出并阐述了一种区室化机制，该机制是触珠蛋白作为血管内和组织溶血下游毒性效应关键调节剂发挥潜在治疗功效的基础。

SCD is a genetic hemolytic anemia with multiple contributing comorbidities and each is affected by numerous molecular and biochemical changes. SCD originates from a mutation in the beta-Hb gene on chromosome 11 (11p15.1) that is characterized by a genotype-CAG codon to GTG. A glutamic acid replacing valine at the sixth amino acid position within the Hb beta chain leads to the sickle cell phenotype (HbS) [79]. This seemingly minor modification facilitates the polymerization of Hb during tense state (T-deoxyHb) to relaxed state (R-oxyHb) transitioning, which promotes ‘sickling’ morphological changes and, ultimately, hemolysis of erythrocytes [80,81]. As a result, drug development efforts have targeted antipolymerization strategies to dilute intraerythrocytic HbS by increasing fetal Hb with oral hydroxyurea therapy [82,83] and to increase Hb oxygen affinity [84., 85., 86., 87.], using curative efforts such as gene therapy [88., 89., 90.], as well as comprehensive strategies to address the acute and chronic progressive debilitating sequelae of SCD [91].
镰状细胞病（SCD）是一种遗传性溶血性贫血，伴随多种共病状态，每种并发症均受众多分子与生化改变的影响。该病源于 11 号染色体（11p15.1）β-血红蛋白基因突变，其特征为 CAG 密码子被 GTG 取代。血红蛋白β链第六位氨基酸的谷氨酸被缬氨酸替代，导致镰状血红蛋白（HbS）表型的形成[79]。这一看似微小的修饰促进了血红蛋白在紧张态（T-脱氧 Hb）向松弛态（R-氧合 Hb）转换过程中的聚合，从而引发红细胞"镰变"形态学改变并最终导致溶血[80,81]。因此，药物研发主要针对以下策略：通过羟基脲口服疗法增加胎儿血红蛋白以稀释红细胞内 HbS 的抗聚合策略[82,83]；提高血红蛋白氧亲和力[84-87]；采用基因治疗等根治性手段[88-90]；以及针对 SCD 急慢性进行性衰弱后遗症的综合治疗策略[91]。

Human SCD patients experience a range of end organ damage that results in whole body pathophysiology [92., 93., 94., 95., 96., 97., 98., 99., 100., 101.]. The end organ complications associated with SCD are multifactorial and the impact of free Hb and its metabolic end-products on disease progression have been debated extensively [102., 103., 104., 105., 106.]. Preclinical proof-of-concept studies have focused on the use of haptoglobin in SCD animal models to prevent end organ injury specific to the vasculature, kidneys, and lungs. In these studies, haptoglobin administration seems to attenuate disease progression in both acute and subchronic settings. Acute studies with haptoglobin therapy suggest potential utility in reducing vascular injury and vaso-occlusive crisis in murine SCD [107,108]. Similarly, haptoglobin dosing in subchronic settings suggests reduction of renal tubule Hb accumulation [109], with no demonstrable improvement in overall kidney function [110]. Human SCD with pulmonary hypertension and lung injury appears to be influenced by Hb, hypoxia, and, subsequently, macrophage reprogramming consistent with both stressors [111]. A rat model designed to mimic the effects of hypoxia and Hb on pulmonary hypertension and right heart failure suggests that progression of pulmonary vascular remodeling and right heart dysfunction are attenuated by haptoglobin dosing, administered multiple times per week [76]. Within these organ systems (i.e., vasculature, kidney, and lung), evidence is emerging that haptoglobin has a positive therapeutic effect in preclinical models of SCD.
