Identification of the factor XII contact activation site enables sensitive coagulation diagnostics.

Contact activation refers to the process of surface-induced activation of factor XII (FXII), which initiates blood coagulation and is captured by the activated partial thromboplastin time (aPTT) assay. Here, we show the mechanism and diagnostic implications of FXII contact activation. Screening of recombinant FXII mutants identified a continuous stretch of residues Gln317-Ser339 that was essential for FXII surface binding and activation, thrombin generation and coagulation. Peptides spanning these 23 residues competed with surface-induced FXII activation. Although FXII mutants lacking residues Gln317-Ser339 were susceptible to activation by plasmin and plasma kallikrein, they were ineffective in supporting arterial and venous thrombus formation in mice. Antibodies raised against the Gln317-Ser339 region induced FXII activation and triggered controllable contact activation in solution leading to thrombin generation by the intrinsic pathway of coagulation. The antibody-activated aPTT allows for standardization of particulate aPTT reagents and for sensitive monitoring of coagulation factors VIII, IX, XI.

Plasma contact system activation drives anaphylaxis in severe mast cell-mediated allergic reactions.

BACKGROUND: Anaphylaxis is an acute, potentially lethal, multisystem syndrome resulting from the sudden release of mast cell-derived mediators into the circulation. OBJECTIVES AND METHODS: We report here that a plasma protease cascade, the factor XII-driven contact system, critically contributes to the pathogenesis of anaphylaxis in both murine models and human subjects. RESULTS: Deficiency in or pharmacologic inhibition of factor XII, plasma kallikrein, high-molecular-weight kininogen, or the bradykinin B2 receptor, but not the B1 receptor, largely attenuated allergen/IgE-mediated mast cell hyperresponsiveness in mice. Reconstitutions of factor XII null mice with human factor XII restored susceptibility for allergen/IgE-mediated hypotension. Activated mast cells systemically released heparin, which provided a negatively charged surface for factor XII autoactivation. Activated factor XII generates plasma kallikrein, which proteolyzes kininogen, leading to the liberation of bradykinin. We evaluated the contact system in patients with anaphylaxis. In all 10 plasma samples immunoblotting revealed activation of factor XII, plasma kallikrein, and kininogen during the acute phase of anaphylaxis but not at basal conditions or in healthy control subjects. The severity of anaphylaxis was associated with mast cell degranulation, increased plasma heparin levels, the intensity of contact system activation, and bradykinin formation. CONCLUSIONS: In summary, the data collectively show a role of the contact system in patients with anaphylaxis and support the hypothesis that targeting bradykinin generation and signaling provides a novel and alternative treatment strategy for anaphylactic attacks.

A factor XIIa inhibitory antibody provides thromboprotection in extracorporeal circulation without increasing bleeding risk.

Currently used anticoagulants prevent thrombosis but increase bleeding. We show an anticoagulation therapy without bleeding risk based on a plasma protease factor XII function-neutralizing antibody. We screened for antibodies against activated factor XII (FXIIa) using phage display and demonstrated that recombinant fully human antibody 3F7 binds into the FXIIa enzymatic pocket. 3F7 interfered with FXIIa-mediated coagulation, abolished thrombus formation under flow, and blocked experimental thrombosis in mice and rabbits. We adapted an extracorporeal membrane oxygenation (ECMO) cardiopulmonary bypass system used for infant therapy to analyze clinical applicability of 3F7 in rabbits. 3F7 provided thromboprotection as efficiently as heparin, and both drugs prevented fibrin deposition and thrombosis within the extracorporeal circuit. Unlike heparin, 3F7 treatment did not impair the hemostatic capacity and did not increase bleeding from wounds. These data establish that targeting of FXIIa is a safe mode of thromboprotection in bypass systems, and provide a clinically relevant anticoagulation strategy that is not complicated by excess bleeding.

BACE-1 inhibition prevents the γ-secretase inhibitor evoked Aß rise in human neuroblastoma SH-SY5Y cells.

BACKGROUND: Accumulation of amyloid ß-peptide (Aß) in the plaques is one of the major pathological features in Alzheimer's disease (AD). Sequential cleavage of amyloid precursor protein (APP) by ß-site APP cleaving enzyme 1 (BACE-1) and γ-secretase results in the formation of Aß peptides. Preventing Aß formation is believed to attenuate AD progression and BACE-1 and γ-secretase are thus attractive targets for AD drug development. METHODS: Combining BACE-1 and γ-secretase inhibition on Aß secretion from human neuroblastoma SH-SY5Y cells was evaluated in this study. Secreted Aß40 and Aß42 levels were measured from SH-SY5Y cells stably transfected with APPwt or APPswe genes. A selective BACE inhibitor and the γ-secretase inhibitor LY450139 (semagacestat) were used to inhibit respective secretase. RESULTS: LY450139 increased Aß40 and Aß42 secretion from SH-SY5Y APPwt cells at low concentrations (by 60% at 3 nM) followed by subsequent inhibition at higher concentrations (IC(50) 90 nM). Washout studies showed that the Aß increase evoked by 3 nM LY450139 was not due to enhanced cleavage following substrate accumulation but rather to activation of Aß formation. By contrast, LY450139 inhibited Aß formation from SH-SY5Y APPswe in a monophasic manner (IC(50) 18 nM). The BACE inhibitor per se inhibited Aß secretion from both SH-SY5Y APPwt and SH-SY5Y APPswe cells with IC(50)s ranging between 7 - 18 nM and also prevented the increased Aß secretion evoked by 3 nM LY450139. Combining the BACE inhibitor with higher inhibitory concentrations of LY450139 failed to demonstrate any clear additive or synergistic effects. CONCLUSION: BACE-1 inhibition attenuates the Aß increase evoked by LY450139 while not providing any obvious synergistic effects on LY450139-mediated inhibition.

