Kininogen Binding Research Articles

OC 70.3 Effect of High-Molecular-Weight Kininogen Binding to Factor XI and Prekallikrein on Thrombosis

OC 70.3 Effect of High-Molecular-Weight Kininogen Binding to Factor XI and Prekallikrein on Thrombosis

The Role of Copper (II) on Kininogen Binding to Tropomyosin in the Presence of a Histidine-Proline-Rich Peptide.

The antiangiogenic activity of the H/P domain of histidine–proline-rich glycoprotein is mediated by its binding with tropomyosin, a protein exposed on endothelial cell-surface during the angiogenic switch, in presence of zinc ions. Although it is known that copper ion serum concentration is significantly increased in cancer patients, its role in the interaction of H/P domain with tropomyosin, has not yet been studied. In this paper, by using ELISA assay, we determined the modulating effect of TetraHPRG peptide, a sequence of 20 aa belonging to H/P domain, on the binding of Kininogen (HKa) with tropomyosin, both in absence and presence of copper and zinc ions. A potentiometric study was carried out to characterize the binding mode adopted by metal ions with TetraHPRG, showing the formation of complex species involving imidazole amide nitrogen atoms in metal binding. Moreover, circular dichroism showed a conformational modification of ternary systems formed by TetraHPRG, HKa and copper or zinc. Interestingly, slight pH variation influenced the HKa-TetraHPRG-tropomyosin binding. All these results indicate that both metal ions are crucial in the interaction between TetraHPRG, tropomyosin and HKa.

Peptidylarginine Deiminase of Porphyromonas gingivalis Modulates the Interactions between Candida albicans Biofilm and Human Plasminogen and High-Molecular-Mass Kininogen.

Microorganisms that create mixed-species biofilms in the human oral cavity include, among others, the opportunistic fungus Candida albicans and the key bacterial pathogen in periodontitis, Porphyromonas gingivalis. Both species use arsenals of virulence factors to invade the host organism and evade its immune system including peptidylarginine deiminase that citrullinates microbial and host proteins, altering their function. We assessed the effects of this modification on the interactions between the C. albicans cell surface and human plasminogen and kininogen, key components of plasma proteolytic cascades related to the maintenance of hemostasis and innate immunity. Mass spectrometry was used to identify protein citrullination, and microplate tests to quantify the binding of modified plasminogen and kininogen to C. albicans cells. Competitive radioreceptor assays tested the affinity of citrullinated kinins to their specific cellular receptors. The citrullination of surface-exposed fungal proteins reduced the level of unmodified plasminogen binding but did not affect unmodified kininogen binding. However, the modification of human proteins did not disrupt their adsorption to the unmodified fungal cells. In contrast, the citrullination of kinins exerted a significant impact on their interactions with cellular receptors reducing their affinity and thus affecting the role of kinin peptides in the development of inflammation.

Plasma Prekallikrein: Its Role in Hereditary Angioedema and Health and Disease.

Plasma prekallikrein (PK) has a critical role in acute attacks of hereditary angioedema (HAE). Unlike C1 inhibitor, its levels fall during HAE attacks with resultant cleaved high-molecular-weight kininogen. Cleavage of high-molecular-weight kininogen liberates bradykinin, the major biologic peptide that promotes the edema. How prekallikrein initially becomes activated in acute attacks of HAE is not known. PK itself is negatively associated with cardiovascular disease. High prekallikrein is associated with accelerated vascular disease in diabetes and polymorphisms of prekallikrein that reduce high-molecular-weight kininogen binding are associated with protection from cardiovascular events. Prekallikrein-deficient mice have reduced thrombosis risk and plasma kallikrein (PKa) inhibition is associated with reduced experimental gastroenterocolitis and arthritis in rodents. In sum, prekallikrein and its enzyme PKa are major targets in HAE providing much opportunity to improve the acute and chronic management of HAE. PKa inhibition also may be a target to ameliorate cardiovascular disease, thrombosis risk, and inflammation as in enterocolitis and arthritis.

