Interaction of high-molecular-weight kininogen with endothelial cell binding proteins suPAR, gC1qR and cytokeratin 1 determined by surface plasmon resonance (BiaCore).

The physiologic activation of the plasma kallikrein-kinin system requires the assembly of its constituents on a cell membrane. High- molecular-weight kininogen (HK) and cleaved HK (HKa) both interact with at least three endothelial cell binding proteins: urokinase plasminogen activator receptor (uPAR), globular C1q receptor (gC1qR,) and cytokeratin 1 (CK1). The affinity of HK and HKa for endothelial cells are KD=7-52 nM. The contribution of each protein is unknown. We examined the direct binding of HK and HKa to the soluble extracellular form of uPAR (suPAR), gC1qR and CK1 using surface plasmon resonance. Each binding protein linked to a CM-5 chip and the association, dissociation and KD (equilibrium constant) were measured. The interaction of HK and HKa with each binding protein was zinc-dependent. The affinity for HK and HKa was gC1qR>CK1>suPAR, indicating that gC1qR is dominant for binding. The affinity for HKa compared to HK was the same for gC1qR, 2.6-fold tighter for CK1 but 53-fold tighter for suPAR. Complex between binding proteins was only observed between gC1qR and CK1 indicating that a binary CK1-gC1qR complex can form independently of kininogen. Although suPAR has the weakest affinity of the three binding proteins, it is the only one that distinguished between HK and HKa. This finding indicates that uPAR may be a key membrane binding protein for differential binding and signalling between the cleaved and uncleaved forms of kininogen. The role of CK1 and gC1qR may be to initially bind HK to the membrane surface before productive cleavage to HKa.

[Increased knowledge of ARIA and GINA guides 2006 to general physicians by an educational intervention]. / Incremento del conocimiento de las guías ARIA y GINA 2006 para médicos generales mediante una intervención educativa.

BACKGROUND: Educational strategies look for the increase of knowledge in physicians; they are a useful resourse for the diffusion among physicians of guides GINA and ARIA. OBJECTIVE: To evaluate a course shop-like for physicians as an educative strategy. PARTICIPANTS AND METHODS: A transversal study was performed, where knowledge was evaluated to primary contact physicians about recent currents of guides of GINA and ARIA 2006. RESULTS: There was a participation of 69 primary contact physicians who applied a questionnaire of 30 questions: 20 about asthma (GINA) and 10 about allergic rhinitis (ARIA) before and after a course shop-like for physicians; there was improvement on calification after educative strategy on knowledge about asthma and allergic rhinitis with a p = < 0.05. CONCLUSION: The educative strategy proposed as course shop-like for primary contact physicians is effective for teaching the guides of GINA and ARIA 2006.

Annexin A2: biology and relevance to the antiphospholipid syndrome.

Antiphospholipid antibodies (aPL), the majority of which are directed against beta(2)-glycoprotein I (beta(2)GPI), are associated with an increased incidence of venous and arterial thrombosis. The pathogenesis of antiphospholipid/anti-beta(2)GPI-associated thrombosis has not been defined, and is likely multifactorial. However, accumulating evidence suggests an important role for endothelial cell activation with the acquisition of a procoagulant phenotype by the activated endothelial cell. Previous work demonstrated that endothelial activation by antiphospholipid/anti-beta(2)GPI antibodies is beta(2)GPI-dependent. We extended these observations by defining annexin A2 as an endothelial beta(2)GPI binding site. We also observed that annexin A2 plays a critical role in endothelial cell activation induced by anti-beta(2)GPI antibodies, and others have described direct endothelial activation by anti-annexin A2 antibodies in patients with aPL . Similar findings have been reported using human monocytes, which also express annexin A2. Because annexin A2 is not a transmembrane protein, how binding of beta(2)GPI/anti-beta(2)GPI antibodies, or anti-annexin A2 antibodies, to endothelial annexin A2 causes cellular activation is unknown. Recent studies, however, suggest an important role for the Toll-like receptor family, particularly TLR4. In this article, we review the role of these interactions in the activation of endothelial cells by aPL . The influence of these antibodies on the ability of annexin A2 to enhance t-PA-mediated plasminogen activation is also discussed.

Intrauterine fetal death in mice caused by cytomegalovirus-derived peptide induced aPL antibodies.

Immunization of mice with beta2 glycoprotein I (beta2GPI) and also with GDKV, a synthetic peptide representing the phospholipid (PL)-binding site of beta2GPI, induced pathogenic aPL antibodies that bind and activate endothelial cells, enhanced thrombus formation and caused fetal death in pregnant mice. TIFI is a PL-binding peptide spanning the Thr101-Thr120 of ulb0-hcmva from human cytomegalovirus (CMV), which shares structural similarity with the PL-binding site of beta2GPI. Immunization with this peptide induced pathogenic aPL and anti-beta2GPI antibodies in mice. These antibodies activated endothelial cells and enhanced thrombus formation in vivo, but whether these antibodies cause fetal death in mice is not known. The objective of this study was to examine the effects of these antibodies on pregnancy outcome in mice. Two groups of pregnant BALB/c mice were injected with either hybridoma supernatant containing D3/AC10, a CMV-peptide-induced monoclonal aPL, at days four, eight and 12 of the pregnancy, 100 microg per mouse (study group) or with culture media alone (control group). The litter size was significantly smaller in the study group (4.80 +/- 1.15 versus 7.28 +/- 0.18, t = - 2.526, P < 0.03). In conclusion, aPL induced by CMV peptides may have pathogenic properties similar to human autoimmune aPL. These findings further support the hypothesis that at least in some patients with APS, pathogenic aPL antibodies may be generated by immunization with CMV products during incidental exposure to the virus via a molecular mimicry mechanism.