镰状细胞病（SCD）患者会出现一系列终末器官损伤，导致全身性病理生理变化[92-101]。与 SCD 相关的终末器官并发症具有多因素性，游离血红蛋白及其代谢终产物对疾病进展的影响一直存在广泛争议[102-106]。临床前概念验证研究主要聚焦于在 SCD 动物模型中使用结合珠蛋白（haptoglobin）预防特定血管系统、肾脏和肺部的终末器官损伤。这些研究表明，无论是急性还是亚慢性状态下，给予结合珠蛋白都能延缓疾病进展。急性期结合珠蛋白治疗研究显示，该疗法可能有助于减轻小鼠 SCD 模型的血管损伤和血管阻塞危象[107,108]；而在亚慢性给药方案中，结合珠蛋白显示出减少肾小管血红蛋白积聚的作用[109]，但未观察到整体肾功能改善[110]。人类 SCD 合并肺动脉高压和肺损伤的病理过程似乎受到血红蛋白、低氧以及随之发生的巨噬细胞重编程（与上述双重应激因素相关）的共同影响[111]。 一项旨在模拟缺氧和血红蛋白对肺动脉高压及右心衰竭影响的大鼠模型研究表明，每周多次给予结合珠蛋白治疗可减轻肺血管重塑进展和右心功能障碍[76]。在这些器官系统（即脉管系统、肾脏和肺部）中，越来越多的证据表明结合珠蛋白在镰状细胞病的临床前模型中具有积极治疗效果。

During sepsis, significant changes in RBC morphology [112], oxidative stress [113], oxygen delivery [114], and circulatory clearance [115] have been reported. Consistent with a process of sepsis-induced hemolysis, plasma levels of cell-free Hb are increased in patients with sepsis [116] and higher plasma concentrations of cell-free Hb coincided with higher mortality [117]. Accordingly, plasma concentrations of haptoglobin and Hpx are often depleted [118], while transferrin saturations are generally increased [119] in critically ill septic patients. Plasma Hb levels in these studies were consistent with a mild hemolysis (10–40 mg/dl, or ~5–25 μM heme in plasma). Sepsis with concomitant transfusion of RBCs increases hemolysis and exposures to cell-free Hb [120,121] and its degradation products, heme [122] and iron [123,124], in experimental models and clinical disease [125,126]. However, the true contribution of hemolysis and cell-free Hb ‘toxicity’ toward end organ injury in sepsis is not fully understood.
脓毒症期间，已报道红细胞形态[112]、氧化应激[113]、氧输送[114]和循环清除率[115]发生显著变化。与脓毒症诱导的溶血过程一致，脓毒症患者血浆中游离血红蛋白水平升高[116]，且较高浓度的血浆游离血红蛋白与更高死亡率相关[117]。相应地，危重脓毒症患者的结合珠蛋白和血凝素血浆浓度常出现耗竭[118]，而转铁蛋白饱和度通常升高[119]。这些研究中的血浆血红蛋白水平符合轻度溶血特征（10-40 mg/dl，或约 5-25 μM 血浆血红素）。在实验模型和临床疾病中[125,126]，脓毒症伴随红细胞输注会加剧溶血，增加游离血红蛋白[120,121]及其降解产物血红素[122]和铁[123,124]的暴露。然而，溶血和游离血红蛋白"毒性"对脓毒症终末器官损伤的真实作用机制尚未完全阐明。

Animal models of endotoxemia and sepsis have been evaluated to assess the contributions of cell-free Hb to acute kidney injury [67,127,128], acute lung injury (ALI), and acute respiratory distress syndrome [129], as well as to cardiac injury [75]. The primary studies that have developed proof-of-concept understanding on the feasibility of haptoglobin administration in sepsis were conducted with concomitant Hb exposure or blood transfusion. Haptoglobin administration was evaluated in sepsis-free animal models with extensive Hb exposure following transfusion of poor quality RBCs. In these studies, haptoglobin attenuated the deleterious effects of Hb and its degradation products on the kidneys and the vasculature [68,74,77]. Haptoglobin also attenuated lipopolysaccharide (LPS)-enhanced Hb cardiomyocyte toxicity in a guinea pig model of endotoxemia, [75] which supports data that LPS increases Hb-mediated end organ injury as well as mortality in sepsis animal models [130., 131., 132.]. These observations are also consistent with lung injury and mortality endpoint studies in septic dogs, where both ALI and survival were improved following haptoglobin administration in the absence and in the presence of concomitant RBC transfusion [133]. Nonetheless, sepsis remains a complex indication and a challenge for therapeutic development. For example, interpretation of human dosing from animal studies is not accurate, because clearance mechanisms in humans differ from species commonly used in preclinical studies [134., 135., 136.]. Further, some bacterial pathogens express receptors that bind Hb–haptoglobin complexes in their efforts to obtain iron for nutrient metabolism, which may impact safety and efficacy of a haptoglobin therapy in human infection, depending on the bacterial strain and its adaptation to acquisition of Hb-derived iron sources [137].