Identification of a Receptor for Neuropeptide VGF and Its Role in Neuropathic Pain

VGF (nonacronymic) is a neuropeptide precursor that plays multiple roles in regulation of energy balance, reproduction, hippocampal synaptic plasticity, and pain. Data from a number of pain models showed significant up-regulation of VGF in sensory neurons. TLQP-21, one of the VGF-derived neuropeptides, has been shown to induce a hyperalgesic response when injected subcutaneously into the hind paw of mice. However, the precise role of VGF-derived neuropeptides in neuropathic pain and the molecular identity of the receptor for VGF-derived peptides are yet to be investigated. Here we identified gC1qR, the globular heads of the C1q receptor, as the receptor for TLQP-21 using chemical cross-linking combined with mass spectrometry analysis. TLQP-21 caused an increase in intracellular Ca(2+) levels in rat macrophages and microglia. Inoculation of TLQP-21-stimulated macrophages into rat hind paw caused mechanical hypersensitivity. The increase in intracellular Ca(2+) levels in macrophages was attenuated by either siRNA or neutralizing antibodies against gC1qR. Furthermore, application of the gC1qR-neutralizing antibody to rats with partial sciatic nerve ligation resulted in a delayed onset of nerve injury-associated mechanical hypersensitivity. These results indicate that gC1qR is the receptor for TLQP-21 and plays an important role in chronic pain through activation of macrophages. Because direct association between TLQP-21 and gC1qR is required for activation of macrophages and causes hypersensitivity, disrupting this interaction may be a useful new approach to develop novel analgesics.

Kinin-generating cellular model obtained from human glioblastoma cell line U-373.

Kinins, a group of important pro-inflammatory peptides, are abundantly found in tissues and biological fluids of cancer patients. Bradykinin, the major representative of kinins, induces vascular permeability and, in consequence, promotes tumor expansion. Additionally, the kinin-induced inflammatory responses, especially those mediated by kinin metabolites without the C-terminal arginine residue, lead to enhanced tumor growth. The present study aimed at analyzing the ability of the human glioblastoma cell line U-373, derived from a malignant tumor, to produce kinin peptides. The proteins involved in kinin generation, i.e., the kininogens and the kallikreins, were shown to be expressed in these cells. Moreover, tumor necrosis factor α, a proinflammatory cytokine that mediates tumorigenesis, was found to enhance the expression of enzymes associated with kinin production. The strong binding of kininogen to the cell surface and the enzymatic degradation of this protein by cells suggest the activation of kinin-generating systems. Indeed, glioblastoma cells, pre-treated with tumor necrosis factor α, released kinin peptides from exogenous kininogen. The expression of kinin receptors in these cells was also shown to increase under the influence of this cytokine. Our results suggest that the human glioblastoma cell line U-373 constitutes a good cellular model that can be helpful in cancer research focused on kinin-induced inflammation. Furthermore, our findings can contribute to new approaches in cancer treatment with the use of kinin receptor antagonists and inhibitors of kinin production.

Different Effect of Hydrogelation on Antifouling and Circulation Properties of Dextran–Iron Oxide Nanoparticles

Premature recognition and clearance of nanoparticulate imaging and therapeutic agents by macrophages in the tissues can dramatically reduce both the nanoparticle half-life and delivery to the diseased tissue. Grafting nanoparticles with hydrogels prevents nanoparticulate recognition by liver and spleen macrophages and greatly prolongs circulation times in vivo. Understanding the mechanisms by which hydrogels achieve this "stealth" effect has implications for the design of long-circulating nanoparticles. Thus, the role of plasma protein absorption in the hydrogel effect is not yet understood. Short-circulating dextran-coated iron oxide nanoparticles could be converted into stealth hydrogel nanoparticles by cross-linking with 1-chloro-2,3-epoxypropane. We show that hydrogelation did not affect the size, shape and zeta potential, but completely prevented the recognition and clearance by liver macrophages in vivo. Hydrogelation decreased the number of hydroxyl groups on the nanoparticle surface and reduced the binding of the anti-dextran antibody. At the same time, hydrogelation did not reduce the absorption of cationic proteins on the nanoparticle surface. Specifically, there was no effect on the binding of kininogen, histidine-rich glycoprotein, and protamine sulfate to the anionic nanoparticle surface. In addition, hydrogelation did not prevent activation of plasma kallikrein on the metal oxide surface. These data suggest that (a) a stealth hydrogel coating does not mask charge interactions with iron oxide surface and (b) the total blockade of plasma protein absorption is not required for maintaining iron oxide nanoparticles' long-circulating stealth properties. These data illustrate a novel, clinically promising property of long-circulating stealth nanoparticles.

