Factor XII deficiency has been postulated to be a risk factor for thrombosis suggesting that factor XII is an antithrombotic protein. The biochemical mechanism leading to this clinical observation is unknown. We have previously reported high molecular weight kininogen (HK) inhibition of thrombin-induced platelet aggregation by binding to the platelet glycoprotein (GP) Ib-IX-V complex. Although factor XII will bind to the intact platelet through GP Ib␣ (glycocalicin) without activation, we now report that factor XIIa (0.37 M), but not factor XII zymogen, is required for the inhibition of thrombin-induced platelet aggregation. Factor XIIa had no significant effect on SFLLRN-induced platelet aggregation. Moreover, an antibody to the thrombin site on protease-activated receptor-1 failed to block factor XII binding to platelets. Inhibition of thrombin-induced platelet aggregation was demonstrated with factor XIIa but not with factor XII zymogen or factor XIIf, indicating that the conformational exposure of the heavy chain following proteolytic activation is required for inhibition. However, inactivation of the catalytic activity of factor XIIa did not affect the inhibition of thrombininduced platelet aggregation. Factor XII showed displacement of biotin-labeled HK (30 nM) binding to gelfiltered platelets and, at concentrations of 50 nM, was able to block 50% of the HK binding, suggesting involvement of the GP Ib complex. Antibodies to GP Ib and GP IX, which inhibited HK binding to platelets, did not block factor XII binding. However, using a biosensor, which monitors protein-protein interactions, both HK and factor XII bind to GP Ib␣. Factor XII may serve to regulate thrombin binding to the GP Ib receptor by colocalizing with HK, to control the extent of platelet aggregation in vivo.Recently, the functions of the contact system have been reevaluated in view of our increasing knowledge of the interactions of factor XII, prekallikrein, and kininogens with platelets, neutrophils, monocytes, and endothelial cells. Prolongation of the activated partial thromboplastin time found with individuals, having deficiencies of the contact proteins, originally suggested a coagulation defect. However, no hemorrhagic diathesis has ever been demonstrated. Recently, anticoagulant (1) and profibrinolytic (2) activities of these proteins have now been documented in vitro and in some cases in vivo (3).The locus of the anticoagulant action of the contact system appears to be the platelet. Puri et al. (4) showed that HK 1 inhibited thrombin-induced platelet aggregation by inhibiting the binding of thrombin to platelets. Domain 3 of HK is responsible (5). Recently, the binding site on platelets, which mediates this effect, was shown to be GP Ib-IX complex (1). This observation helps to explain the fact that patients with BernardSoulier disease require 10-fold the concentration of thrombin needed to stimulate normal platelets (6). Kininogens also shift the dose-response curve for thrombin activation of platelets about 10-fold (1, 4). These s...

Objective-Plasma high-molecular-weight kininogen (HK) is cleaved in inflammatory diseases by kallikrein to HKa with release of bradykinin (BK). We postulated a direct link between HKa and cytokine/chemokine release. Methods and Results-HKa, but not BK, releases cytokines tumor necrosis factor (TNF)-␣, interleukin (IL)-1␤, IL-6, and chemokines IL-8 and MCP-1 from isolated human mononuclear cells. At a concentration of 600 nM, glutathione-Stransferase (GST) fusion proteins of kininogen domain 3 (D3), a fragment of domain 3, E7P (aaG255-Q292), HK domain 5 (D5), the D5 recombinant peptides HG (aa K420-D474) and HGK (aa H475-S626) stimulated secretion of IL-1␤ from mononuclear cells. Monoclonal antibodies (MAbs) specific for D5 or specific for D3 blocked release of IL-1␤ by HKa, supporting the importance of both domains. Antibodies to HK receptors on leukocytes including Mac-1, LFA-1, uPAR, and C1qR inhibited IL-1␤ secretion induced by tKa 98%, 89%, 85%, and 62%, respectively. Fractionation of mononuclear cells identified the responsible cell, a blood monocyte. Inhibitors of signaling pathways NFkB, JNK, and p38 but not extracellular signal-regulated kinase (ERK) decreased cytokine release from mononuclear cells. HKa increased the synthesis of IL-1␤ as deduced by an increase of IL-1␤ mRNA at 1 to 2 hours. Conclusions-HKa domains 3 and 5 may contribute to the pathogenesis of inflammatory diseases by releasing IL-1␤ from human monocytes using intracellular signaling pathways initiated by uPAR, ␤2 integrins and gC1qR. Key Words: chemokines Ⅲ cytokines Ⅲ kininogen Ⅲ monocytes Ⅲ uPAR F rom the discovery of kininogen, 1 the kallikrein-kinin system (KKS) has been intimately involved with inflammation. Plasma kallikrein cleaves HK to form BK and cleaved HK (HKa), which differs from HK because of major conformational changes. 2 BK increases capillary permeability by opening the tight junctions between endothelial cells and directly stimulates nerve endings causing pain, and is a potent vasodilator directly relaxing smooth muscles by releasing PGI 2 . The sum of these effects of BK reproduces many but not all aspects of inflammation. Emphasis in the past decade has shifted from contributions of HK to the humoral aspects of inflammation to its cellular participation, particularly interactions of HKa with leukocytes and endothelial cells. 3 Receptors on either or both of these cell types include selectins, which mediate leukocyte rolling and integrins, which mediate cell adhesion. Cellular proteases such as matrix metalloproteases degrade extracellular matrix protein in the basement membrane, facilitating neutrophil and mononuclear cell migration and emigration into tissues. The activation and participation of the KKS has been documented in inflammatory bowel disease 4 and arthritis 5 in rodents, which are models for human diseases such as rheumatoid arthritis and Crohns disease. Cytokines and chemokines released primarily but not exclusively from monocytes and tissue macrophages are known to play a central role in human inflamm...

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.

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