Integrin-Mediated Cell-Matrix Interaction in Physiological and Pathological Blood Vessel Formation

Physiological as well as pathological blood vessel formation are fundamentally dependent on cell-matrix interaction. Integrins, a family of major cell adhesion receptors, play a pivotal role in development, maintenance, and remodeling of the vasculature. Cell migration, invasion, and remodeling of the extracellular matrix (ECM) are integrin-regulated processes, and the expression of certain integrins also correlates with tumor progression. Recent advances in the understanding of how integrins are involved in the regulation of blood vessel formation and remodeling during tumor progression are highlighted. The increasing knowledge of integrin function at the molecular level, together with the growing repertoire of integrin inhibitors which allow their selective pharmacological manipulation, makes integrins suited as potential diagnostic markers and therapeutic targets

Phosphorylation of CLEC-2 is dependent on lipid rafts, actin polymerization,secondary mediators, and Rac

The C-type lectin-like receptor 2 (CLEC-2)activates platelets through Src and Syk tyrosine kinases via a single cytoplasmic YxxL motif known as a hem immunoreceptor tyrosine-based activation motif (hemITAM).Here, we demonstrate using sucrose gradient ultracentrifugation and methyl--cyclodextrin treatment that CLEC-2 translocates to lipid rafts upon ligand engagement and that translocation is essential for hemITAM phosphorylation and signal initiation. HemITAM phosphorylation, but not translocation, is also critically dependent on actin polymerization,Rac1 activation, and release of ADP and thromboxane A2 (TxA2). The role of ADP and TxA2 in mediating hosphorylation is dependent on ligand engagement and rac activation but is independent of platelet aggregation. In contrast,tyrosine phosphorylation of the GPVIFcR -chain ITAM, which has 2 YxxL motifs,is independent of actin polymerization and secondary mediators. These results reveal a unique series of proximal events in CLEC-2 phosphorylation involving actin polymerization, secondary mediators,and Rac activation

Preserved bioactivity and tunable release of a SDF1-GPVI bi-specific protein using photo-crosslinked PEGda hydrogels

AbstractChemokine-induced stem cell recruitment is a promising strategy for post myocardial infarction treatment. Injection of stromal cell-derived factor 1 (SDF1) has been shown to attract bone marrow-derived progenitor cells (BMPCs) from the blood that have the potential to differentiate into cardiovascular cells, which support angiogenesis, enabling the improvement of myocardial function. SDF1-GPVI bi-specific protein contains a glycoprotein VI (GPVI)-domain that serves as an anchor for collagen type I (Col I) and III, which are exposed in the wall of injured vasculature. In this study, we generated a cytocompatible hydrogel via photo-crosslinking of poly(ethylene glycol) diacrylate that serves as a reservoir for SDF1-GPVI. Controlled and sustained release of SDF1-GPVI was demonstrated over a period of 7 days. Release features were modifiable depending on the degree of the crosslinking density. Functionality of the GPVI-domain was investigated using a GPVI-binding ELISA to Col I. Activity of the SDF1-domain was tested for its CXCR4 binding potential. Preserved functionality of SDF1-GPVI bi-specific protein after photo-crosslinking and controllable release was successfully demonstrated in vitro supporting the implementation of this drug delivery system as a powerful tool for therapeutic protein delivery in the treatment of cardiovascular ischemic disease

A Comprehensive Tyrosine Phosphoproteomic Analysis Reveals Novel Components of the Platelet CLEC-2 Signaling Cascade