已通过内毒素血症和脓毒症动物模型评估了游离血红蛋白对急性肾损伤[67,127,128]、急性肺损伤（ALI）及急性呼吸窘迫综合征[129]以及心脏损伤[75]的作用机制。关于结合珠蛋白在脓毒症中应用可行性的概念验证研究，主要在同时存在血红蛋白暴露或输血条件下开展。研究人员还在无脓毒症的动物模型中评估了结合珠蛋白的疗效，这些模型通过输注劣质红细胞造成大量血红蛋白暴露。研究表明，结合珠蛋白能有效减轻血红蛋白及其降解产物对肾脏和血管系统的损害[68,74,77]。 在豚鼠内毒素血症模型中，结合珠蛋白同样减轻了脂多糖（LPS）增强的血红蛋白心肌细胞毒性[75]，该结果支持了 LPS 会增加血红蛋白介导的终末器官损伤及脓毒症动物模型死亡率的数据[130-132]。这些观察结果也与脓毒症犬的肺损伤和死亡率终点研究一致——无论是否伴随红细胞输注，给予结合珠蛋白后均能改善急性肺损伤（ALI）和生存率[133]。然而脓毒症仍是治疗开发的复杂适应症和重大挑战。例如，从动物研究推算人类给药剂量并不准确，因为人体的清除机制与临床前研究常用物种存在差异[134-136]。此外，某些细菌病原体为获取铁营养代谢会表达结合血红蛋白-结合珠蛋白复合物的受体，这可能影响结合珠蛋白疗法在人类感染中的安全性和有效性，具体取决于细菌菌株及其对血红蛋白来源铁获取的适应能力[137]。

SAH typically results from an aneurysmal malformation of a large cerebral artery. This type of intracranial bleeding accounts for 5–10% of all strokes in the USA [138]. The loss of productive life years due to aneurysmal SAH (aSAH) is similar to the cumulative morbidity caused by cerebral infarction, since it often affects patients younger than 65 years of age [139]. A major contributor to poor outcome in patients with SAH is delayed ischemia with secondary brain damage and irreversible neurological deficit [140]. This complication occurs between 4 and 14 days after SAH and is caused by transient deficits in brain perfusion. This hypoperfusion is caused in part by vasospasm of large cerebral arteries and by a dysregulated microcirculation resulting from small vessel vasoconstriction or thrombotic occlusion [141]. The pathophysiology of delayed ischemia is complex, multifactorial, and incompletely understood. However, lysis of RBC and release of cell-free Hb into the subarachnoid space have been suspected as potential drivers of delayed neurological damage for decades [142]. A recent study demonstrated that injection of cell-free Hb into the subarachnoid space of sheep was sufficient to cause severe spasm of all major cerebral arteries, as well as of small arteries within the brain parenchyma [20]. These effects of Hb were correlated with the delocalization of Hb deep into the neuronal brain tissue and into the interstitial space compartment of the arterial vascular smooth muscle layer of cerebral arteries. Haptoglobin completely blocked this cell-free Hb delocalization from the CSF compartment into the brain parenchyma and the NO-sensitive compartments of cerebral arteries. This confinement of cell-free Hb within the CSF prevented spasm of large and small cerebral arteries and it may also protect susceptible neuronal structures and microglia against oxidative and proinflammatory effects of cell-free Hb. The protective activity of haptoglobin was reproducible in an ex vivo model of basilar artery vasospasm that was induced by exposure of porcine basilar arteries to Hb-rich CSF collected from patients with delayed ischemia after an aSAH [20]. Also in the CSF environment, haptoglobin did not alter the NO reaction kinetics of bound Hb, reinforcing the exclusion of NO-reactive Hb from NO-sensitive tissue compartments as the primary mechanism of protection. In a mouse model, haptoglobin was also effective in restricting brain parenchymal delocalization and mitigating neuronal toxicity that was induced by continuous intrathecal administration of cell-free Hb for 2 weeks [143].
