Modulation of VEGF-induced migration and network formation by lymphatic endothelial cells: Roles of platelets and podoplanin.

Lymphatic endothelial cells (LEC) express the transmembrane receptor podoplanin whose only known endogenous ligand CLEC-2 is found on platelets. Both podoplanin and CLEC-2 are required for normal lymphangiogenesis as mice lacking either protein develop a blood-lymphatic mixing phenotype. We investigated the roles of podoplanin and its interaction with platelets in migration and tube formation by LEC.

Bimodal Expansion of the Lymphatic Vessels Is Regulated by the Sequential Expression of IL-7 and Lymphotoxin α1β2 in Newly Formed Tertiary Lymphoid Structures.

Lymphangiogenesis associated with tertiary lymphoid structure (TLS) has been reported in numerous studies. However, the kinetics and dynamic changes occurring to the lymphatic vascular network during TLS development have not been studied. Using a viral-induced, resolving model of TLS formation in the salivary glands of adult mice we demonstrate that the expansion of the lymphatic vascular network is tightly regulated.

The expression of mouse CLEC-2 on leucocyte subsets varies according to their anatomical location and inflammatory state.

Expression of mouse C-type lectin-like receptor 2 (CLEC-2) has been reported on circulating CD11b(high) Gr-1(high) myeloid cells and dendritic cells (DCs) under basal conditions, as well as on a variety of leucocyte subsets following inflammatory stimuli or in vitro cell culture. However, previous studies assessing CLEC-2 expression failed to use CLEC-2-deficient mice as negative controls and instead relied heavily on single antibody clones. Here, we generated CLEC-2-deficient adult mice using two independent approaches and employed two anti-mouse CLEC-2 antibody clones to investigate surface expression on hematopoietic cells from peripheral blood and secondary lymphoid organs.

Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling.

Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow.

Syk and Src family kinases regulate C-type lectin receptor 2 (CLEC-2)-mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells.

The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin.

CLEC-2-dependent activation of mouse platelets is weakly inhibited by cAMP but not by cGMP.

Background: The activation of platelet CLEC-2 by podoplanin on lymphatic endothelial cells (LECs) has a critical role in prevention of mixing of lymphatic and blood vasculatures during embryonic development. Paradoxically, LECs release cAMP and cGMP-elevating agents, prostacyclin (PGI2 ) and nitric oxide (NO), respectively, which are powerful inhibitors of platelet activation. This raises the question of how podoplanin is able to activate CLEC-2 in the presence of the inhibitory cyclic nucleotides.

The physiological and pathophysiological roles of platelet CLEC-2.

CLEC-2 is a C-type lectin receptor which is highly expressed on platelets but also found at low levels on different immune cells. CLEC-2 elicits powerful platelet activation upon engagement by its endogenous ligand, the mucin-type glycoprotein podoplanin. Podoplanin is expressed in a variety of tissues, including lymphatic endothelial cells, kidney podocytes, type I lung epithelial cells, lymph node stromal cells and the choroid plexus epithelium.

Protein kinase Cε and protein kinase Cθ double-deficient mice have a bleeding diathesis.

Background: In comparison to the classical isoforms of protein kinase C (PKC), the novel isoforms are thought to play minor or inhibitory roles in the regulation of platelet activation and thrombosis.

Objectives: To measure the levels of PKCθ and PKCε and to investigate the phenotype of mice deficient in both novel PKC isoforms.

Methods: Tail bleeding and platelet activation assays were monitored in mice and platelets from mice deficient in both PKCθ and PKCε.

Platelet CLEC-2 and podoplanin in cancer metastasis.

It has long been recognised that the function of platelets in health and disease span far beyond their roles in haemostasis and thrombosis. The observation that tumour cells induce platelet aggregation was followed by extensive experimental evidence linking platelets to cancer progression. Aggregated platelets coat tumour cells during their transit through the bloodstream and mediate adherence to vascular endothelium, protection from shear stresses, evasion from immune molecules, and release of an array of bioactive molecules that facilitate tumour cell extravasation and growth at metastatic sites.

CLEC-2 and Syk in the megakaryocytic/platelet lineage are essential for development.

The C-type lectin receptor CLEC-2 signals through a pathway that is critically dependent on the tyrosine kinase Syk. We show that homozygous loss of either protein results in defects in brain vascular and lymphatic development, lung inflation, and perinatal lethality. Furthermore, we find that conditional deletion of Syk in the hematopoietic lineage, or conditional deletion of CLEC-2 or Syk in the megakaryocyte/platelet lineage, also causes defects in brain vascular and lymphatic development, although the mice are viable.

Rare homozygous status of P43 β1-tubulin polymorphism causes alterations in platelet ultrastructure.