Factor XI Activation In Tissue Factor-Initiated Blood Coagulation

Abstract 2239 Introduction.In in vitro models of tissue factor (TF)-initiated coagulation, FXI activation has been linked to increased thrombin generation. However the effects of FXI in experimental models of normal hemostasis have often been subtle, prompting ongoing investigations to define contributing cofactors, potential collaborating activators and/or reaction conditions (e.g. low TF concentrations). In this study, predictions from a computational model of TF-initiated thrombin generation that includes thrombin dependent FXI activation are used to direct an investigation of the role of FXI in two empirical models of TF-initiated coagulation. Methods. FXI activation and FXIa interactions were computationally modeled by adding the appropriate sets of equations describing thrombin activation of FXI, FXIa activation of FIX, antithrombin inhibition of FXIa, and high molecular weight kininogen binding to FXI to the existing framework of differential equations. The efficacy of FXIa in promoting thrombin generation (α- thrombin-antithrombin, αTAT) was assessed via titration in contact pathway inhibited whole blood and compared to activation by TF, FIXa, FXa and α-thrombin. TF-initiated reactions and their resupply were performed as described previously (Orfeo T et al. J. Biol. Chem., 2008) in either contact pathway inhibited blood ± an inhibitory anti-FXI antibody) or in synthetic coagulation proteome (SCP) mixtures (± FXI), allowing FXI effects on both the TF dependent phase and the resulting procoagulant pool of catalysts to be evaluated. Results. The computational model (± FXI pathway) was validated by showing congruence between computational thrombin generation profiles initiated with 5 pM TF and corresponding SCP reconstructions, which showed the lag phase shortened by 30 to 60 s and maximum thrombin levels increased when FXI was present. Similarly there was good correspondence between computational and SCP thrombin generation when FXIa (4 pM by active site) was the initiator. A computational analysis provided the following ranking of effectiveness in initiating thrombin generation: FXIa>TF>FIXa>FXa>α-thrombin. When tested in contact pathway inhibited blood, addition of 5 pM FXIa (by active site) resulted in clot times and 20 min αTAT levels similar to those observed with 5 pM TF, while similar outcomes required 25 pM FIXa, 100 pM FXa or between 10–100 nM α-thrombin. The computational modeling made clear two consequences of FXI feedback activation. The first was mechanistic, demonstrating that the amplification of thrombin generation was achieved by better coordinating the initial activations of FIX and FVIII; limited early initiation phase activation of FXI (<0.01% zymogen activation) increases the amount of FIXa available during the activation of FVIII, yielding higher concentrations of intrinsic tenase earlier in the initiation phase. The second consequence was increased FIXa accumulation as the reaction proceeds. This prediction was confirmed in two ways: 1) Western blot analysis of SCP reactions containing FXI showed ∼20% consumption of FIX over 20 min compared to no detectable FIX consumption in reactions without FXI; and 2) Resupply of TF-initiated SCP reactions (± 30 nM FXI) demonstrated that both the FVIII dependence and the long-term stability of the observed re-initiation of thrombin generation depended on the presence of FXI. Resupply studies were then performed in contact pathway inhibited blood (N=4 individuals) by using an inhibitory anti-FXI antibody to negate FXI contributions to both the TF-initiated and resupply dynamics. No effect of blocking FXI function on the time courses of αTAT formation was observed in the TF initiated phase. Two of the four individuals showed attenuated αTAT formation upon resupply when FXI function was blocked from the onset of TF initiation. Conclusions. FXIa is a potent initiator of coagulation, and the computational and SCP analyses predict the potential for feedback activation by thrombin after TF initiation to somewhat enhance the propagation phase of thrombin generation and more dramatically enhance the resupply response. However in contact pathway inhibited blood, FXI contributions to Tf-initiated thrombin generation are not discernible and the effect on the resupply response is variable between individuals suggesting additional mechanisms suppressing FXI activation in blood. Disclosures:Mann:Haematologic Technologies: Chairman of the Board, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; corn trypsin inhibitor: Patents & Royalties; NIH, DOD, Baxter: Research Funding; Merck, Daiichi Sankyo, Baxter, GTI: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Speakers Bureau.

