Prekallikrein and high-molecular-weight kininogen were found associated in normal human plasma at a molecular weight of 285,000, as assessed by gel filtration on Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was 115,000. This prekallikrein could be isolated at a molecular weight of 285,000 after plasma deficient in high-molecularweight kininogen was combined with plasma that is congenitally deficient in prekallikrein. Addition of purified 125I-labeled prekallikrein and high-molecular-weight kininogen to the respective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated by incubating prekallikrein with high-molecular weight kininogen. This study demonstrates that prekallikrein and highmolecular-weight kininogen are physically associated in plasma as a noncovalently linked complex and may therefore be adsorbed together during surface activation of Hageman factor. The complex is disrupted when these proteins are isolated by ion exchange chromatography.Activation of Hageman factor upon a negatively charged surface initiates the intrinsic coagulation pathway, the fibrinolytic pathway, and the generation of the vasoactive peptide bradykinin. Recent investigations from several laboratories have shown that the proteins of the kinin-forming pathway, namely, prekallikrein (1, 2) and high-molecular-weight (HMW) kininogen (3-5), are both required for optimal activation and function of Hageman factor. Since prekallikrein and HMW kininogen are intimately associated functionally, it appeared possible that they might be physically associated in plasma. In this paper we demonstrate that prekallikrein and HMW kininogen circulate in plasma as a noncovalently linked complex. Formation of this complex was observed: (a) when prekallikrein-deficient plasma was combined with plasma deficient in HMW kininogen, (b) after prekallikrein-deficient plasma and plasma deficient in HMW kininogen were reconstituted with prekallikrein and HMW kininogen, respectively, and (c) when purified prekallikrein was incubated with HMW kininogen. Preparation of Plasma Proteins. Fresh plasma used for the isolation of prekallikrein and HMW kininogen was collected in 0.38% sodium citrate. Hexadimethrine bromide (3.6 mg) in 0.1 ml of 0.15 M saline was added for each 10 ml of blood drawn. The tubes were then centrifuged at 900 X g for 20 min at 40 and the plasma was separated with plastic pipettes. Plastic columns and test tubes were used throughout the chromatographic procedures to minimize contact activation of Hageman factor and nonspecific adsorption to glass surfaces. Samples were concentrated by ultrafiltration (Amicon Corp., Lexington, Mass.) through a UM-10 membrane. MATERIALS AND METHODSHageman Factor Fragments. Prealbumin fragments of Hageman factor were purified by chromatography of plasma on QAE-Sephadex twice, Sephadex G-100, and SP-Sephadex, and elution from alkaline disc gels after electrophoresis as rep...

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Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 X 107 M-1 and 4.2 X 108 M-l, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMWkininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity. High molecular weight (HMW)-kininogen functions as a coagulation cofactor by forming complexes with the Hageman factor substrates, prekallikrein and Factor XI (1, 2). These complexes bind to certain negatively charged surfaces and then interact with surface-bound Hageman factor to initiate pathways of coagulation, fibrinolysis, and kinin formation. We have previously shown that digestion of human HMW-kininogen by plasma kallikrein does not diminish its ability to act as a coagulation cofactor (3, 4). Furthermore, when kinin-free HMW-kininogen was reduced, alkylated, and separated into heavy and light chains, all of the coagulant activity was associated with the light chain (4). In this study, we have compared the binding affinity of prekallikrein and Factor XI for HMW-kininogen and present evidence that they compete for binding to a site that is present on the light chain derived from kinin-free HMW-kininogen. In Protein Purification. Prekallikrein was prepared from 4 liters of human plasma by the method of Mandle and Kaplan (5). Factor was prepared from human plasma by a modification of published procedures (5, 6). Plasma was desalted on Sephadex G-25 and fractionated by sequential chromatography on QAE-Sephadex A-50 and CM-Sepharose. The conditions and buffers were the same as those described in refs. 5 and 6. The Factor XI pool was dialyzed and fractionated on SP-Sephadex C-25 and concanavalin A-Sepharose as in ref. 7. HMW-kininogen was isolated from citrated human plasma as described by Thompson et al. (4). In order to isolate the heavy and light chains derived from kinin-free HMW-kininogen, purified HMW-kininogen was digested for 1 hr with 1% (wt/wt) plasma kallikrein. After reduction and alkylation (4) with dithiothreitol and iodoacetamide, respectively, the mixture was fractionated on a 2.5 X 100 cm column of Sephadex G-2Q0 equilibrated with 6 M guanid...

High molecular weight (HMW) 1 kininogen has been shown to be a critical factor which functions at the initial step of the Hageman factor-dependent pathways. Thus, plasmas deficient in HMW kininogen have a markedly prolonged partial thromboplastin time and diminished kaolin-activatable fibrinolysis (1-4). The Hageman factor substrates, prekallikrein and factor XI, circulate bound to HMW kininogen (5, 6) and are adsorbed to negatively charged surfaces where they interact with surface-bound Hageman factor. HMW kininogen augments the ability of activated Hageman factor or Hageman factor fragments to activate prekallikrein (7-10) and factor XI (7-11), and it also augments the rate of Hageman factor activation (8) and cleavage (7, 12) by kallikrein. Kallikrein also cleaves the HMW kininogen to liberate the vasoacrive peptide bradykinin.We have previously reported that kinin-free HMW kininogen could still function as a coagulation factor (2) and this observation was confirmed by Schiffman et al. (13). However, Chan et al. (14) reported that kallikrein digestion of human HMW kininogen progressively diminished its coagulant activity, while Matheson et al. (15) assessed bovine HMW kininogen in human HMW kininogen-deficient plasma and found that the kinin-free protein possessed <1% of the coagulant activity of the native molecule. In this report we investigate the structural changes that occur in purified human HMW kininogen as a consequence of kallikrein digestion. The critical portion of the molecule that is responsible for its coagulant activity has been isolated, as reported in preliminary form (6), and is shown to possess an antigenic determinant that distinguishes HMW kininogen from low molecular weight (LMW) kininogen.

Human monocyte/neutrophil elastase inhibitor (M/NEI) is a fast-acting stoichiometric inhibitor of neutrophil elastase (NE), cathepsin-G, and proteinase-3. Recombinant M/NEI (rM/NEI) was evaluated with a rat model of NE-induced lung damage. rM/NEI was found to protect against pulmonary injury caused by instilled human NE or by a preparation from airway secretions (sputum) of cystic fibrosis patients (CF sol). Human NE instilled into rat lungs produced dose-dependent hemorrhage and increased epithelial permeability, whereas NE incubated in vitro with rM/NEI did neither. Similarly, hemorrhage was induced by CF sol, but not by CF sol incubated in vitro with rM/NEI. To examine its distribution and survival time in airways, rM/NEI was labeled with the fluorochrome Texas Red (rM/NEI-TR) and instilled into rat lungs. Confocal microscopy showed that rM/NEI-TR could be detected on large airways (300 microm) at 5 min, 1 h, 4 h, and 24 h after instillation. Pretreating rats with rM/NEI was found to provide extended protection upon subsequent NE challenge, reducing hemorrhage by 98, 96, and 73%, respectively, at 1, 4, and 24 h after rM/NEI pretreatment. Pretreating rats with rM/NEI similarly conferred protection against subsequent exposure to CF sol, reducing hemorrhage by 95, 86, and 87%, respectively, at 1, 4, and 24 h after pretreatment. The findings that rM/NEI (1) mitigates protease-induced lung injury and (2) remains present and active in the lungs for 24 h after instillation strongly support its potential for treating patients with neutrophil protease-induced inflammatory lung damage, such as occurs in CF and other diseases.

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