Platelets are required for enhanced activation of the endothelium and fibrinogen in a mouse thrombosis model of APS

Antiphospholipid syndrome (APS) is defined by thrombosis, fetal loss, and the presence of antiphospholipid antibodies, including anti-beta 2-glycoprotein-1 autoantibodies (anti-beta 2GP1) that have a direct role in the pathogenesis of thrombosis in vivo. The cellular targets of the anti-beta 2GP1autoantibody/beta 2GP1complex in vivo were studied using a laser-induced thrombosis model of APS in a live mouse and human anti-beta 2GP1 autoantibodies affinity-purified from APS patients. Cell binding of fluorescently labeled beta 2GP1 and anti-beta 2GP1 autoantibodies revealed their colocalization on the platelet thrombus but not the endothelium. Anti-beta 2GP1 autoantibodies enhanced platelet activation, monitored by calcium mobilization, and endothelial activation, monitored by intercellular adhesion molecule-1 expression. When eptifibatide was infused to block platelet thrombus formation, enhanced fibrin generation and endothelial cell activation were eliminated. Thus, the anti-beta 2GP1 autoantibody/beta 2GP1 complex binds to the thrombus, enhancing platelet activation, and platelet secretion leads to enhanced endothelium activation and fibrin generation. These results lead to a paradigm shift away from the concept that binding of the anti-beta 2GP1 autoantibody/beta 2GP1 complex activates both endothelial cells and platelets toward one in which activation of platelets in response to anti-beta 2GP1 autoantibody/beta 2GP1 complex binding leads to subsequent enhanced endothelium activation and fibrin generation

Studies on the Structure of Human Fibronectin

Plasma fibronectin is a glycoprotein that consists of two polypeptide chains of approximately 250,000 and 245,000 daltons, joined by disulfide bonds located near one end of the molecule. Fibronectin was isolated from human plasma by gelatin-agarose affinity chromatography and DEAE-cellulose ion exchange chromatography. The two subunit chains were found to be similar or identical with respect to primary structure, using a one-dimensional peptide mapping technique

Thrombus Formation: Direct Real‐Time Observation and Digital Analysis of Thrombus Assembly in a Living Mouse by Confocal and Widefield Intravital Microscopy

We have developed novel instrumentation using confocal and widefield microscopy to image and analyze thrombus formation in real time in the microcirculation of a living mouse. This system provides high-speed, near-simultaneous acquisition of images of multiple fluorescent probes and a brightfield channel, and supports laser-induced injury through the microscope optics. Although this imaging facility requires interface of multiple hardware components, the primary challenge in vascular imaging is careful experimental design and interpretation. This system has been used to localize tissue factor during thrombus formation, to observe defects in thrombus assembly in genetically altered mice, to study the kinetics of platelet activation and P-selectin expression following vascular injury, to analyze leukocyte rolling on arterial thrombi, to generate three-dimensional models of thrombi, and to analyze the effect of antithrombotic agents in vivo

Lessons learned from bone marrow failure in systemic lupus erythematosus: Case reports and review of the literature

© 2017 Elsevier Inc. Objective: In the present review, four new cases of bone marrow failure are presented and the potential contribution of systemic lupus erythematosus (SLE) is discussed. Furthermore, a comprehensive literature review of cases of autoimmune myelofibrosis (AIMF), aplastic anemia (AA), and paroxysmal nocturnal hemoglobinuria (PNH) with concurrent SLE aims to allow their direct comparison. Based on a clearer characterization of reported cases and our own experience, diagnostic and therapeutic strategies of these disorders in SLE are proposed based on lessons learned from the present and previous cases. Methods: A literature search was done in PubMed, accessed via the National Library of Medicine PubMed interface (http://www.ncbi.nlm.nih.gov/pubmed). Using PubMed, a Boolean search of the literature was performed by crossing the keywords “systemic lupus erythematosus,” AND [“bone marrow fibrosis” or “bone marrow failure” or “myelofibrosis” or “aplastic anemia” or “paroxysmal nocturnal hemoglobinuria”]. Results: After a stringent selection of previous cases with a clear diagnosis of SLE, we summarized in the present review 31 cases of AIMF, 26 cases of AA, and 3 cases of PNH. In addition, four new cases illustrate the problem of attribution of bone marrow failure to SLE. Conclusions: The attribution of SLE to bone marrow failure is challenging due to a lack of biomarkers, which complicates treatment decisions. Autoimmune myelofibrosis is likely underreported, but corticosteroids and intravenous immunoglobulin appear to be effective immediate therapies. In AA attributable to SLE, a serum inhibitor of bone marrow precursors should be tested, since plasma exchange has been universally successful in these cases, and a PNH clone should be tested for in the setting of ongoing hemolysis, as complement inhibition may be effective. Further research is warranted to elucidate pathophysiological mechanisms of bone marrow failure in SLE

