The role of plasma high molecular weight kininogen in experimental intestinal and systemic inflammation.

Inflammation is accompanied by activation of the plasma kallikrein-kinin system (KKS). KKS activation has been demonstrated in a variety of inflammatory human diseases. To further explore the participation of KKS in arthritis and inflammatory bowel disease, we used two experimental animal models in arthritis and enterocolitis.

Differential requirements for calcium and Src family kinases in platelet GPIIb/IIIa activation and thromboxane generation downstream of different G-protein pathways.

G(12/13) or G(q) signaling pathways activate platelet GPIIb/IIIa when combined with G(i) signaling. We tested whether combined G(i) and G(z) pathways also cause GPIIb/IIIa activation and compared the signaling requirements of these events. Platelet aggregation occurred by combined stimulation of G(i) and G(z) pathways in human platelets and in P2Y1-deficient and G alpha(q)-deficient mouse platelets, confirming that the combination of G(i) and G(z) signaling causes platelet aggregation.

Platelet coagulation-protein interactions.

The biochemical mechanisms by which activated platelets participate in exposing receptors for the assembly of enzyme-cofactor-substrate complexes at all stages of the blood coagulation cascade are reviewed. Information derived from studies conducted during the last 30 years supports the concept that the initiation of blood coagulation is triggered by exposure of tissue factor at injury sites, leading to the generation of minute quantities of thrombin (limited by tissue factor pathway inhibitor), sufficient to activate platelets, factors XI, VIII, and V, and trigger the consolidation pathway (i.e.

Platelet cyclic adenosine monophosphate phosphodiesterases: targets for regulating platelet-related thrombosis.

Platelets contain two cyclic adenosine monophosphate (cAMP) phosphodiesterases (PDEs) that regulate the level of cAMP, the major inhibitor of platelet activation pathways. PDE3A hydrolyzes cAMP to 5' AMP with a low K (m). PDE3A is inhibited by cyclic guanosine monophosphate (cGMP), which provides a feedback control and controls basal levels of cAMP.

A monoclonal antibody to high-molecular weight kininogen is therapeutic in a rodent model of reactive arthritis.

We reported that high-molecular weight kininogen is proangiogenic by releasing bradykinin and that a monoclonal antibody to high-molecular weight kininogen, C11C1, blocked its binding to endothelial cells. We now test if this antibody can prevent arthritis and systemic inflammation in a Lewis rat model. We studied 32 animals for 16 days.

Allosteric modification of factor XIa functional activity upon binding to polyanions.

The effects of several polyanions on the hydrolysis of the chromogenic substrate L-pyroglutamyl-L-prolyl-L-arginyl-p-nitroaniline (S-2366) and on the activation of factor IX by factor XIa have been investigated. Two forms of dextran sulfate (M(r) approximately 500000 and M(r) approximately 10000, DX10) and two forms of heparin (64 disaccharide units, M(r) approximately 14000, and hypersulfated heparin, S-Hep, M(r) approximately 12000) inhibited both factor XIa amidolytic activity and factor IX activation in a concentration-dependent manner. The inhibitory effect was not due to binding of either substrate by the polyanions since only a decrease in V(max) without any effect on K(m) was observed in kinetic assays.

The contact system and angiogenesis: potential for therapeutic control of malignancy.

We have demonstrated that domain 5 (D5, kininostatin) and cleaved high-molecular-weight kininogen (HKa) inhibit endothelial proliferation, migration, and neovascularization in the in ovo chicken chorioallantoic membrane (CAM) assay, and that D5 and HKa act by stimulating apoptosis and interfering with the cell cycle at the G (1)-S transition. Both intact high-molecular-weight kininogen (HK) and low-molecular-weight kininogen induce angiogenesis in the CAM assay by releasing bradykinin. A monoclonal antibody, mAb C11C1, targeted to HK D5, inhibits FGF2- (fibroblast growth factor-2) and vascular endothelial growth factor-stimulated angiogenesis in the CAM assay by interfering with the binding of HK to endothelial cells.

Interactions between bradykinin (BK) and cell adhesion molecule (CAM) expression in peptidoglycan-polysaccharide (PG-PS)-induced arthritis.

