Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma.

Abstract

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...

Expression of Plasma Prekallikrein mRNA in Human Nonhepatic Tissues and Cell Lineages Suggests Special Local Functions of the Enzyme

“…The active enzyme is generated in the contact system, the early phase of the intrinsic pathway of blood coagulation. The contact system is activated when plasma is exposed to negatively charged surfaces: the PPK-HK complex is bound via cationic regions of domain 5 of HK to the anionic surface (Mandle et al, 1976), and activated factor XII (FXIIa) generates plasma kallikrein which in turn produces FXIIa (Mandle and Kaplan, 1977). Thus, by a positive feedback reaction, a local increase in the concentration of both PK and FXIIa is achieved.…”

The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc‐dependent binding to an anionic surface

“…Cleavage of HK is required for optimal expression of its procoagulant activity in the plasmatic environment (Scott et al, 1984). The light chain of HK contains the procoagulant activity (Mandle et al, 1976), which depends in part on its ability to associate with the zymogens, PK, through residues 556-595 of HK, or FXI, through residues 556-613 (Tait & Fujikawa, 1987). The coagulant activity of HK also depends on the binding of cleaved HK (Sugo et al, 1980) to anionic surfaces.…”

Activation of Rabbit Hageman Factor by Homogenates of Cultured Rabbit Endothelial Cells

“…However, if the HF-cleaving activity is exposed when EC or deeper layers are damaged, HF activation should occur. Furthermore, damage to the EC layer might expose subcellular structures with a sufficient density of negative charges to facilitate assembly of the surfacebound HF-high Mr kininogen-Factor XI-prekallikrein complexes similar to the one demonstrated for kaolin (12,39,40). Under these circumstances, the EC enzyme could initiate HF cleavage, an essential step for the reciprocal activation between HF and prekallikrein (41).…”