β1-tubulin is the main constituent of the platelet marginal band and studies with deficient mice showed that it maintains discoid shape and it is required for normal platelet formation. TUBB1 Q43P polymorphism is associated with decreased β1-tubulin expression, diminished platelet reactivity, and partial loss of discoid shape in heterozygous carriers. However, to date no studies have been carried out on homozygous PP individuals.

Differential effects of quercetin, apigenin and genistein on signalling pathways of protease-activated receptors PAR(1) and PAR(4) in platelets.

Background And Purpose: The modulation by flavonoids of platelet responses induced by thrombin has been little investigated, and the antiplatelet activity, as well as possible inhibitory mechanisms of these compounds on thrombin signalling, has not yet been elucidated. We explored whether flavonoids affect platelet signalling pathways triggered by thrombin and by the selective activation of its protease-activated receptors (PARs) 1 and 4, and analysed the antagonism of these polyphenols at thrombin receptors.

Experimental Approach: We investigated the effect of a range of polyphenolic compounds on platelet aggregation, 5-HT secretion, intracellular calcium mobilization, protein kinase activity and tyrosine phosphorylation, triggered by thrombin and PAR agonist peptides (PAR-APs).

Effect of quercetin on platelet spreading on collagen and fibrinogen and on multiple platelet kinases.

alpha(2)beta(1) and alpha(IIb)beta(3) integrins, that support platelet adhesion to collagen and fibrinogen, respectively, share common signaling molecules. The effect of quercetin on platelet static adhesion to collagen and fibrinogen was assessed and correlated with its kinase inhibitory activity. Quercetin strongly abrogated PI3K and Src kinases, mildly inhibited Akt1/2, and slightly affected PKC, p38 and ERK1/2.

Genotype-phenotype relationship for six common polymorphisms in genes affecting platelet function from 286 healthy subjects and 160 patients with mucocutaneous bleeding of unknown cause.

Polymorphisms affecting platelet receptors and intracellular proteins have been extensively studied in relation to their potential influence in thrombosis and haemorrhages. However, few reports have addressed their impact on platelet function, with contradictory results. Limitations of these studies include, among others, small number of patients, the platelet functional parameters analyzed and their known variability in the healthy population.

Platelet receptors and signaling in the dynamics of thrombus formation.

Hemostasis and pathological thrombus formation are dynamic processes that require a co-ordinated series of events involving platelet membrane receptors, bidirectional intracellular signals, and release of platelet proteins and inflammatory substances. This review aims to summarize current knowledge in the key steps in the dynamics of thrombus formation, with special emphasis on the crucial participation of platelet receptors and signaling in this process. Initial tethering and firm adhesion of platelets to the exposed subendothelium is mediated by glycoprotein (GP) Ib/IX/V complex and collagen receptors, GP VI and alpha(2)beta(1) integrin, in the platelet surface, and by VWF and fibrillar collagen in the vascular site.

Thromboxane A2 receptor antagonism by flavonoids: structure-activity relationships.

Thromboxane A2 (TxA2) is a strong platelet agonist involved in the pathogenesis of thrombotic diseases that elicits platelet aggregation and vasoconstriction through the activation of its specific membrane receptor (TP). Previous studies have demonstrated that certain flavonoids, naturally occurring phytochemicals, inhibit platelet function through several mechanisms, including antagonism of TP in these cells. However, the steric and inductive or mesomeric requirements underlying this effect are not fully understood.

Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway.

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548.

Evaluation of four rapid methods for hemoglobin screening of whole blood donors in mobile collection settings.

Introduction: Predonation hemoglobin measurement is a problematic requirement in mobile donation settings, where accurate determination of venous hemoglobin by hematology analyzers is not available.

Objective: We have evaluated hemoglobin screening in prospective donors by the semiquantitative copper sulphate test and by capillary blood samples analyzed by three portable photometers, HemoCue, STAT-Site MHgb, and the CompoLab HB system.

Methods: Capillary blood samples were obtained from 380 donors and tested by the copper sulphate test and by at least one of the named portable photometers.

The association of the beta1-tubulin Q43P polymorphism with intracerebral hemorrhage in men.

Background And Objectives: Platelets play a fundamental role in hemostasis and alterations of their function can be determinant in the onset of stroke. A polymorphism in beta1-tubulin (TUBB1 Q43P), a protein specifically expressed in the megakaryocytic line, has been described as a protective factor in cardiovascular disease. The potential effect of this variant in the pathogenesis of hemorrhagic stroke has not yet been investigated.

Flavonoids inhibit the platelet TxA(2) signalling pathway and antagonize TxA(2) receptors (TP) in platelets and smooth muscle cells.

Aims: Flavonoids may affect platelet function by several mechanisms, including antagonism of TxA(2) receptors (TP). These TP are present in many tissues and modulate different signalling cascades. We explored whether flavonoids affect platelet TP signalling, and if they bind to TP expressed in other cell types.