Bradykinin (BK), a vasoactive, proinflammatory nonapeptide, promotes cell adhesion molecule (CAM) expression, leukocyte sequestration, inter-endothelial gap formation, and protein extravasation in postcapillary venules. These effects are mediated by bradykinin-1 (B1R) and-2 (B2R) receptors. We delineated some of the mechanisms by which BK could influence chronic inflammation by altering CAM expression on leukocytes, endothelium, and synovium in joint sections of peptidoglycan-polysaccharide-injected Lewis rats.

Binding studies of the enzyme (factor IXa) with the cofactor (factor VIIIa) in the assembly of factor-X activating complex on the activated platelet surface.

Activated platelet membranes expose binding sites for the enzyme factor (F)IXa, the substrate (FX) and the cofactor (FVIIIa) that colocalize to assemble the FX-activating complex and promote optimal rates of FX activation. To determine the stoichiometry and affinity of binding to activated platelets, coordinate, equilibrium binding studies with enzyme (125I-FIXa) and cofactor (131I-FVIII or 131I-FVIIIa) were carried out in the presence of saturating concentrations of substrate (FX). Results of these studies indicate that in the presence of FX (1.

Inhibition of angiogenesis by antibody blocking the action of proangiogenic high-molecular-weight kininogen.

Previously we demonstrated that domain 5 (D5) of high-molecular-weight kininogen (HK) inhibits neovascularization in the chicken chorioallantoic membrane (CAM) assay and further found that kallikrein cleaved HK (HKa) inhibited FGF2-and VEGF-induced neovascularization, and thus was antiangiogenic. In this study, we sought to demonstrate whether uncleaved HK stimulates neovascularization and thus is proangiogenic. The chick chorioallantoic membrane was used as an in ovo assay of angiogenesis.

The assembly of the factor X-activating complex on activated human platelets.

Platelet membranes provide procoagulant surfaces for the assembly and expression of the factor X-activating complex and promote the proteolytic activation and assembly of the prothrombinase complex resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable, receptors for factors XI, XIa, IX, IXa, X, VIII, VIIIa, V, Va and Xa, prothrombin, and thrombin. Studies described in this review support the hypothesis that the factor X-activating complex on the platelet surface consists of three receptors (for the enzyme, factor IXa; the substrate, factor X; and the cofactor, factor VIIIa), the colocalization of which results in a 24 million-fold acceleration of the rate of factor X activation.

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation.

Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma.

P2 receptors and platelet activation.

Adenosine diphosphate (ADP) plays a crucial role in hemostasis and thrombosis by activating platelets. In platelets, the classical P2T receptor is now resolved into three P2 receptor subtypes: the P2Y1, the P2Y12, and the P2X1 receptors. Both pharmacological and molecular biological approaches have confirmed the role of the P2Y1 and P2Y12 receptors in the ADP-induced platelet fibrinogen receptor activation.

Regulation of CD11b/CD18 (Mac-1) adhesion to fibrinogen by urokinase receptor (uPAR).

Objective And Design: The goal of this study is to investigate the consequence of the interaction between Mac-1 and uPAR and determine the mechanisms by which uPAR regulates Mac-1 dependent adhesion to fibrinogen.

Material: Human embryonic kidney 293 cells transfected with Mac-1 or uPAR or co-transfected with both Mac-1 and uPAR.

Methods: Cell adhesion and binding assays and Western Blotting for protein tyrosine phosphorylation analysis.

Factor XI, but not prekallikrein, blocks high molecular weight kininogen binding to human umbilical vein endothelial cells.

Previous studies on the interaction of high molecular weight kininogen (HK) with endothelial cells have reported a large number of binding sites (106-107 sites/cell) with differing relative affinities (KD = 7-130 nm) and have implicated various receptors or receptor complexes. In this study, we examined the binding of HK to human umbilical vein endothelial cells (HUVEC) with a novel assay system utilizing HUVEC immobilized on microcarrier beads, which eliminates the detection of the high affinity binding sites found nonspecifically in conventional microtiter well assays. We report that HK binds to 8.

Kininostatin as an antiangiogenic inhibitor: what we know and what we do not know.

