High Molecular Weight Kininogen Inhibits Fibrinogen Binding to Cytoadhesins of Neutrophfls and Platelets

Fibrinogen inhibited ~25I-high molecular weight kininogen (HMWK) binding and displaced bound J25I-HMWK from neutrophils. Studies were performed to determine whether fibrinogen could bind to human neutrophils and to describe the HMWK-fibrinogen interaction on cellular surfaces. At 4°C, the binding of ~2H-fibrinogen to neutrophils reached a plateau by 30 min and did not decrease. At 23 and 37°C, the amount of t25I-fibrinogen bound peaked by 4 min and then decreased over time because of proteolysis of fibrinogen by human neutrophil elastase (HNE). Zn++ (50 #M) was required for binding of ~25I-fibrinogen to neutrophils at 4°C and the addition of Ca ++ (2 mM) increased the binding twofold. Excess unlabeled fibrinogen or HMWK completely inhibited binding of ~25I-fibrinogen. Fibronectin degradation products (FNDP) partially inhibited binding, but prekallikrein and factor XII did not. The binding of ~25I-fibrinogen at 4°C was reversible with a 50-fold molar excess of fibrinogen or HMWK. Binding of ~25I-fibrinogen, at a concentration range of 5-200/~g/ml of added radioligand, was saturable with an apparent Kd of 0.17/~M and 140,000 sites/cell. The binding of ~25I-fibrinogen to neutrophils was not inhibited by the peptide RGDS derived from the ot chain of fibrinogen or by the mAb 10E5 to the platelet glycoprotein IIb/IIIa heterodimer. Fibrinogen binding was inhibited by a 3,-chain peptide CYGHHLGGAKQAGDV and by mAb OKM1 but was not inhibited by OKM10, an mAb to a different domain of the adhesion glycoprotein Mac-1 (complement receptor type 3 [CR3]). HMWK binding to neutrophils was not inhibited by OKM1. These observations were consistent with a further finding that fibrinogen is a noncompetitive inhibitor of tzSI-HMWK binding to neutrophils. Fibrinogen binding to ADP-stimulated platelets was increased twofold by Zn +÷ (50 #M) and was inhibited by HMWK. These studies indicate that fibrinogen specifically binds to the C3R receptor on the neutrophil surface through the carboxy terminal of the 3,-chain and that HMWK interferes with the binding of fibrinogen to integrins on both neutrophils and activated platelets. H UMAN kininogens are multifunctional proteins (48) coded for by a gene containing 11 exons (58). The first nine are expressed as a heavy chain-containing domain with cysteine protease inhibitory activity (43). Exon 10 codes for bradykinin and 12 additional amino acids which, like domains 1-9, are common to both high (120,000) and low (67,000) molecular weight kininogen (HMWK and LMWK, respectively). ~ By differential splicing of the mRNA one obtains HMWK with the rest of domain 10, specifying a light chain (56 kD) with surface binding (51) and prekallikrein and factor XI binding sites (57). Together, these domains confer on HMWK the ability to accelerate activation of the contact H. Lukasiewicz's permanent address is Children's Memorial Hospital, Warsaw, Poland. 1. Abbreviations used in this paper: CR3, complement receptor type 3; FNDP, fibronectin degradation product; GP, glycoprotein; HBSS, Hanks' balanced salt solution; HMWK, high molecular weight kininogen; HNE, human neutrophil elastase; LMWK, low molecular weight kininogen. phase of blood coagulation. Alternatively, a different splicing site allows attachment of domain 11, supplying an alternate light chain which has no known biologic activity and is contained within LMWK. Vroman et al. (60) noted that HMWK can displace fibrinogen from artificial hydrophilic surfaces such as glass. This effect is specifically due to the light chain of HMWK since purified LMWK did not alter the surface expression of fibrinogen (47). To date no evidence exists to indicate whether such a phenomenon exists on biologic surfaces. Fibrinogen has been shown to specifically bind to platelets (7, 31) as well as to integrins on endothelial cells (14) and monocytes (1). Similarly, HMWK has been demonstrated to bind to platelets (19, 20), neutrophils (21), and endothelial cells (49). The binding of fibrinogen to platelet membrane receptors (7, 28, 35) and to neutrophil surfaces (8) may result in the aggregation of these cells. Since neutrophils can ingest fibrin (5), accumulate within thrombi, and penetrate preformed blood clots (22), we sought to determine whether ~25I-fibrinogen © The Rockefeller University Press, 0021-9525/89/07/377/11 $2.00 The Journal of Cell Biology, Volume 109, July 1989 377-387 377 on M ay 4, 2017 D ow nladed fom Published July 1, 1989