Selective factor VIII activation by the tissue factor-factor VIIa-factor Xa complex.

Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.

The M protein of group A Streptococcus is a key virulence factor and a clinically relevant strain identification marker.

The M protein coats group A streptococci (GAS) and acts as the primary antigen and determinant of type-specific immunity. M is essential for GAS virulence, providing antiphagocytic functions critical to survival in human tissues and fluids. Specific regions of M protein also serve as shared antigens, and cross-reactivity between these epitopes and human proteins may be the source of autoimmune sequelae such as rheumatic heart disease. The M protein is hypervariable, and has long served as the primary target for epidemiological typing of GAS. Though other markers or genotyping methods may be necessary to increase strain resolution when clones of a given M type differ in clinically critical ways, M typing remains the most directly informative and well-documented method for tracking outbreaks of GAS, predicting clinical outcomes during those outbreaks, and measuring the general threat presented by GAS at any given time and place.

Statins inhibit cyclooxygenase-2 and matrix metalloproteinase-9 in human endothelial cells: anti-angiogenic actions possibly contributing to plaque stability.

AIMS: Cyclooxygenase (COX)-2 expression is increased in inflammation and angiogenesis and also in atherosclerotic plaques, where it co-localizes with metalloproteinases (MMPs) involved in the fibrous cap weakening. Insight into the regulation of COX-2 and MMP-9 expression suggests the involvement of a Rho-dependent pathway. Because statins interfere with Rho activation, we investigated the statin effect on COX-2 and MMP expressions in the human endothelium. METHODS AND RESULTS: Simvastatin and atorvastatin were incubated with endothelial cells for 12 h before stimulation with phorbol myristate acetate or tumour necrosis factor-alpha, for times suitable to assess the endothelial tube differentiation on Matrigel and COX-2 and MMPs activities, proteins, and mRNA expressions. At 0.1-10 micromol/L, both statins reduced COX-2 expression and activity, without affecting COX-1. The statin effect was reversed by mevalonate and geranylgeranyl-pyrophosphate and mimicked by the Rho inhibitor C3 transferase, indicating the involvement of Rho in the signal transduction pathway leading to COX-2 expression. In parallel, statins, as well as COX-2 inhibitors, reduced the MMP-9 stimulated release and the endothelial tubular differentiation. CONCLUSION: In the human vascular endothelium, statins reduce COX-2 and MMP-9 expression and activity. Through this mechanism, statins exert an anti-angiogenic effect possibly contributing to the cholesterol-lowering-unrelated protective effects of statins against plaque inflammatory angiogenesis and rupture.

Prominent role of NF-kappaB in the induction of endothelial activation by endogenous nitric oxide inhibition.

Decreased endothelial nitric oxide (NO) production and increased expression of vascular cell adhesion molecule-1 (VCAM-1) are early features of atherosclerosis. We investigated the effects of suppressing endogenous NO production by the NO synthase inhibitor l-mono-methyl-arginine (L-NMMA), given alone or in combination with interleukin(IL)-1alpha, on VCAM-1 expression by human umbilical vein endothelial cells (HUVEC). VCAM-1 expression (by enzyme immunoassay), barely detectable at baseline, was significantly increased by L-NMMA (by no more than 20% over control compared with IL-1alpha induction). This was paralleled by an increase in U937 monocytoid cell adhesion. When HUVEC incubated with L-NMMA were stimulated with low concentrations of IL-1alpha (0.05-0.5ng/mL), these determined a higher VCAM-1 expression than in the presence of L-NMMA or IL-1alpha alone. Northern analysis indicated that VCAM-1 mRNA was induced by L-NMMA alone, and that the effects of L-NMMA and IL-1alpha were, again, at least additive. Nuclear factor-kappaB (NF-kappaB), GATA, activator protein-1 (AP-1) and interferon regulatory factor-1 (IRF-1), transcription factors all involved in VCAM-1 gene expression, were all activated at electrophoretic mobility shift assay and at chromatin immunoprecipitation assay by L-NMMA, but additive effects with the combined administration of L-NMMA and IL-1alpha only occurred for NF-kappaB. These results support the view that endogenous NO mantains a normal endothelial non-reactivity towards circulating monocytes, and that suppression of this endogenous brake for endothelial activation results in the activation of multiple transcription factors even in the absence of other endothelial activators, with a prominent role of NF-kappaB in the presence or absence of other inflammatory mediators.

Mice with mutations of Dock7 have generalized hypopigmentation and white-spotting but show normal neurological function.

