High molecular weight kininogen is an inhibitor of platelet calpain.

Abstract

Recent studies from our laboratory indicate that a high concentration of platelet-derived calcium-activated cysteine protease (calpain) can cleave high molecular weight kininogen (HMWK). On immunodiffusion and immunoblot, antiserum directed to the heavy chain of HMWK showed immunochemical identity with alpha-cysteine protease inhibitor-a major plasma inhibitor of tissue calpains. Studies were then initiated to determine whether purified or plasma HMWK was also an inhibitor of platelet calpain. Purified alpha-cysteine protease inhibitor, alpha-2-macroglobulin, as well as purified heavy chain of HMWK or HMWK itself inhibited purified platelet calpain. Kinetic analysis revealed that HMWK inhibited platelet calpain noncompetitively (Ki 5 nM). Incubation of platelet calpain with HMWK, alpha-2-macroglobulin, purified heavy chain of HMWK, or purified alphacysteine protease inhibitor under similar conditions resulted in an ICso of 36, 500, 700, and 1,700 nM, respectively. The contribution of these proteins in plasma towards the inhibition of platelet calpain was investigated next. Normal plasma contained a protein that conferred a five to sixfold greater IC5. of purified platelet calpain than plasma deficient in either HMWK or total kininogen. Reconstitution of total kininogen deficient plasma with purified HMWK to normal levels (0.67 gM) completely corrected the subnormal inhibitory activity. However, reconstitution of HMWK deficient plasma to normal levels of low molecular weight kininogen (2.4 uM) did not fully correct the subnormal calpain inhibitory capacity of this plasma. These studies indicate that HMWK is a potent inhibitor as well as a substrate of platelet calpain and that the plasma and cellular kininogens may function as regulators of cytosolic, calcium-activated cysteine proteases.

Kininogen deficiency protects from ischemic neurodegeneration in mice by reducing thrombosis, blood-brain barrier damage, and inflammation

“…Mice were controlled for physiologic parameters that can critically affect stroke outcome (cerebral blood flow, blood pressure, heart rate, brain structure, blood gases; supplemental Figures 1-3; supplemental Table 1). For reconstitution experiments, high-molecular-weight kininogen from human plasma (Calbiochem) was injected intravenously in a subgroup of Kng Ϫ/Ϫ mice at a dosage of 4 g/g of body weight 16 immediately before the induction of tMCAO. To reconstitute bradykinin levels in the central nervous system of Kng Ϫ/Ϫ mice, Kng Ϫ/Ϫ mice received an intrathecal 17 injection of bradykinin (Sigma-Aldrich; 100 ng dissolved in 5 L of PBS) 18,19 immediately after the induction of tMCAO and again at the time of reperfusion.…”

Kininogens Are Antithrombotic Proteins In Vivo

“…A deficiency of plasma high-(HK) and low-(LK) molecular-weight kininogens 2 in humans does not lead to a hemorrhagic disorder, despite a marked prolongation of the activated partial thromboplastin time. In fact, cloning 3 and delineation of the structurefunction relationships of kininogen have revealed new properties of this protein, including cysteine protease inhibition, 4 modulation of thrombin-induced platelet aggregation, 5 antiadhesive properties, 6 and profibrinolytic potential. 7 HK and LK have been divided into domains D1, D2, and D3, which compose the common heavy-chain N-terminal to domain D4, which contains the bradykinin sequence.…”

“…LK contains a small domain, D5 L , whereas HK contains the large domains D5 H and D6, which compose the light chains. 3 The ability of D2 of the kininogens to inhibit platelet calpain 4 has been linked to an inhibition of thrombin-induced platelet aggregation, 8 whereas inhibition of the binding of thrombin to platelets by D3 of kininogens has led to a 10-fold shift in the doseresponse curve of thrombin-induced platelet activation. 9 HK can compete for fibrinogen binding to neutrophils 10 by competing for a site on the integrin Mac-1 11 and thus, limit the adhesion of neutrophils to artificial and biological surfaces.…”

p-Benzoquinone, a reactive metabolite of benzene, prevents the processing of pre-interleukins-1 alpha and -1 beta to active cytokines by inhibition of the processing enzymes, calpain, and interleukin-1 beta converting enzyme.

“…The cells were fed every 7 days and diluted to the initial seeding density. Cells growing at a density of 4 x 106/ml secrete approximately 25 (28) and was assayed by measuring the hydrolysis of a specific peptide substrate, 3-carboxypropionyl-leutyr-NH-7-(4-methyl) coumarylamide, which liberates a fluorescent product (29). The dialyzed enzyme and buffer (or inhibitor) were mixed and immediately added to a rectangular quartz cuvette at 25°C containing 1 mM peptide substrate in 60 mM Tris/HCl buffer, pH 7.5, 2.5% (v/v) dimethylsulfoxide (DMSO) and 5 mM CaCl2.…”