High-molecular-weight kininogen (HK) is a plasma protein consisting of six domains (designated D1-D6). It was first characterized as a precursor of bradykinin, a bioactive peptide that regulates many cardiovascular processes. HK can bind to endothelial cells where it can be cleaved by plasma kallikrein to release bradykinin contained within domain 4.

Chronic intestinal inflammation and angiogenesis in genetically susceptible rats is modulated by kininogen deficiency.

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis associated with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK), we backcrossed Brown Norway rats having an HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a approximately 97% Lewis genome.

Inhibition of angiogenesis by a monoclonal antibody to kininogen as well as by kininostatin which block proangiogenic high molecular weight kininogen.

High molecular weight kininogen (HK) exhibits two activities with respect to angiogenesis after cleavage by plasma kallikrein. Cleaved HK (HKa) and its cell-binding domain 5 (D5), kininostatin, are potent antiangiogenic polypeptides. They inhibit endothelial cell migration, proliferation and tube formation.

Coordinate binding studies of the substrate (factor X) with the cofactor (factor VIII) in the assembly of the factor X activating complex on the activated platelet surface.

The assembly of the factor X activating complex on the platelet surface requires the occupancy of three receptors: (1) enzyme factor IXa, (2) cofactor factor VIII(a), and (3) substrate factor X. To further evaluate this three-receptor model, simultaneous binding isotherms of (125)I-factor X and (131)I-factor VIII(a) to activated platelets were determined as a function of time and also as a function of the concentrations of both ligands in the presence of active site-inhibited factor IXa (45 nM) and 5 mM CaCl(2). In the presence of active site-inhibited factor IXa and factor VIIIa there are two independent factor X binding sites: (1) low affinity, high capacity (approximately 9000 sites/platelet; K(d) approximately 380 nM) and (2) low capacity, high affinity (1700 sites/platelet; K(d) approximately 30 nM).

Identification of interaction sites of cyclic nucleotide phosphodiesterase type 3A with milrinone and cilostazol using molecular modeling and site-directed mutagenesis.

To identify amino acid residues involved in PDE3-selective inhibitor binding, we selected eight presumed interacting residues in the substrate-binding pocket of PDE3A using a model created on basis of homology to the PDE4B crystal structure. We changed the residues to alanine using site-directed mutagenesis technique, expressed the mutants in a baculovirus/Sf9 cell system, and analyzed the kinetic characteristics of inhibition of the mutant enzymes by milrinone and cilostazol, specific inhibitors of PDE3. The mutants displayed differential sensitivity to the inhibitors.

The interaction of factor XIa with activated platelets but not endothelial cells promotes the activation of factor IX in the consolidation phase of blood coagulation.

We have previously shown that the zymogen factor XI (FXI) binds to activated platelets but not to human umbilical vein endothelial cells (HUVEC), a conclusion that is in conflict with previous reports stating that FXI binds to 2.7-13 x 10(6) high affinity sites per HUVEC (Berrettini, M., Schleef, R.

Molecular cloning and biochemical characterization of rabbit factor XI.

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160 kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80 kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer.

Kininogen deficiency modulates chronic intestinal inflammation in genetically susceptible rats.

Genetically susceptible Lewis rats injected in the intestinal wall with peptidoglycan-polysaccharide (PG-APS) polymers develop chronic granulomatous enterocolitis concomitant with activation of the kallikrein-kinin system. To elucidate the role of high-molecular-weight kininogen (HK) in chronic enterocolitis, we back crossed Brown-Norway rats having a HK deficiency with Lewis rats for five generations. Two new strains were produced, wild-type F5 (F5WT) and HK deficient (F5HKd), each with a approximately 97% Lewis genome.

Protease-activated receptors 1 and 4 do not stimulate G(i) signaling pathways in the absence of secreted ADP and cause human platelet aggregation independently of G(i) signaling.

Thrombin is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Thrombin activates platelets mainly through protease-activated receptor 1 (PAR1), PAR4, and glycoprotein Ib. Because adenosine diphosphate and thromboxane A(2) have been shown to cause platelet aggregation by concomitant signaling through G(q) and G(i) pathways, we investigated whether coactivation of G(q) and G(i) signaling pathways is the general mechanism by which PAR1 and PAR4 agonists also activate platelet fibrinogen receptor (alphaIIbbeta3).