Co-localization Of Von Willebrand Factor Research Articles

To be EndMT or not to be, that is the question in pulmonary hypertension.

To be EndMT or not to be, that is the question in pulmonary hypertension.

Siglecs as Novel Cellular Partners for Von Willebrand Factor

AbstractAbstract 4306 Introduction:Von Willebrand factor (VWF) is a multimeric protein that is pertinent to the haemostatic process by promoting platelet recruitment to the growing thrombus and acting as a carrier-protein for factor VIII. It is well established that VWF is able to interact with several cellular receptors present on the surface of platelets and leukocytes. In search for novel cellular partners for VWF present on these cells, we made use of the notion that VWF is heavily glycosylated, with the mature molecule containing 12 N-linked glycans and 10 O-linked glycans. Importantly, the vast majority of these glycans (>90%) are sialylated. The human proteome contains a family of sialic acid-binding receptors, known as Sialic acid-binding Immunoglobulin-like Lectins (Siglecs), which are expressed on cells of hematopoietic origin. The current study was designed to investigate the potential of Siglecs to interact with VWF. Methods:Stable human HEK293 cell lines were established that express human Siglec-5, Siglec-7 or Siglec-9. In addition, soluble variants of Siglecs were obtained: monomeric HPC4-tagged Siglec-5 and Siglec-7 (sSg5/HPC4 & sSg7/HPC4) and dimeric Fc-fusion proteins for human Siglec-3, -5, -7, -9, -10, -11 and murine Siglec-F (sSg-3/Fc etc). These Siglecs were used to study their interaction with purified plasma-derived (pdVWF) or recombinant BHK-cell derived VWF (rVWF). Protein-protein interactions were investigated via immuno-sorbent assays and via surface plasmon resonance (SPR) using Octet QK equipment. Binding of VWF to Siglec-expressing cells was assessed via immuno-fluorescence microscopy. Results:We observed consistent association of VWF (irrespective whether immuno-sorbent or SPR assays were used) to all of the immobilized Siglecs tested, with the exception of sSg-11/Fc that did not interact with pdVWF or with rVWF. Inversely, all soluble Siglecs but sSg-11/Fc efficiently bound to immobilized pdVWF or rVWF. Half-maximal binding in immuno-sorbent assays was between 97 and 645 nM (binding of VWF to immobilized Siglecs) versus 166 and 270 nM (binding of Siglecs to immobilized VWF). More detailed studies on the determination of the kinetic parameters using SPR technology are in progress. Specificity of the interaction was confirmed by treating VWF with sialidase before testing Siglec binding, and such treatment resulted in a strongly reduced (up to 80%) ability of Siglecs to bind to immobilized VWF. VWF was further found to bind to Siglec-5, -7 or -9 expressing HEK293 cells as assessed via immuno-fluorescence microscopy. In general, 10–15 % of the Siglec-expressing cells were covered with VWF after incubation, with the fluorescent intensity increasing dose-dependently with the applied VWF concentration. No fluorescent signal was observed upon incubation of VWF with non-transfected cells or when VWF was omitted from the incubation. Since Siglecs may be masked by the presence of endogenous sialylated proteins at the cellular surface, we also tested VWF binding to these cells following sialidase treatment. Sialidase treatment of cells resulted in a marked increase in the number of VWF-binding cells (up to 80% of the Siglec-positive cells) as well as an increase in intensity of the fluorescent signal. Co-localization of VWF with Siglecs at the cellular surface was confirmed by DuoLink-analysis, which allows the detection of proteins that are in the vicinity of ≤40 nm. Emerging evidence demonstrates that endothelial. Conclusions:We identified one murine and six human Siglec-family members as novel partners for VWF. Interactions between VWF and Siglecs are mediated by sialic acid structures present on VWF. In addition, the cellular accessibility of Siglecs for VWF is modulated by cis-acting sialylated proteins at the cell surface. In conclusion, our data demonstrate that the VWF glycome allows the interaction with a novel family of cellular receptors, potentially opening previously unrecognized avenues in the biology of VWF. Disclosures:No relevant conflicts of interest to declare.

Co-localization of von Willebrand factor with platelet thrombi, tissue factor and platelets with fibrin, and consistent presence of inflammatory cells in coronary thrombi obtained by an aspiration device from patients with acute myocardial infarction.

Detailed histochemical analysis of coronary thrombi obtained freshly from acute phase of myocardial infarction patients may provide information necessary to understand the mechanism of coronary occlusive thrombus formation. Coronary thrombi causing myocardial infarction were obtained from 10 consecutive patients of myocardial infarction in the acute phase, using a newly developed aspiration catheter. All the fixed specimens of coronary thrombi, by hematoxylin and eosin staining, were found to contain three major constituents, namely, platelets, densely packed fibrin and inflammatory cells, including polymorphonuclear and mononuclear cells, although their distribution in each specimen is totally heterogeneous. Immunohistochemical staining revealed the prominent presence of von Willebrand factor (VWF) at the sites of platelet accumulation, presence of tissue factor and platelets at the sites of deposition of fibrin fibrils. It also revealed the presence of CD16-, CD45- and CD34-positive cells, yet the functional roles of these cells have still to be elucidated. There are weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain. In spite of various limitations, our results contain information suggesting the possible role of VWF in platelet-thrombus formation, possible important role played by tissue factor and activated platelets in the formation of fibrin fibrils, and the positive relationship between inflammatory cells migration and the formation of occlusive thrombi in human coronary arteries.

Brain dystrophin-glycoprotein complex: Persistent expression of beta-dystroglycan, impaired oligomerization of Dp71 and up-regulation of utrophins in animal models of muscular dystrophy

BackgroundAside from muscle, brain is also a major expression site for dystrophin, the protein whose abnormal expression is responsible for Duchenne muscular dystrophy. Cognitive impairments are frequently associated with this genetic disease, we therefore studied the fate of brain and skeletal muscle dystrophins and dystroglycans in dystrophic animal models.ResultsAll dystrophin-associated glycoproteins investigated were reduced in dystrophic muscle fibres. In Dp427-deficient mdx brain and Dp71-deficient mdx-3cv brain, the expression of α-dystroglycan and laminin was reduced, utrophin isoforms were up-regulated and β-dystroglycan was not affected. Immunofluorescence localization of β-dystroglycan in comparison with glial, endothelial and neuronal cell markers revealed co-localization of von Willebrand factor with β-dystroglycan. Its expression at the endothelial-glial interface was preserved in dystrophin isoform-deficient brain from mdx and mdx-3cv mice. In addition, chemical crosslinking revealed that the Dp71 isoform exists in mdx brain predominantly as a monomer.ConclusionsThis suggests an association of β-dystroglycan with membranes at the vascular-glial interface in the forebrain. In contrast to dystrophic skeletal muscle fibres, dystrophin deficiency does not trigger a reduction of all dystroglycans in the brain, and utrophins may partially compensate for the lack of brain dystrophins. Abnormal oligomerization of the dystrophin isoform Dp71 might be involved in the pathophysiological mechanisms underlying abnormal brain functions.