High molecular weight kininogen utilizes heparan sulfate proteoglycans foraccumulation on endothelial cells

Kininogens, the high molecular weight precursor of vasoactive kinins, bind to a wide variety of cells in a specific, reversible, and saturable manner. The cell docking sites have been mapped to domains D3 and D5(H) of kininog ens; however, the corresponding cellular acceptor sites are not fully estab lished. To characterize the major cell binding sites for kininogens exposed by the endothelial cell line EA.hy926, we digested intact cells with tryps in and other proteases and found a time- and concentration-dependent loss o f I-125-labeled high molecular weight kininogen (H-kininogen) binding capac ity (up to 82%), indicating that proteins are crucially involved in kininog en cell attachment. Cell surface digestion with heparinases similarly reduc ed kininogen binding capacity (up to 78%), and the combined action of hepar inases and trypsin almost eliminated kininogen binding (up to 85%), suggest ing that proteoglycans of the heparan sulfate type are intimately involved. Consistently, inhibitors such as p-nitrophenyl-beta -D-xylopyranoside and chlorate interfering with heparan sulfate proteoglycan biosynthesis reduced the total number of kininogen binding sites in a time- and concentration-d ependent manner (up to 67%). In vitro binding studies demonstrated that bio tinylated H-kininogen binds to heparan sulfate glycosaminoglycans via domai ns D3 and D5(H) and that the presence of Zn2+ promotes this association. Cl oning and over-expression of the major endothelial heparan sulfate-type pro teoglycans syndecan-1, syndecan-2, syndecan-4, and glypican in HEK293t cell s significantly increased total heparan sulfate at the cell surface and thu s the number of kininogen binding sites (up to 3.3-fold). This gain in kini nogen binding capacity was completely abolished by treating transfected cel ls with heparinases. We conclude that heparan sulfate proteoglycans on the surface of endothelial cells provide a platform for the local accumulation of kininogens on the vascular lining. This accumulation may allow the circu mscribed release of short-lived kinins from their precursor molecules in cl ose proximity to their sites of action.