This is an Accepted Manuscript of an article published by Thieme Publishing Group in Thrombosis and Haemostasis on 04 January 2020, available online at https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0039-3400295C-type lectin-like receptor 2 (CLEC-2) plays a crucial role in different platelet-related physiological and pathological processes. It signals through a tyrosine kinase-mediated pathway that is highly dependent on the positive feedback exerted by the platelet-derived secondary mediators, adenosine diphosphate (ADP) and thromboxane A2 (TXA2). Here, we aimed to analyze the tyrosine phosphoproteome of platelets activated with the CLEC-2 agonist rhodocytin to identify relevant phosphorylated tyrosine residues (p-Tyr) and proteins involved in platelet activation downstream of this receptor. We identified 363 differentially p-Tyr residues, corresponding to the majority of proteins previously known to participate in CLEC-2 signaling and also novel ones, including adaptors (e.g., DAPP1, Dok1/3, CASS4, Nck1/2), kinases/phosphatases (e.g., FAK1, FES, FGR, JAK2, SHIP2), and membrane proteins (e.g., G6F, JAM-A, PECAM-1, TLT-1). To elucidate the contribution of ADP and TXA2 at different points of the CLEC-2 signaling cascade, we evaluated p-Tyr levels of residues identified in the analysis and known to be essential for the catalytic activity of kinases Syk(p-Tyr525+526) and Src(p-Tyr419), and for PLCγ2 activity (p-Tyr759). We demonstrated that Syk phosphorylation at Tyr525+526 also happens in the presence of ADP and TXA2 inhibitors, which is not the case for Src-pTyr419 and PLCγ2-pTyr759. Kinetics studies for the three phosphoproteins show some differences in the phosphorylation profile. Ca2+ mobilization assays confirmed the relevance of ADP and TXA2 for full CLEC-2-mediated platelet activation. The present study provides significant insights into the intracellular events that take place following CLEC-2 activation in platelets, contributing to elucidate in detail the CLEC-2 signalosomeThis study was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [grant No. SAF2016-79662-R], co-funded by the European Regional Development Fund (ERDF); and the Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia [ED431C 2018/21; predoctoral grant Plan I2C 2014; and Centro Singular de investigación de Galicia accreditation 2016-2019, ED431G/05], co-funded by the European Regional Development Fund (ERDF). The study also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 766118. J.A.E. is supported by Deutsche Forschungsgemeinschaft [DFG grant: EB177/13-1]S

Experimental validation of computerised models of clustering of platelet glycoprotein receptors that signal via tandem SH2 domain proteins

The clustering of platelet glycoprotein receptors with cytosolic YxxL and YxxM motifs, including GPVI, CLEC-2 and PEAR1, triggers activation via phosphorylation of the conserved tyrosine residues and recruitment of the tandem SH2 (Src homology 2) domain effector proteins, Syk and PI 3-kinase. We have modelled the clustering of these receptors with monovalent, divalent and tetravalent soluble ligands and with transmembrane ligands based on the law of mass action using ordinary differential equations and agent-based modelling. The models were experimentally evaluated in platelets and transfected cell lines using monovalent and multivalent ligands, including novel nanobody-based divalent and tetravalent ligands, by fluorescence correlation spectroscopy. Ligand valency, receptor number, receptor dimerisation, receptor phosphorylation and a cytosolic tandem SH2 domain protein act in synergy to drive receptor clustering. Threshold concentrations of a CLEC-2-blocking antibody and Syk inhibitor act in synergy to block platelet aggregation. This offers a strategy for countering the effect of avidity of multivalent ligands and in limiting off-target effects

Comparison of the GPVI inhibitors losartan and honokiol

<p>Losartan and honokiol are small molecules which have been described to inhibit aggregation of platelets by collagen. Losartan has been proposed to block clustering of GPVI but not to affect binding of collagen. Honokiol has been reported to bind directly to GPVI but only at a concentration that is three orders of magnitude higher than that needed for inhibition of aggregation. The mechanism of action of both inhibitors is so far unclear. In the present study, we confirm the inhibitory effects of both agents on platelet aggregation by collagen and show that both also block the aggregation induced by the activation of CLEC-2 or the low affinity immune receptor FcγRIIa at similar concentrations. For GPVI and CLEC-2, this inhibition is associated with a reduction in protein tyrosine phosphorylation of multiple proteins including Syk. In contrast, on a collagen surface, spreading of platelets and clustering of GPVI (measured by single molecule localisation microscopy) was not altered by losartan or honokiol. Furthermore, in flow whole-blood, both inhibitors suppressed the formation of multi-layered platelet thrombi at arteriolar shear rates at concentrations that hardly affect collagen-induced platelet aggregation in platelet rich plasma. Together, these results demonstrate that losartan and honokiol have multiple effects on platelets which should be considered in the use of these compounds as anti-platelet agents.</p