Identification of non-muscle myosin heavy chain as a substrate for Cdk5 and tool for drug screening.

BACKGROUND: Deregulated activation of cyclin-dependent kinase-5 (Cdk5) is implicated in neurodegenerative disorders such as Alzheimer's disease. One of the restricting factors for developing specific Cdk5 inhibitors is the lack of reproducible and well-characterized cellular in vitro assay systems. METHODS: HEK293 cells were transfected with Cdk5 and its activator p25 as a starting point for an assay to screen for Cdk5 kinase inhibitors. To identify suitable substrates for Cdk5 we utilized an antibody that recognizes phospho serine in a consensus motif for Cdk substrates. RESULTS: Western blot analysis of transfected cells detected a 200 kDa band that was identified, by mass spectrometry, as non-muscle myosin heavy chain, type B (NMHC-B). Phosphorylation of NMHC-B was evident only in cells that were double transfected with Cdk5/p25 and was dose-dependently inhibited by Roscovitine and other Cdk5 inhibitors. Cdk5 was found to phosphorylate NMHC-B also in the human neuroblastoma SH-SY5Y cell line. CONCLUSION: A novel Cdk5 substrate NMHC-B was identified in this study. A cellular assay for screening of Cdk5 inhibitors was established using NMHC-B phosphorylation as a read-out in Cdk5/p25 transfected HEK293 cells. A novel Cdk5 inhibitor was also pharmacologically characterized in this assay system.

Increased tau phosphorylation at the Ser396 epitope after amyloid beta-exposure in organotypic cultures.

The hallmarks of Alzheimer's disease include extracellular plaques primarily consisting of amyloid-beta peptide and intracellular neurofibrillary tangles composed of highly phosphorylated tau protein. We report that exposure of organotypic hippocampal cultures to synthetic amyloid-beta peptide(25-35) (50 microM, 96 h) causes neurodegeneration concomitant with a significant increase in tau phosphorylation at the Ser epitope (+60%). Furthermore, the level of active glycogen synthase kinase-3beta (GSK-3beta [pTyr]) was increased (+55%) after amyloid-beta peptide(25-35) exposure. These findings support the role of amyloid-beta peptide as a mediator of tau phosphorylation and demonstrate the usefulness of organotypic cultures for investigating the link between amyloid-beta peptide-induced neurotoxicity and tau phosphorylation. Our results also confirm that amyloid-beta peptide induces activation of glycogen synthase kinase-3beta.

Glutamate treatment and p25 transfection increase Cdk5 mediated tau phosphorylation in SH-SY5Y cells.

Neurofibrillary tangles (NFT) of hyperphosphorylated tau protein are a major pathological hallmark of Alzheimer's disease (AD). One of the tau phosphorylating kinases with pathological relevance in AD has been suggested to be the cyclin-dependent kinase 5 (Cdk5). The proposed mechanism leading to pathological Cdk5 activity is through induced cleavage of p35 to a proteolytic product, p25. To further study activation of Cdk5 and its role in tau phosphorylation in vitro, we used differentiated SH-SY5Y cells treated with neurotoxic stimuli or transfected with p25. We show that glutamate increased tau phosphorylation, concomitant with an increased Cdk5 activity achieved by upregulation of Cdk5 and p35 protein levels. Treatment with the calcium ionophore A23187 generated the calpain cleaved p25 fragment but only in toxic conditions that caused dephosphorylation and loss of tau. When p25 was transfected to the cells, increased tau phosphorylation was achieved. However, application of the Cdk5 inhibitor Roscovitine did not result in inhibition of tau phosphorylation possibly due to activation of extracellular regulated kinase 1/2 (Erk1/2), which also is capable of phosphorylating tau. Cdk5 and Erk1/2 kinases share some common substrates but impact of their cross talk on tau phosphorylation has not previously been demonstrated. We also show that p25 is degraded via the proteasome in Roscovitine treated cells.

The retinoic acid and brain-derived neurotrophic factor differentiated SH-SY5Y cell line as a model for Alzheimer's disease-like tau phosphorylation.

The paired helical filaments of highly phosphorylated tau protein are the main components of neurofibrillary tangles (NFT) in Alzheimer's disease (AD). Protein kinases including glycogen synthase kinase 3 beta (GSK3beta), cyclin-dependent kinase 5 (Cdk5), and c-Jun N-terminal kinase (JNK) have been implicated in NFT formation making the use of selective kinase inhibitors an attractive treatment possibility in AD. When sequentially treated with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF), the human neuroblastoma SH-SY5Y differentiates to neuron-like cells. We found that coincident with morphologically evident neurite outgrowth, both the content and phosphorylation state of tau increased in RA-BDNF differentiated SH-SY5Y cells. Tau phosphorylation increased at all the examined sites ser-199, ser-202, thr-205, ser-396, and ser-404, all of which are hyperphosphorylated in AD brain. We also investigated whether GSK3beta, Cdk5 or JNK was involved in tau phosphorylation in the differentiated SH-SY5Y cells. We found that GSK3beta contributed most and that Cdk5 made a minor contribution. JNK was not involved in tau phosphorylation in this system. The GSK3beta-inhibitor, lithium, inhibited tau phosphorylation in a concentration-dependent manner and with good reproducibility, which enables ranking of substances in this cell model. RA-BDNF differentiated SH-SY5Y cells could serve as a suitable model for studying the mechanisms of tau phosphorylation and for screening potential GSK3beta inhibitors.