The classical recessive coat color mutation misty (m) arose spontaneously on the DBA/J background and causes generalized hypopigmentation and localized white-spotting in mice, with a lack of pigment on the belly, tail tip, and paws. Here we describe moonlight (mnlt), a second hypopigmentation and white-spotting mutation identified on the C57BL/6J background, which yields a phenotypic copy of m/m coat color traits. We demonstrate that the 2 mutations are allelic. m/m and mnlt/mnlt phenotypes both result from mutations that truncate the dedicator of cytokinesis 7 protein (DOCK7), a widely expressed Rho family guanine nucleotide exchange factor. Although Dock7 is transcribed at high levels in the developing brain and has been implicated in both axon development and myelination by in vitro studies, we find no requirement for DOCK7 in neurobehavioral function in vivo. However, DOCK7 has non-redundant role(s) related to the distribution and function of dermal and follicular melanocytes.

Hyperantithrombotic, noncytoprotective Glu149Ala-activated protein C mutant.

Activated protein C (APC) reduces mortality in severe sepsis patients. APC exerts anticoagulant activities via inactivation of factors Va and VIIIa and cytoprotective activities via endothelial protein C receptor and protease-activated receptor-1. APC mutants with selectively altered and opposite activity profiles, that is, greatly reduced anticoagulant activity or greatly reduced cytoprotective activities, are compared here. Glu149Ala-APC exhibited enhanced in vitro anticoagulant and in vivo antithrombotic activity, but greatly diminished in vitro cytoprotective effects and in vivo reduction of endotoxin-induced murine mortality. Thus, residue Glu149 and the C-terminal region of APC's light chain are identified as functionally important for expression of multiple APC activities. In contrast to Glu149Ala-APC, 5A-APC (Lys191-193Ala + Arg229/230Ala) with protease domain mutations lacked in vivo antithrombotic activity, although it was potent in reducing endotoxin-induced mortality, as previously shown. These data imply that APC molecular species with potent antithrombotic activity, but without robust cytoprotective activity, are not sufficient to reduce mortality in endotoxemia, emphasizing the need for APC's cytoprotective actions, but not anticoagulant actions, to reduce endotoxin-induced mortality. Protein engineering can provide APC mutants that permit definitive mechanism of action studies for APC's multiple activities, and may also provide safer and more effective second-generation APC mutants with reduced bleeding risk.

Omega-3 fatty acids, atherogenesis, and endothelial activation.

Omega-3 or n-3 polyunsaturated fatty acids (PUFAs) display a variety of beneficial effects on various organ systems and diseases. Evidence for cardiovascular protective effects was among the first reasons for biomedical interest in these fats. Following these initial reports, evidence has continued to emerge demonstrating the cardioprotective effects of n-3 PUFAs and elucidating the underlying biological mechanisms. Decreased atherogenesis is currently thought to account at least in part for cardiovascular protection by n-3 PUFAs. We summarise the evidence for such effects and the putative mechanisms involved.

Nutritional mechanisms that influence cardiovascular disease.

Current evidence suggests that most significant risk factors for heart disease have been identified. Although age, sex, and genetics are important unmodifiable risk factors, most new cases of acute myocardial infarctions today can be predicted by the presence and level of 9 risk (or cardioprotective) factors that can easily be assessed and, most importantly, modified. These risk factors are the same in almost every geographic region and in every racial/ethnic group worldwide and are consistent in men and women. Eight of these 9 risk factors are influenced by diet, and most act by promoting atherogenesis, which is the most important background condition for cardiovascular disease. Dietary interventions mostly affect atherogenesis by modulating, at the cellular level, proinflammatory processes that initiate and perpetuate endothelial dysfunction, plaque formation, and, eventually, plaque rupture. For example, there is now enough evidence, both epidemiologic and clinical, of the beneficial effects of n-3 fatty acids. Either as part of a normal low-fat diet or as supplements, these fatty acids are now recommended to prevent cardiovascular disease. This review will summarize the mechanisms by which diet may influence atherogenesis through the early inception, progression, and clinical emergence of atherosclerosis, with a special focus on n-3 fatty acids.

Antiatherogenic effects of n-3 fatty acids - evidence and mechanisms.