Pharmacological Aspects of Vipera xantina palestinae Venom

In Israel, Vipera xantina palestinae (V.x.p.) is the most common venomous snake, accounting for several hundred cases of envenomation in humans and domestic animals every year, with a mortality rate of 0.5 to 2%. In this review we will briefly address the research developments relevant to our present understanding of the structure and function of V.x.p. venom with emphasis on venom disintegrins. Venom proteomics indicated the presence of four families of pharmacologically active compounds: (i) neurotoxins; (ii) hemorrhagins; (iii) angioneurin growth factors; and (iv) different types of integrin inhibitors. Viperistatin, a α1β1selective KTS disintegrin and VP12, a α2β1 selective C-type lectin were discovered. These snake venom proteins represent promising tools for research and development of novel collagen receptor selective drugs. These discoveries are also relevant for future improvement of antivenom therapy towards V.x.p. envenomation

Low dose Btk inhibitors selectively block platelet activation by CLEC-2

Inhibitors of the tyrosine kinase Btk have been proposed as novel antiplatelet agents. In this study we show that low concentrations of the Btk inhibitor ibrutinib block CLEC-2-mediated activation and tyrosine phosphorylation including Syk and PLCγ2 in human platelets. Activation is also blocked in patients with X-linked agammaglobulinaemia (XLA) caused by a deficiency or absence of Btk. In contrast, the response to GPVI is delayed in the presence of low concentrations of ibrutinib or in patients with XLA, and tyrosine phosphorylation of Syk is preserved. A similar set of results is seen with the second-generation inhibitor, acalabrutinib. The differential effect of Btk inhibition in CLEC-2 relative to GPVI signalling is explained by the positive feedback role involving Btk itself, as well as ADP and thromboxane A2 mediated activation of P2Y12 and TP receptors, respectively. This feedback role is not seen in mouse platelets and, consistent with this, CLEC-2-mediated activation is blocked by high but not by low concentrations of ibrutinib. Nevertheless, thrombosis was absent in 8 out of 13 mice treated with ibrutinib. These results show that Btk inhibitors selectively block activation of human platelets by CLEC-2 relative to GPVI suggesting that they can be used at ‘low dose’ in patients to target CLEC-2 in thrombo-inflammatory disease

Significant Hypo-Responsiveness to GPVI and CLEC-2 Agonists in Pre-Term and Full-Term Neonatal Platelets and following Immune Thrombocytopenia

Neonatal platelets are hypo-reactive to the tyrosine kinase-linked receptor agonist collagen. Here, we have investigated whether the hypo-responsiveness is related to altered levels of glycoprotein VI (GPVI) and integrin α2β1, or to defects in downstream signalling events by comparison to platelet activation by C-type lectin-like receptor 2 (CLEC-2). GPVI and CLEC-2 activate a Src- and Syk-dependent signalling pathway upstream of phospholipase C (PLC) γ2. Phosphorylation of a conserved YxxL sequence known as a (hemi) immunotyrosine-based-activation-motif (ITAM) in both receptors is critical for Syk activation. Platelets from human pre-term and full-term neonates display mildly reduced expression of GPVI and CLEC-2, as well as integrin αIIbβ3, accounted for at the transcriptional level. They are also hypo-responsive to the two ITAM receptors, as shown by measurement of integrin αIIbβ3 activation, P-selectin expression and Syk and PLCγ2 phosphorylation. Mouse platelets are also hypo-responsive to GPVI and CLEC-2 from late gestation to 2 weeks of age, as determined by measurement of integrin αIIbβ3 activation. In contrast, the response to G protein-coupled receptor agonists was only mildly reduced and in some cases not altered in neonatal platelets of both species. A reduction in response to GPVI and CLEC-2, but not protease-activated receptor 4 (PAR-4) peptide, was also observed in adult mouse platelets following immune thrombocytopenia, whereas receptor expression was not impaired. Our results demonstrate developmental differences in platelet responsiveness to GPVI and CLEC-2, and also following immune platelet depletion leading to reduced Syk activation. The rapid generation of platelets during development or following platelet depletion is achieved at the expense of signalling by ITAM-coupled receptors.Research by the group of F.F.-M. and J.R. is supported by grants from Instituto de Salud Carlos III and Feder (PI14/ 01956) and Fundación Séneca (19873/GERM/15). V.P.-B. holds a research fellowship from CIBERER (CB15/00055). Research by the group of S.P.W. is supported by the British Heart Foundation (RG/ PG/13/36/30275; RG/09/007)