Human high molecular weight kininogen. Effects of cleavage by kallikrein on protein structure and procoagulant activity.

Abstract

Circulating human high molecular weight kininogen functions not only as a source of bradykinin but also as an important contact phase clotting factor.The bradykinin peptide is released from it by kallikrein in a reaction significantly altering the structure of the molecule without affecting clotting function.These experiments were designed to define the sequence of kallikrein modification and the kallikrein-resistant procoagulant portion.Kallikrein converted the native molecule (Mr = 121,000) to an intermediate of M, = 102,000 and finally to the end product of M, = 95,000.Each form retained procoagulant activity.On reduction, they appeared as: untreated, M, = 111,000; intermediate, M, = 65,000; end product, almost equal amounts of M, = 65,000 and 54,000.The sequence of events is consistent with the interpretation that part of the chains of M, = 65,000 of the intermediate gave rise to the M , = 54,000 chain.Reduced forms retained procoagulant activity which appeared sequentially at M, = 111,000, 65,000, and 54,000.The heavier chain of the end product (Mr = 65,000) had no procoagulant activity.Total activity eventually resided in the chain of M, = 54,000.Carbohydrate was associated with all nonreduced forms of the molecule, but on reduction of the end product carbohydrate was detected only on the M, = 65,000 chain.The active chain of M, = 54,000 lacked detectable carbohydrate.We conclude that kallikrein initially cleaves the kininogen molecule to two chains of Mr a 65,000 connected by disulfide bonds, probably with liberation of a small peptide.Further cleavage degrades one of the chains of M, = 65,000 so that the active end product is comprised of two chains of M, = 65,000 and 54,000.Activity is conferred by the chain of M, = 54,000, which lacks carbohydrate.Kininogen was first recognized as a physiologically important protein as the source of the small vasoactive peptide, bradykinin.Several different fractions containing kininogen activity could be isolated from human plasma (1, 2), and these have been shown to be separable into two circulating classes according to their size: low molecular weight kininogen and high molecular weight kininogen (HMWK)' (3-5).In the past