蛛网膜下腔出血（SAH）通常由大脑动脉瘤性血管畸形导致。此类颅内出血占美国所有脑卒中病例的 5%-10%[138]。由于动脉瘤性 SAH（aSAH）好发于 65 岁以下患者，其造成的有效生命年损失与脑梗死累积发病率相当[139]。SAH 患者预后不良的主要原因是迟发性脑缺血引发的继发性脑损伤及不可逆神经功能缺损[140]。该并发症发生于 SAH 后 4-14 天，由脑灌注短暂性不足引起。这种低灌注部分源于大脑血管痉挛，以及小血管收缩或血栓性闭塞导致的微循环失调[141]。迟发性缺血的病理生理机制复杂多元，目前尚未完全阐明。但数十年来，红细胞溶解导致游离血红蛋白释放至蛛网膜下腔，始终被视为迟发性神经损伤的潜在驱动因素[142]。 最近一项研究表明，将游离血红蛋白注射入绵羊蛛网膜下腔足以引发所有主要脑动脉及脑实质内小动脉的严重痉挛[20]。血红蛋白的这些效应与其向神经元脑组织深部及脑动脉血管平滑肌层间质空间的迁移密切相关。触珠蛋白能完全阻断游离血红蛋白从脑脊液腔室向脑实质及脑动脉一氧化氮敏感区域的迁移。将游离血红蛋白限制在脑脊液腔内可预防大小脑动脉痉挛，同时可能保护易损神经元结构和微胶质细胞免受游离血红蛋白的氧化及促炎作用。触珠蛋白的保护作用在基底动脉血管痉挛的离体模型中得到重现，该模型通过将猪基底动脉暴露于 aSAH 后迟发性脑缺血患者富含血红蛋白的脑脊液而诱导产生[20]。 同样在脑脊液环境中，结合珠蛋白并未改变与血红蛋白结合的 NO 反应动力学，这进一步证实了其保护机制主要是将具有 NO 反应活性的血红蛋白隔离于 NO 敏感组织区室之外。在小鼠模型中，持续两周鞘内注射游离血红蛋白诱导的脑实质扩散和神经元毒性，结合珠蛋白也能有效限制其扩散并减轻毒性作用[143]。

In patients with aSAH, the haptoglobin concentrations in CSF are orders of magnitudes below the cell-free Hb concentrations that accumulate within a few days after the initial bleeding [144]. However, because hemolysis in the subarachnoid space occurs with a delay of several days after the bleeding event [145], it may be possible to administer enough purified or recombinant haptoglobin into the CSF compartment via a ventricular drainage catheter to supplement sufficient Hb scavenger capacity to prevent Hb-driven complications.
在动脉瘤性蛛网膜下腔出血（aSAH）患者中，脑脊液（CSF）中的结合珠蛋白浓度比出血后数日内积累的游离血红蛋白浓度低数个数量级[144]。然而，由于蛛网膜下腔的溶血现象在出血事件后数日才会延迟发生[145]，因此有可能通过脑室引流导管向 CSF 腔隙内输注足量纯化或重组结合珠蛋白，以补充足够的血红蛋白清除能力，从而预防血红蛋白驱动的并发症。