High molecular weight kininogen: localization in the unstimulated and activated platelet and activation by a platelet calpain(s)

Abstract

High mol wt kininogen (HMWK), the major cofactor-substrate of the contact phase of coagulation, is contained within and secreted by platelets. Studies have been performed to localize platelet HMWK in both the unstimulated and activated platelet and to ascertain the effect of platelet enzymes on HMWK itself. On platelet subcellular fractionation, platelet HMWK was localized to alpha-granules, and platelets from a patient with a deficiency of these granules (gray platelet syndrome) had 28% normal platelet HMWK. Platelet HMWK, in addition to being secreted from the platelet, was also localized to the surface of the platelet when activated. Using a competitive enzyme- linked immunosorbent assay for HMWK as an indirect antibody consumption assay, the external membrane of thrombin-activated platelets as well as the releasate from these stimulated platelets had 17 ng HMWK antigen/10(8) platelets available, whereas unstimulated platelets and their supernatant had only 4.9 and 4.2 ng HMWK/10(8) platelets present, respectively. The anti-HMWK antibody consumption by activated normal platelets was specific for membrane-expressed platelet HMWK, since activated platelets from a patient with total kininogen deficiency did not adsorb the anti-HMWK antibody. Enzymes in the cytosolic fraction of platelets cleaved 125I-HMWK (mol wt 120,000) into a mol wt 100,000 polypeptide as well as smaller products at mol wt 74,000, mol wt 62,000, mol wt 47,000, and a few components below mol wt 45,000. No cleavage products were observed when DFP and leupeptin were present. The cleavage of HMWK was specifically prevented by inhibitors of calcium-activated cysteine proteases (leupeptin, N-ethylmaleimide, iodoacetamide, and EDTA) but not by inhibitors of serine proteases (DFP, benzamidine, soybean trypsin inhibitor, or aprotinin). Platelet cytosol increased the coagulant activity of exogenous purified HMWK with maximum HMWK coagulant activity (35-fold) occurring within ten minutes of exposure to platelet cytosol. Treatment of platelet cytosol with leupeptin prevented the increase in the coagulant activity of exogenous HMWK. These studies indicate that activated platelets express platelet HMWK on their external membrane and platelet enzymes can cleave and increase the coagulant activity of exogenous HMWK.

Antiphospholipid Antibodies and Kininogens in Pathologic Pregnancies: A Review

“…Both HK and LK bind to platelets 51–53 and both can inhibit platelet calpain 54 . Domains 2 (D2) and 3 (D3) of the kininogen heavy chain contain a conserved pentapeptide sequence, QVVAG, which is found throughout the superfamily of mammalian cysteine protease inhibitors 55 .…”

The Role of Regioselectively Sulfated and Acetylated Polysaccharide Coatings of Biomaterials for Reducing Platelet and Plasma Protein Adhesion

“…42 HK, a contact cofactor, is contained within platelet granules and is secreted by and expressed on the surface of the activated platelets. 43 Small cationic proteins (platelet factor 4) secreted from platelets inhibit FXII and prekallikrein (PK) activation on artificial surfaces. 44 The degree of spreading has been interpreted as an expression of the reactivity of surfaces.…”

Kinetics of inhibition of platelet calpain II by human kininogens

“…Human HK was purified from fresh plasma by a modification (Schmaier et al, 1986b) of the procedure of Kerbiriou & Griffin (1979). This preparation of HK on reduced SDS/PAGE was essentially a single band of 120 kDa with greater than 98 % purity and has a specific activity of 12.5 units/mg (Schmaier et al, 1986b). HK was radiolabelled with Na125I with the use of lodogen (Schmaier et al, 1983).…”