Bradykinin and the Pathogenesis of Hereditary Angioedema

Bradykinin and the Pathogenesis of Hereditary Angioedema

Localization of Ligand-Binding Exosites In the Catalytic Domain of Factor XIa.

AbstractAbstract 1148Coagulation factor XI (FXI) is a plasma zymogen that is activated to FXIa, the catalytic domain of which contains exosites that interact with its normal macromolecular substrate (FIX), and its major regulatory inhibitor (protease nexin-2 kunitz protease inhibitor, PN2KPI). To localize the catalytic domain residues involved in active site architecture and in various ligand-binding exosites, we aligned the sequence of the FXI catalytic domain with that of the prekallikrein (PK) catalytic domain which is highly homologous (64% identity) in sequence, but functionally very different from FXI. Six distinct regions (R1-R6) of dissimilarity between the two proteins were identified as possible candidates for FXIa-specific ligand binding exosites. FXI/PK chimeric proteins (FXI-R1, FXI-R2, FXI-R3, FXI-R4, FXI-R5, and FXI-R6) containing substitutions with PK residues within the six regions were prepared and characterized. FXIa-R1, R2, R3 displayed enhanced proteolysis after activation suggesting that the residues within R1, R2 and R3 regions may be important to maintain proper folding of the enzyme. Comparisons of amidolytic assays vs. activated partial thromboplastin time assays showed similar activities for all chimeras except FXI-R6, which displayed 60% of the normal amidolytic activity but only 28% of clotting activity suggesting the possibility that the R6 region (autolysis loop) of FXIa may comprise an exosite involved in the interaction with its macromolecular substrate FIX. This hypothesis was further confirmed experiments showing that the proteolytic activation of FIX by FXIa-R6 was significantly impaired compared with that achieved by FXIawt. Although FXIa-R5 and FXIa-R6 were defective (50-60%) in amidolytic assays, these chimeras were very similar to FXIawt in heparin and high molecular weight kininogen binding assays, suggesting that residues within the R5 and R6 regions are involved in active-site architecture. These chimeras were further investigated to determine whether any of them had acquired kallikrein activity. After activation all except FXIa-R4 showed insignificant activity using a kallikrein-specific substrate. FXIa-R4 displayed 87% of the activity of kallikrein using the kallikrein-specific substrate but only 3% of the activity of FXIawt using the FXIa chromogenic substrate. Moreover the cleavage pattern and cleavage rate of high molecular weight kininogen by FXIa-R4 were similar to those achieved by kallikrein but not by FXIawt. Therefore substitutions in the R4 region of FXI with the corresponding residues of PK resulted in loss of activity for the FXIa substrates and gain of activity for the kallikrein substrates suggesting that the R4 region (99-loop) of FXIa plays a role in determining the substrate specificity. From the co-crystal structure of the FXIa catalytic domain with PN2KPI, the residues R3704, Y5901, E98, Y143, I151, and K192 (chymotrypsin numbering) in the FXIa catalytic domain have been identified to be possibly involved in the interactions with its inhibitors. A single mutation comprising Y5901A in the R2 region of FXIa does not affect folding however this mutant displayed resistance to inhibition by PN2KPI indicating that Y5901 is involved in the interaction of FXIa with PN2KPI. In conclusion, these studies of FXI/PK chimeric and mutant proteins implicate residues within the R4 region (99-loop) of FXIa in the determination of amidolytic substrate specificity; residues within the R6 region (autolysis loop) of FXIa in the interaction with the macromolecular substrate, FIX; and the residue Y5901 in the R2 region of FXIa in the interaction of FXIa with PN2KPI. Disclosures:No relevant conflicts of interest to declare.