Endothelial diaphragmed fenestrae: in vitro modulation by phorbol myristate acetate

Cultured microvascular endothelial cells isolated from fenestrated capillaries have been shown to express many properties of their in vivo differentiated phenotype, yet they contain very few diaphragmed fenestrae. We show here that treatment of capillary endothelial cells with the tumor promoter, 4 beta-phorbol 12-myristate 13-acetate, induces more than a fivefold increase in the frequency of fenestrae per micron 2 of cell surface, as determined from a quantitative evaluation on freeze-fracture replicas. In quick-frozen, deep-etched preparations, the endothelial fenestrae appeared to be bridged by a diaphragm composed of radial fibers interweaving in a central mesh, as previously observed in vivo. These results indicate that diaphragmed fenestrae are inducible structures, and provide an opportunity to study them in vitro

Factor IX San Dimas. Substitution of glutamine for Arg-4 in the propeptide leads to incomplete gamma-carboxylation and altered phospholipid binding properties.

DNA sequence analysis of the Factor IX gene from a hemophilia B patient (98% Factor IX antigen; less than 0.01 unit/ml clotting activity) has identified a point mutation in exon II. A guanine to adenine transition causes the substitution of a glutamine codon for an arginine codon at -4 in the propeptide of Factor IX. This variant, termed Factor IX San Dimas, circulates in the plasma as proFactor IX with a mutant 18-amino acid propeptide still attached. Like Factor IX Cambridge (Arg-1----Ser), Factor IX San Dimas is unable to express metal-induced epitopes recognized by conformation-specific polyclonal antibodies. Amino acid analysis of the alkaline hydrolysate indicates that purified Factor IX San Dimas contains a reduced number of gamma-carboxyglutamyl residues compared to Factor IX. However, this protein undergoes metal-induced quenching of the intrinsic fluorescence. In addition, Factor IX San Dimas is unable to interact with phospholipid vesicles. The absence of coagulant activity in Factor IX San Dimas can be attributed to impaired calcium-induced conformational changes and loss in the ability to bind phospholipid vesicles in the presence of calcium ions

Methods for high-dimensonal analysis of cells dissociated from cyropreserved synovial tissue

© 2018 The Author(s). Background: Detailed molecular analyses of cells from rheumatoid arthritis (RA) synovium hold promise in identifying cellular phenotypes that drive tissue pathology and joint damage. The Accelerating Medicines Partnership RA/SLE Network aims to deconstruct autoimmune pathology by examining cells within target tissues through multiple high-dimensional assays. Robust standardized protocols need to be developed before cellular phenotypes at a single cell level can be effectively compared across patient samples. Methods: Multiple clinical sites collected cryopreserved synovial tissue fragments from arthroplasty and synovial biopsy in a 10% DMSO solution. Mechanical and enzymatic dissociation parameters were optimized for viable cell extraction and surface protein preservation for cell sorting and mass cytometry, as well as for reproducibility in RNA sequencing (RNA-seq). Cryopreserved synovial samples were collectively analyzed at a central processing site by a custom-designed and validated 35-marker mass cytometry panel. In parallel, each sample was flow sorted into fibroblast, T-cell, B-cell, and macrophage suspensions for bulk population RNA-seq and plate-based single-cell CEL-Seq2 RNA-seq. Results: Upon dissociation, cryopreserved synovial tissue fragments yielded a high frequency of viable cells, comparable to samples undergoing immediate processing. Optimization of synovial tissue dissociation across six clinical collection sites with ~ 30 arthroplasty and ~ 20 biopsy samples yielded a consensus digestion protocol using 100 μg/ml of Liberase™ TL enzyme preparation. This protocol yielded immune and stromal cell lineages with preserved surface markers and minimized variability across replicate RNA-seq transcriptomes. Mass cytometry analysis of cells from cryopreserved synovium distinguished diverse fibroblast phenotypes, distinct populations of memory B cells and antibody-secreting cells, and multiple CD4+ and CD8+ T-cell activation states. Bulk RNA-seq of sorted cell populations demonstrated robust separation of synovial lymphocytes, fibroblasts, and macrophages. Single-cell RNA-seq produced transcriptomes of over 1000 genes/cell, including transcripts encoding characteristic lineage markers identified. Conclusions: We have established a robust protocol to acquire viable cells from cryopreserved synovial tissue with intact transcriptomes and cell surface phenotypes. A centralized pipeline to generate multiple high-dimensional analyses of synovial tissue samples collected across a collaborative network was developed. Integrated analysis of such datasets from large patient cohorts may help define molecular heterogeneity within RA pathology and identify new therapeutic targets and biomarkers

High-levelexpression of functional recombinant human coagulation factor VII in insect cells

Abstract: Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future. Keywords: Baculovirus; γ-carboxylase; Coagulation FVII; Factor VII; Insect cel