N-3 (omega-3) (polyunsaturated) fatty acids are thought to display a variety of beneficial effects for human health. Clues to the occurrence of cardiovascular protective effects have been, however, the spur for the first biomedical interest in these compounds, and are the best documented. Historically, the epidemiologic association between dietary consumption of n-3 fatty acids and cardiovascular protection was first suggested by Bang and Dyerberg, who identified the high consumption of fish, and therefore, of fish oil-derived n-3 fatty acids, as the likely explanation for the strikingly low rate of coronary heart disease events reported in the Inuit population. Since their initial reports, research has proceeded in parallel to provide further evidence for their cardioprotection and to understand underlying mechanisms. Decreased atherogenesis is currently thought to be a part of the cardiovascular protection by n-3 fatty acids. This article summarizes the evidence for such a claim and the mechanisms putatively involved.

Contrasting effect of fish oil supplementation on the development of atherosclerosis in murine models.

OBJECTIVE: Increased fish oil intake is associated with protection against coronary heart disease and sudden death, while effects on atherosclerosis are controversial. We explored the effects of supplementing fish oil (rich in n-3 polyunsaturated fatty acids, PUFA) or corn oil (rich in n-6 PUFA) in two different models of atherosclerosis. METHODS AND RESULTS: Sixty-three low density lipoprotein receptor-deficient (LDLR(-/-)) mice and sixty-nine apolipoprotein E-deficient (apoE(-/-)) mice were fed diets without supplementations or supplemented with either 1% fish oil or 1% corn oil. In apoE(-/-) mice, neither fish oil nor corn oil had any major impact on plasma lipids or atherosclerosis. In LDLR(-/-) mice, conversely, the fish oil and the corn oil group had lower levels of LDL-cholesterol and triglycerides and had lesser atherosclerosis in the aortic root and in the entire aorta (p < 0.01 versus unsupplemented group). Atherosclerosis was significantly less in the fish oil group compared with the corn oil group when evaluated en face in the aortic arch (area positive to lipid staining: 32% with fish oil versus 38% with corn oil; 48% with unsupplemented diet). CONCLUSIONS: n-3 and n-6 PUFA supplementation retarded the development of atherosclerosis in LDLR(-/-) mice, with a stronger effect seen with n-3 PUFA. There was an important strain-dependence of the effect, with no protection against atherosclerosis in apoE(-/-) mice.

Capillary electrophoresis-electrospray mass spectrometry and HR-ICP-MS for the detection and quantification of 10B-boronophenylalanine (10B-BPA) used in boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is a bimodal radiotherapeutic treatment based on the irradiation of neoplastic tissues with neutrons after the tissues have selectively accumulated molecules loaded with nuclides with large neutron capture cross-sections (such boron-10). Boron-10 carriers have been tested to a limited extent, and clinical trials have been conducted on sulfhydryl borane (10B-BSH) and boronophenylalanine (10B-BPA). However, precise and accurate measurements of boron-10 concentrations (0.1-100 microg/g) in specimens and samples of limited size (microg scale) are needed in order to be able to biologically characterise new compounds in predictive tissue dosimetry, toxicology and pharmacology studies as well as in clinical investigations. A new approach based on fast separation and detection of 10B-BPA performed by coupling capillary electrophoresis to electrospray mass spectrometry is reported. This method allows the quantitative analysis and characterisation of 10B-BPA in a short time with a high separation efficiency. Detection limits of 3 microM for 10B-BPA and 30 ng/mL for 10B were obtained with CE-ESI-MS. A quantification limit of 10 microM for 10B-BPA (100 ng/mL for 10B) was attained. The total boron-10 concentration was determined by high-resolution inductively coupled mass spectrometry in order to validate the method. Boron-10 isotope measurements were carried out by HR-ICP-MS at medium resolution (R=4000) due to the presence of an isobaric interference at mass 10. Good agreement was obtained between the values from CE-ESI-MS and those from HR-ICP-MS. The method has been successfully used to determine the 10B-BPA in two lines of cultured cells.

New cardiovascular risk factors: homocysteine and vitamins involved in homocysteine metabolism.

Among various cardiovascular risk factors, hyperhomocysteinemia has recently emerged as an important one. While there are currently no doubts on the relationship between severe hyperhomocysteinemia and vascular disease, some uncertainty still persists on the relationship between mild hyperhomocysteinemia and vascular disease. Several group B vitamins, namely vitamin B6, vitamin B12, and folate, influence homocysteine metabolism, being cofactors of the main metabolic pathways which allow the disposal of this amino acid. There are also, however, suggestions from the literature that group B vitamins, and in particular vitamin B6 (pyridoxine/pyridoxal-phosphate), are modulators of cardiovascular risk independent of homocysteine. The results of a recent study of ours, with a long follow-up, indeed suggest that homocysteine and vitamin B6 are independent and additive cardiovascular risk factors.