Kininogen binding to the surfaces of macrophages

Kinin generation may be initiated on the cell surfaces via a primary kininogen docking which has been characterized for endothelial cells, platelets, neutrophils, astrocytes and smooth muscle cells. In this work we describe the adsorption of biotin-labeled human kininogens by murine RAW 264.7 macrophages and human U-937 monocytes/macrophages. Both cell types strongly bound high molecular mass kininogen (HK) in a zinc–ion dependent manner with the dissociation constants of 9.1 nM and 3.3 nM, respectively, and the binding capacities of 46 fmol and 71 fmol per million of respective cells. The HK binding was quenched by 50% by antibodies against Mac-1, gC1qR and uPAR proteins indicating that these macrophage surface receptors are involved in the HK adsorption. A significant increase of HK binding was observed after cell activation with phorbol myristate acetate. Our results suggest that macrophages, similarly to neutrophils, may supply kininogens to the inflammatory foci to support the local kinin production at these sites.

Identification and characterization of plasma kallikrein–kinin system inhibitors from salivary glands of the blood‐sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.

A Monoclonal Antibody to High Molecular Weight Kininogen Reduces Inflammation in the HLA-B27 Transgenic Rat Model of Inflammatory Bowel Disease and Arthritis.

The HLA-B27 rat is a well-characterized model of human bowel disease, rheumatoid arthritis and psoriasis. Previous studies of chronic inflammation in other rat models of inflammatory disease have demonstrated activation of the kallilrein-kinin system (KKS) as well as modulation by a kallikrein inhibitor and HK deficiency. The effects of C11C1, a monoclonal antibody acting to inhibit cellular binding of high molecular weight kininogen (HK), were studied in the HLA-B27 transgenic rat. Thrice weekly intraperitoneal injections of C11C1 (1.9 mg/kg) or IgG1 (6mg/kg) were given to male HLA-B27 transgenic rats for 3 weeks, beginning when the rats were 23 weeks old. Stool character was scored daily as a measure of intestinal inflammation, and the rear limbs were scored daily for clinical signs of arthritis, tarsal joint swelling and erythema. At the end of the experiment the animals were euthanized, and the colon and tarsal joint histologic lesions were examined. The histology sections were assigned a numerical score for colonic inflammation, synovitis, and cartilage damage. Activation of KKS was assessed by assays of plasma prekallikrein, HK, factor XI, and factor XII. Administration of the monoclonal antibody directed against HK rapidly decreased the clinical scores of inflammatory bowel disease from 3.0 ± 0 to 1.2 ± 0.2 (p<0.005) and arthritis from 12.0 ± 0 to 0.8 ± 0.8 (p<0.001). Histologic analyses confirmed significant reductions in colonic lesions from 7.7 ± 0.8 to 2.8 ± 0.9 (p=0.004) and synovitis from 9.5 ± 0.8 to 5.0 ± 1.0 (p=0.009). Decreased prekallikrein and HK levels were reversed by monoclonal antibody C11C1, providing evidence of KKS activation. A monoclonal antibody to kininogen appears to have therapeutic potential in human inflammatory bowel disease and arthropathies.

Inhibition of tumor angiogenesis in vivo by a monoclonal antibody targeted to domain 5 of high molecular weight kininogen

AbstractWe have shown that human high molecular weight kininogen is proangiogenic due to release of bradykinin. We now determined the ability of a murine monoclonal antibody to the light chain of high molecular weight kininogen, C11C1, to inhibit tumor growth compared to isotype-matched murine IgG. Monoclonal antibody C11C1 efficiently blocks binding of high molecular weight kininogen to endothelial cells in a concentration-dependent manner. The antibody significantly inhibited growth of human colon carcinoma cells in a nude mouse xenograft assay and was accompanied by a significant reduction in the mean microvascular density compared to the IgG control group. We also showed that a hybridoma producing monoclonal antibody C11C1 injected intramuscularly exhibited markedly smaller tumor mass in a syngeneic host compared to a hybridoma producing a monoclonal antibody to the high molecular weight kininogen heavy chain or to an unrelated plasma protein. In addition, tumor inhibition by purified monoclonal antibody C11C1 was not due to direct antitumor effect because there was no decrease of tumor cell growth in vitro in contrast to the in vivo inhibition. Our results indicate that monoclonal antibody C11C1 inhibits angiogenesis and human tumor cell growth in vivo and has therapeutic potential for treatment of human cancer. (Blood. 2004;104:2065-2072)