E-Selectin mediates pathogenic effects of antiphospholipid antibodies.

Antiphospholipid (aPL) antibodies, detected in patients with antiphospholipid syndrome (APS) are associated with thrombosis, pregnancy loss and thrombocytopenia. Studies have shown that aPL are thrombogenic in vivo, but the mechanism(s) involved are not completely understood. Several studies have demonstrated that aPL antibodies activate endothelial cells (ECs) in vitro, as determined by up-regulation of adhesion molecules: E-selectin (E-sel); intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and in vivo. The objectives of these study were to determine the effects of aPL antibodies on the expression of E-selectin on ECs, on the adhesion of monocytes to ECs and to study the role of E-selectin on aPL antibodies enhanced thrombus formation and activation of ECs in vivo. We demonstrated that the surface expression of E-selectin on HUVEC by ELISA was increased 400-fold when treated with tumor necrosis factor-alpha (TNF-alpha) and 421-fold when treated with aPL antibodies during 4 h. APL antibodies also induced activation of the nuclear factor-kappa B (NF-kappaB). APL antibodies increased significantly the number of adhering leukocytes to ECs in vivo in C57BL/6 J mice when compared to IgG-NHS treated mice. This effect was abrogated in E-selectin-deficient mice. The thrombus size was significantly increased in C57BL/6 J mice treated with aPL antibodies when compared to mice treated with IgG-NHS. This enhancement in thrombus size by aPL antibodies was abrogated in E-selectin-deficient mice treated with aPL antibodies.

Thrombogenic effects of antiphospholipid antibodies are mediated by intercellular cell adhesion molecule-1, vascular cell adhesion molecule-1, and P-selectin.

Recent studies have shown that antiphospholipid (aPL) enhances expression of intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin on endothelial cells (ECs) and that these effects are correlated with increased adhesion of leukocytes to endothelium in cremaster muscle in vivo and with thrombosis in a mouse model. Activation of ECs by aPL may create a hypercoagulable state that precedes and contributes to thrombosis in patients with aPL syndrome (APS). This study proposed to examine whether this in vivo activation of ECs and enhanced thrombosis by aPL are mediated by ICAM-1, P-selectin, or VCAM-1. The dynamics of thrombus formation and the number of adhering leukocytes were studied in ICAM-1-deficient (ICAM-1(-/-)) mice or ICAM-1-/P-selectin-deficient (ICAM-1(-/-)/P-selectin(-/-)) mice treated with affinity-purified aPL antibodies (ap IgG-APS) or with control IgG and compared with wild-type mice treated in a similar fashion. In another set of experiments, the adhesion of leukocytes to cremaster muscle and the dynamics of thrombus formation were studied in CD1 mice treated with aPL or control IgG before and 30 minutes after intravenous infusion with 100 microg monoclonal antibody anti-VCAM-1. The results indicate that the enhanced adhesion of leukocytes to endothelium in wild-type mice was significantly reduced in ICAM-1(-/-) and completely abrogated in ICAM-1(-/-)/P-selectin(-/-) mice treated with ap IgG-APS compared with wild-type mice treated with ap IgG-APS (6.9+/-2.3, 0.4+/-0.4 versus 35+/-12, respectively). More importantly, this correlated with a significant reduction in thrombus size compared with wild-type mice treated with ap IgG-APS (895+/-259 microm(2), 859+/-243 microm(2) versus 3816+/-672 microm(2), respectively). Infusion of the mice with anti-VCAM-1 antibodies significantly reversed the enhanced adhesion of leukocytes (14.9+/-3 to 11.3+/-2.1) and thrombus size 3830+/-1008 microm(2) versus 876+/-548 microm(2)) in mice treated with ap IgG-APS. The data indicate that ICAM-1, P-selectin, and VCAM-1 expression are important in thrombotic complications by aPL antibodies and may provide novel targets for therapy in patients with APS.

GDKV-induced antiphospholipid antibodies enhance thrombosis and activate endothelial cells in vivo and in vitro.

Antiphospholipid (aPL) Abs are associated with thrombosis, pregnancy loss, and thrombocytopenia in patients with systemic lupus erythematosus or primary antiphospholipid syndrome (APS). beta2-Glycoprotein I (beta2GPI), a phospholipid-binding serum protein, is involved in aPL binding to phospholipids. aPL can be generated in mice by immunization with beta2GPI, and these Abs are thrombogenic and cause pregnancy loss in mice. The objective of this study is to determine whether aPL induced by immunization with the phospholipid-binding site of beta2GPI are thrombogenic and whether they activate endothelial cells (EC) in vivo and in vitro. Murine monoclonal aPL were generated from spleen cells of a mouse immunized with GDKV, a synthetic 15-aa peptide spanning Gly274-Cys288 in the fifth domain of human beta2GPI, which represents the phospholipid-binding site of beta2GPI. The Abs generated had aPL and anti-beta2GPI activities. The effect of these Abs on thrombus formation and on EC activation in vivo was determined using a mouse model of thrombosis and microcirculation that enables examination of the adhesion of leukocyte to EC as an indication of EC activation as well as adhesion molecule expression using in vitro ELISA analysis. Mice injected with this monoclonal aPL showed a significant increase in leukocyte sticking and also produced larger thrombi that persisted longer. Exposure to GDKV-induced aPL for 4 h significantly increased surface Ag expression of E-selectin, ICAM-1, and VCAM-1. These data indicate that aPL induced by immunization with the phospholipid binding site of beta2GPI are thrombogenic and activate endothelial cells.