The procoagulant activity of tissue factor expressed on fibroblasts is increased by tissue factor-negative extracellular vesicles.

Tissue factor (TF) is critical for the activation of blood coagulation. TF function is regulated by the amount of externalised phosphatidylserine (PS) and phosphatidylethanolamine (PE) on the surface of the cell in which it is expressed. We investigated the role PS and PE in fibroblast TF function.

Metabolic Dysregulation of the Lysophospholipid/Autotaxin Axis in the Chromosome 9p21 Gene SNP rs10757274.

Background: Common chromosome 9p21 single nucleotide polymorphisms (SNPs) increase coronary heart disease risk, independent of traditional lipid risk factors. However, lipids comprise large numbers of structurally related molecules not measured in traditional risk measurements, and many have inflammatory bioactivities. Here, we applied lipidomic and genomic approaches to 3 model systems to characterize lipid metabolic changes in common Chr9p21 SNPs, which confer ≈30% elevated coronary heart disease risk associated with altered expression of ANRIL, a long ncRNA.

Phospholipid membranes drive abdominal aortic aneurysm development through stimulating coagulation factor activity.

Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA.

Proline provides site-specific flexibility for in vivo collagen.

Fibrillar collagens have mechanical and biological roles, providing tissues with both tensile strength and cell binding sites which allow molecular interactions with cell-surface receptors such as integrins. A key question is: how do collagens allow tissue flexibility whilst maintaining well-defined ligand binding sites? Here we show that proline residues in collagen glycine-proline-hydroxyproline (Gly-Pro-Hyp) triplets provide local conformational flexibility, which in turn confers well-defined, low energy molecular compression-extension and bending, by employing two-dimensional C-C correlation NMR spectroscopy on C-labelled intact ex vivo bone and in vitro osteoblast extracellular matrix. We also find that the positions of Gly-Pro-Hyp triplets are highly conserved between animal species, and are spatially clustered in the currently-accepted model of molecular ordering in collagen type I fibrils.

Networks of enzymatically oxidized membrane lipids support calcium-dependent coagulation factor binding to maintain hemostasis.

Blood coagulation functions as part of the innate immune system by preventing bacterial invasion, and it is critical to stopping blood loss (hemostasis). Coagulation involves the external membrane surface of activated platelets and leukocytes. Using lipidomic, genetic, biochemical, and mathematical modeling approaches, we found that enzymatically oxidized phospholipids (eoxPLs) generated by the activity of leukocyte or platelet lipoxygenases (LOXs) were required for normal hemostasis and promoted coagulation factor activities in a Ca- and phosphatidylserine (PS)-dependent manner.

LipidFinder: A computational workflow for discovery of lipids identifies eicosanoid-phosphoinositides in platelets.

Accurate and high-quality curation of lipidomic datasets generated from plasma, cells, or tissues is becoming essential for cell biology investigations and biomarker discovery for personalized medicine. However, a major challenge lies in removing artifacts otherwise mistakenly interpreted as real lipids from large mass spectrometry files (>60 K features), while retaining genuine ions in the dataset. This requires powerful informatics tools; however, available workflows have not been tailored specifically for lipidomics, particularly discovery research.

DioxolaneA3-phosphatidylethanolamines are generated by human platelets and stimulate neutrophil integrin expression.

Activated platelets generate an eicosanoid proposed to be 8-hydroxy-9,10-dioxolane A3 (DXA). Herein, we demonstrate that significant amounts of DXA are rapidly attached to phosphatidylethanolamine (PE) forming four esterified eicosanoids, 16:0p, 18:0p, 18:1p and 18:0a/DXA-PEs that can activate neutrophil integrin expression. These lipids comprise the majority of DXA generated by platelets, are formed in ng amounts (24.

Collagen cross-linking: insights on the evolution of metazoan extracellular matrix.

Collagens constitute a large family of extracellular matrix (ECM) proteins that play a fundamental role in supporting the structure of various tissues in multicellular animals. The mechanical strength of fibrillar collagens is highly dependent on the formation of covalent cross-links between individual fibrils, a process initiated by the enzymatic action of members of the lysyl oxidase (LOX) family. Fibrillar collagens are present in a wide variety of animals, therefore often being associated with metazoan evolution, where the emergence of an ancestral collagen chain has been proposed to lead to the formation of different clades.

Mapping the Human Platelet Lipidome Reveals Cytosolic Phospholipase A2 as a Regulator of Mitochondrial Bioenergetics during Activation.

Human platelets acutely increase mitochondrial energy generation following stimulation. Herein, a lipidomic circuit was uncovered whereby the substrates for this are exclusively provided by cPLA2, including multiple fatty acids and oxidized species that support energy generation via β-oxidation. This indicates that acute lipid membrane remodeling is required to support energetic demands during platelet activation.

Hydroxyproline Ring Pucker Causes Frustration of Helix Parameters in the Collagen Triple Helix.

Collagens, the most abundant proteins in mammals, are defined by their triple-helical structures and distinctive Gly-Xaa-Yaa repeating sequence, where Xaa is often proline and Yaa, hydroxyproline (Hyp/O). It is known that hydroxyproline in the Yaa position stabilises the triple helix, and that lack of proline hydroxylation in vivo leads to dysfunctional collagen extracellular matrix assembly, due to a range of factors such as a change in hydration properties. In addition, we note that in model peptides, when Yaa is unmodified proline, the Xaa proline has a strong propensity to adopt an endo ring conformation, whilst when Yaa is hydroxyproline, the Xaa proline adopts a range of endo and exo conformations.

