Joseph Kusumam, Tholanikunnel Baby G, Kaplan Allen P
Department of Medicine, Division of Pulmonary and Critical Care, Allergy and Clinical Immunology and Konishi-MUSC, Institute for Inflammation Research, 96 Jonathan Lucas Street, P.O. Box 250623, Charleston, SC 29425, USA.
Int Immunopharmacol. 2002 Dec;2(13-14):1851-9. doi: 10.1016/s1567-5769(02)00186-8.
Bradykinin is a major mediator of swelling in C1 inhibitor deficiency as well as the angioedema seen with ACE inhibitors and may contribute to bronchial hyper-reactivity in asthma. Formation of bradykinin occurs in the fluid phase and along cell surfaces requiring interaction of Factor XII, prekallikrein and high molecular weight kininogen (HK). The mechanism by which initiation occurs is uncertain. Recent data suggest that activation of the kinin-forming cascade can occur on the surface of endothelial cells, even in the absence of Factor XII. We demonstrate herein that during a 2-h incubation time, plasma deficient in either Factor XII or high molecular weight kininogen (HK) fail to activate kinin-forming cascade as compared to normal plasma. With more prolonged incubation, Factor XII deficient plasma gradually activates and HK deficient plasma does not. Our data support both Factor XII-dependent (rapid) and Factor XII-independent (slow) mechanisms; the latter may require a cell-derived protein (possibly protease) to activate prekallikrein in the presence of zinc ion and HK. To further define this cellular factor, we demonstrated that both cytosolic and membrane fractions from endothelial cells possessed the ability to catalyze prekallikrein conversion to kallikrein in the presence of HK and zinc ion. We purified this factor from cytosol by affinity chromatography employing corn trypsin inhibitor (CTI) as ligand. The fractions with peak activity were subjected to SDS-PAGE analysis, ligand blotted with biotinylated CTI, and positive bands were sequenced. Heat shock protein 90 (Hsp90) was identified as one of the proteins. Zinc-dependent activation of the prekallikrein-HK complex on endothelial cells was inhibited upon the addition of polyclonal antibody to Hsp90 in a dose-dependent manner. Although the mechanism by which Hsp90 activates the kinin-forming cascade is not yet clear, this protein represents the cellular contribution to the reaction and may become the dominant mechanism in pathologic circumstances in which Hsp90 is highly expressed or secreted.
缓激肽是C1抑制剂缺乏时肿胀以及使用血管紧张素转换酶（ACE）抑制剂时出现血管性水肿的主要介质，并且可能导致哮喘中的支气管高反应性。缓激肽的形成发生在液相以及细胞表面，需要因子XII、前激肽释放酶和高分子量激肽原（HK）相互作用。起始发生的机制尚不确定。最近的数据表明，即使在没有因子XII的情况下，激肽形成级联反应也可在内皮细胞表面发生激活。我们在此证明，在2小时的孵育时间内，与正常血浆相比，缺乏因子XII或高分子量激肽原（HK）的血浆无法激活激肽形成级联反应。随着孵育时间延长，缺乏因子XII的血浆逐渐激活，而缺乏HK的血浆则不会。我们的数据支持因子XII依赖性（快速）和因子XII非依赖性（缓慢）机制；后者可能需要一种细胞衍生蛋白（可能是蛋白酶）在锌离子和HK存在的情况下激活前激肽释放酶。为了进一步确定这种细胞因子，我们证明内皮细胞的胞质和膜部分在HK和锌离子存在下均具有催化前激肽释放酶转化为激肽释放酶的能力。我们使用玉米胰蛋白酶抑制剂（CTI）作为配体，通过亲和色谱从胞质溶胶中纯化了该因子。对具有峰值活性的部分进行SDS-PAGE分析，用生物素化的CTI进行配体印迹，对阳性条带进行测序。热休克蛋白90（Hsp9）被鉴定为其中一种蛋白质。向内皮细胞上的前激肽释放酶-HK复合物中加入Hsp90多克隆抗体后，锌依赖性激活以剂量依赖性方式受到抑制。尽管Hsp90激活激肽形成级联反应的机制尚不清楚，但该蛋白代表了细胞对该反应的贡献，并且可能在Hsp90高度表达或分泌的病理情况下成为主要机制。