Tissue factor pathway inhibitor-2 (TFPI-2) recognizes the complement and kininogen binding protein gC1qR/p33 (gC1qR): implications for vascular inflammation.

Evidence is accumulating to suggest that TFPI-2 is involved in regulating pericellular proteases implicated in a variety of physiologic and pathologic processes including cancer cell invasion, vascular inflammation, and atherosclerosis. Recent immunohistochemical studies of advanced atherosclerotic lesions, demonstrated a similar tissue distribution for TFPI-2, High Molecular Weight Kininogen (HK), and gC1qR/p33 (gC1qR), a ubiquitously expressed, multicompartmental cellular protein involved in modulating complement, coagulation, and kinin cascades. Further studies to evaluate TFPI-2 interactions with gC1qR demonstrated direct interactions between gC1qR and TFPI-2 using immunoprecipitation and solid phase binding studies. Specific and saturable binding between TFPI-2 and gC1qR (estimated Kd: approximately 70 nM) was observed by ELISA and surface plasmon resonance (Biacore) binding assays. Binding was inhibited by antibodies to gC1qR, and was strongly dependent on the Kunitz-2 domain of TFPI-2, as deletion of this domain reduced gC1qR-TFPI-2 interactions by approximately 75%. Deletion of gC1qR amino acids 74-95, involved in C1q binding, had no effect on gC1qR binding to TFPI-2, although antibodies to this region and purified C1q both inhibited binding, most likely via allosteric effects. In contrast, HK did not affect TFPI-2 binding to gC1qR. Binding of TFPI-2 to gC1qR produced statistically significant but modest reductions in TFPI-2 inhibition of plasmin, but had no effect on kallikrein inhibition in fluid phase chromogenic assays. Taken together, these data suggest that gC1qR may participate in tissue remodeling and inflammation by localizing TFPI-2 to the pericellular environment to modulate local protease activity and regulate HK activation.

Interaction of high molecular weight kininogen binding proteins on endothelial cells.

Cell surface proteins reported to participate in the binding and activation of the plasma kinin-forming cascade includes gC1qR, cytokeratin 1 and u-PAR. Each of these proteins binds high molecular weight kininogen (HK) as well as Factor XII. The studies on the interaction of these proteins, using dot-blot analysis, revealed that cytokeratin 1 binds to both gC1qR and u-PAR while gC1qR and u-PAR do not bind to each other. The binding properties of these proteins were further analyzed by gel filtration. When biotinylated cytokeratin 1 was incubated with either gC1qR or u-PAR and gel filtered, a new, higher molecular weight peak containing biotin was observed indicating complex formation. The protein shift was also similar to the biotin shift. Further, immunoprecipitation of solubilized endo-thelial cell plasma membrane proteins with anti-gC1qR recovered both gC1qR and cytokeratin 1, but not u-PAR. Immunoprecipitation with anti-u-PAR recovered only u-PAR and cytokeratin 1. By competitive ELISA, gC1qR inhibits u-PAR from binding to cytokeratin 1; u-PAR inhibits gC1qR binding to a lesser extent and requires a 10-fold molar excess. Our data suggest that formation of HK (and Factor XII) binding sites along endothelial cell membranes consists of bimolecular com-plexes of gC1qR-cytokeratin 1 and u-PAR-cytokeratin 1, with gC1qR binding being favored.