Structural constraints on the evolution of the collagen fibril: convergence on a 1014-residue COL domain.

Type I collagen is the fundamental component of the extracellular matrix. Its α1 gene is the direct descendant of ancestral fibrillar collagen and contains 57 exons encoding the rod-like triple-helical COL domain. We trace the evolution of the COL domain from a primordial collagen 18 residues in length to its present 1014 residues, the limit of its possible length.

The recognition of collagen and triple-helical toolkit peptides by MMP-13: sequence specificity for binding and cleavage.

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly(775)-Leu(776) in collagen II.

NMR spectroscopy of native and in vitro tissues implicates polyADP ribose in biomineralization.

Nuclear magnetic resonance (NMR) spectroscopy is useful to determine molecular structure in tissues grown in vitro only if their fidelity, relative to native tissue, can be established. Here, we use multidimensional NMR spectra of animal and in vitro model tissues as fingerprints of their respective molecular structures, allowing us to compare the intact tissues at atomic length scales. To obtain spectra from animal tissues, we developed a heavy mouse enriched by about 20% in the NMR-active isotopes carbon-13 and nitrogen-15.

Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation.

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets.

The properties conferred upon triple-helical collagen-mimetic peptides by the presence of cysteine residues.

Recently, the ability of polymeric collagen-like peptides to regulate cell behavior has generated great interest. A triple-helical peptide known as collagen-related peptide (CRP) contains the sequence (Gly-Pro-Hyp)(10). With Gly-Pro-Cys triplets appended to both of its termini, designated CRP(cys), chemical cross-linking using heterobifunctional reagents generates CRP(cys)-XL, a potent, widely used, polymeric agonist for platelet Glycoprotein VI, whereas non-cross-linked, monomeric CRP(cys) antagonizes Glycoprotein VI.

OSCAR is a collagen receptor that costimulates osteoclastogenesis in DAP12-deficient humans and mice.

Osteoclasts are terminally differentiated leukocytes that erode the mineralized bone matrix. Osteoclastogenesis requires costimulatory receptor signaling through adaptors containing immunoreceptor tyrosine-based activation motifs (ITAMs), such as Fc receptor common γ (FcRγ) and DNAX-activating protein of 12 kDa. Identification of these ITAM-containing receptors and their ligands remains a high research priority, since the stimuli for osteoclastogenesis are only partly defined.

The effect of purity upon the triple-helical stability of collagenous peptides.

Collagen is the fundamental structural protein, comprising 25-35% of the total body protein, its rod-like triple helix providing support in many tissues. Our laboratory has synthesised 113 Toolkit peptides, each 63 residues long, covering the entirety of the homotrimeric helix sequence of collagen II and collagen III. These are used primarily to investigate protein-collagen interactions, from which biomedical applications are under development.

Identification and structural analysis of type I collagen sites in complex with fibronectin fragments.

Collagen and fibronectin are major components of vertebrate extracellular matrices. Their association and distribution control the development and properties of diverse tissues, but thus far no structural information has been available for the complex formed. Here, we report binding of a peptide, derived from the alpha(1) chain of type I collagen, to the gelatin-binding domain of human fibronectin and present the crystal structure of this peptide in complex with the (8-9)FnI domain pair.

Collagen in its fibrillar state is protected from glycation.

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis.

Rapid synthesis of a register-specific heterotrimeric type I collagen helix encompassing the integrin alpha2beta1 binding site.

We report a rapid method to synthesize cystine cross-linked heterotrimeric collagenous peptides. They can be engineered to favour one particular axial alignment of the strands, called the register of the helix. Here, the sequence of the constituent peptides contains 18 residues of "guest" collagen type I sequence flanked by N and C-terminal (Gly-Pro-Pro)5 "host" modules which ensure helicity.

Use of synthetic peptides to locate novel integrin alpha2beta1-binding motifs in human collagen III.

A set of 57 synthetic peptides encompassing the entire triplehelical domain of human collagen III was used to locate binding sites for the collagen-binding integrin alpha(2)beta(1). The capacity of the peptides to support Mg(2+)-dependent binding of several integrin preparations was examined. Wild-type integrins (recombinant alpha(2) I-domain, alpha(2)beta(1) purified from platelet membranes, and recombinant soluble alpha(2)beta(1) expressed as an alpha(2)-Fos/beta(1)-Jun heterodimer) bound well to only three peptides, two containing GXX'GER motifs (GROGER and GMOGER, where O is hydroxyproline) and one containing two adjacent GXX'GEN motifs (GLKGEN and GLOGEN).

Integrin activation state determines selectivity for novel recognition sites in fibrillar collagens.

Only three recognition motifs, GFOGER, GLOGER, and GASGER, all present in type I collagen, have been identified to date for collagen-binding integrins, such as alpha(2)beta(1). Sequence alignment was used to investigate the occurrence of related motifs in other human fibrillar collagens, and located a conserved array of novel GER motifs within their triple helical domains. We compared the integrin binding properties of synthetic triple helical peptides containing examples of such sequences (GLSGER, GMOGER, GAOGER, and GQRGER) or the previously identified motifs.