Assessment of the role of heparan sulfate in high molecular weight kininogen binding to human umbilical vein endothelial cells

The assembly and activation of the kinin forming system components on human umbilical vein endothelial cells (HUVEC) have been studied in great detail. Proteins such as gC1qR, cytokeratin-1 and u-PAR have been identified to be responsible for Zn2+-dependent binding of high molecular weight kininogen (HK) to HUVEC. Heparan sulfate has also been shown to have a major role in Zn2+-dependent binding of HK to the endothelial cell line, Ea.hy 926. In this study, we have analyzed the possible contribution of heparan sulfate to high molecular weight kininogen binding to HUVEC using multiple approaches. The presence of heparan sulfate on HUVEC was analyzed by staining with an antibody specific for heparan sulfate. Incubation of the cells with bacterial heparinases removed the heparan sulfate from the cell surface to the level seen with a control antibody, however, the Zn2+-dependent binding of HK was not affected. Further, blocking of heparan sulfate with a specific antibody to heparan sulfate even after digestion with heparinases did not reduce HK binding whereas antibodies to the proteins gC1qR and cytokeratin-1 consistently reduced the binding of HK to the endothelial cells. The binding intensities of FITC-labeled HK were similar in heparinase-treated and -untreated HUVEC. The rate of kallikrein formation by the assembly of factor XII, HK and PK were similar in both heparinase-treated and non-treated HUVEC. All of these data indicate that heparan sulfate does not contribute significantly to HK binding to HUVEC.

Therapeutic immunoglobulin reduces Ca2+ mobilization and von Willebrand factor secretion, and increases nitric oxide release in human endothelial cells.

Intravenous gamma-immunoglobulin (i.v.Ig) is commonly used in the treatment of autoimmune and inflammatory vascular disorders to prevent thrombotic complications. The mechanism of action of i.v.Ig is, however, not yet elucidated. In view of this, we investigated the ability of i.v.Ig to modulate i) Ca(2+) signals of fura-2 loaded endothelial cells, and ii) the associated release of nitric oxide (NO) and von Willebrand factor (vWf). NO was measured either indirectly by radioimmunoassay of cGMP in unstimulated cells or directly by electrochemistry at the surface of stimulated endothelial cells from human umbilical cord veins (HUVEC). Short-term treatment of unstimulated HUVEC with intact i.v.Ig decreased the basal cytosolic Ca(2+) concentration by 20% while it activated the NO/cGMP synthesis. Following i.v.Ig treatment of HUVEC, the Ca(2+) liberation from internal stores and the vWf secretion induced by ATP, thrombin or histamine were significantly reduced by 38 and 60%, respectively. The effects on Ca(2+) signals were observed with intact i.v.Ig as well as with the F(ab')2 or the Fc fragments indicating that both portions are involved in the mechanism of action. The i.v.Ig treatment of HUVECs had no effect on the NO release induced by thrombin or histamine. By contrast, the i.v.Ig treatment increased the ATP-activated NO release by amplifying the Ser1177-eNOS phosphorylation. The i.v.Ig also activated the NO-dependent cGMP release in resting and collagen-stimulated platelets. Since NO is a potent inhibitor of platelet activation and vWF is a platelet adhesion cofactor, the beneficial effects of therapeutic i.v.Ig may lie in the inhibition of platelet adhesion to damaged endothelium.

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

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

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 8.5 x 104 high affinity (KD = 21 nm) sites per HUVEC, i.e. 10-100-fold fewer than previously reported. Although HK binding is unaffected by the presence of a physiological concentration of prekallikrein, factor XI abrogates HK binding to HUVEC in a concentration-dependent manner. Disruption of the naturally occurring complex between factor XI and HK by the addition of a 31-amino acid peptide mimicking the factor XI-binding site on HK restored HK binding to HUVEC. Furthermore, HK inhibited thrombin-stimulated von Willebrand factor release by HUVEC but not thrombin receptor activation peptide (SFLLRN-amide)-stimulated von Willebrand factor release. Factor XI restored the ability of thrombin to stimulate von Willebrand factor release in the presence of low HK concentrations. These results suggest that free HK, or HK in complex with prekallikrein but not in complex with factor XI, interacts with the endothelium and can maintain endothelial cell quiescence by preventing endothelial stimulation by thrombin.