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. Addition of platelets or antibody-mediated crosslinking of podoplanin inhibited LEC migration induced by vascular endothelial growth factors (VEGF-A or VEGF-C), but did not modify basal migration or the response to basic fibroblast growth factor or epidermal growth factor. In addition, platelets and podoplanin crosslinking disrupted networks of LEC formed in co-culture with fibroblasts. Depletion of podoplanin in LEC using siRNA negated the pro-migratory effect of VEGF-A and VEGF-C. Inhibition of RhoA or Rho-kinase reduced LEC migration induced by VEGF-C, but had no further effect after crosslinking of podoplanin, suggesting that podoplanin is required for signaling downstream of VEGF-receptors but upstream of RhoA. Together, these data reveal for the first time that podoplanin is an intrinsic specific regulator of VEGF-mediated migration and network formation in LEC and identify crosslinking of podoplanin by platelets or antibodies as mechanisms to modulate this pathway.

Bimodal Expansion of the Lymphatic Vessels Is Regulated by the Sequential Expression of IL-7 and Lymphotoxin α1ß2 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. Lymphatic vessel expansion occurs in two distinct phases. The first wave of expansion is dependent on IL-7. The second phase, responsible for leukocyte exit from the glands, is regulated by lymphotoxin (LT)ßR signaling. These findings, while highlighting the tight regulation of the lymphatic response to inflammation, suggest that targeting the LTα1ß2/LTßR pathway in TLS-associated pathologies might impair a natural proresolving mechanism for lymphocyte exit from the tissues and account for the failure of therapeutic strategies that target these molecules in diseases such as rheumatoid arthritis.

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. We rule out constitutive CLEC-2 expression on resting DCs and show that CLEC-2 is upregulated in response to LPS-induced systemic inflammation in a small subset of activated DCs isolated from the mesenteric lymph nodes but not the spleen. Moreover, we demonstrate for the first time that peripheral blood B lymphocytes present exogenously derived CLEC-2 and suggest that both circulating B lymphocytes and CD11b(high) Gr-1(high) myeloid cells lose CLEC-2 following entry into secondary lymphoid organs. These results have significant implications for our understanding of CLEC-2 physiological functions.

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. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbß3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.

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. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the center of the platelet to form a single structure. Fluorescence lifetime imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.

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. Animal models have shown that the correct separation of the lymphatic and blood vasculatures during embryonic development is dependent on CLEC-2-mediated platelet activation. Additionally, podoplanin-deficient mice show abnormalities in heart, lungs, and lymphoid tissues, whereas absence of CLEC-2 affects brain development. This review summarises the current understanding of the molecular pathways regulating CLEC-2 and podoplanin function and suggests other physiological and pathological processes where this molecular interaction might exert crucial roles.

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. The sialyated membrane glycoprotein podoplanin is found on the leading edge of tumour cells and is thought to influence their migratory and invasive properties. Podoplanin elicits powerful platelet aggregation and is the endogenous ligand for the platelet C-type lectin receptor, CLEC-2, which itself regulates podoplanin signalling. Here, the bidirectional relationship between CLEC-2 and podoplanin is described and considered in the context of tumour growth and metastasis.

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. In contrast, conditional deletion of Syk in other hematopoietic lineages had no effect on viability or brain vasculature and lymphatic development. We show that platelets, but not platelet releasate, modulate the migration and intercellular adhesion of lymphatic endothelial cells through a pathway that depends on CLEC-2 and Syk. These studies found that megakaryocyte/platelet expression of CLEC-2 and Syk is required for normal brain vasculature and lymphatic development and that platelet CLEC-2 and Syk directly modulate lymphatic endothelial cell behavior in vitro.

Syk-dependent phosphorylation of CLEC-2: a novel mechanism of hem-immunoreceptor tyrosine-based activation motif signaling.

The C-type lectin-like receptor CLEC-2 signals via phosphorylation of a single cytoplasmic YXXL sequence known as a hem-immunoreceptor tyrosine-based activation motif (hemITAM). In this study, we show that phosphorylation of CLEC-2 by the snake toxin rhodocytin is abolished in the absence of the tyrosine kinase Syk but is not altered in the absence of the major platelet Src family kinases, Fyn, Lyn, and Src, or the tyrosine phosphatase CD148, which regulates the basal activity of Src family kinases. Further, phosphorylation of CLEC-2 by rhodocytin is not altered in the presence of the Src family kinase inhibitor PP2, even though PLCγ2 phosphorylation and platelet activation are abolished. A similar dependence of phosphorylation of CLEC-2 on Syk is also seen in response to stimulation by an IgG mAb to CLEC-2, although interestingly CLEC-2 phosphorylation is also reduced in the absence of Lyn. These results provide the first definitive evidence that Syk mediates phosphorylation of the CLEC-2 hemITAM receptor with Src family kinases playing a critical role further downstream through the regulation of Syk and other effector proteins, providing a new paradigm in signaling by YXXL-containing receptors.

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. Quercetin or the combined use of adenosine diphosphate and thromboxane A(2) inhibitors abrogated platelet spreading on these surfaces to a similar extent. We suggest that the inhibitory effect of quercetin on platelet kinases blocks early signaling events preventing a complete platelet spreading.

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. We studied the effect of six polymorphisms [ITGB3 1565T > C (HPA-1), GPIBA variable number tandem repeat and 524C > T (HPA-2), ITGA2 807C > T, ADRA2A 1780A > G, and TUBB1 Q43P] on platelet function in 286 healthy subjects and their potential pathogenetic role in 160 patients with hereditary mucocutaneous bleeding of unknown cause. We found no effect of any of these polymorphisms on platelet aggregation, secretion, PFA-100, and thrombin generation in platelet rich plasma. Furthermore, patients and controls showed no significant differences in the frequency of any of these polymorphisms. Thus, our study demonstrated that polymorphisms in genes affecting platelet function do not influence significantly major platelet functions and appear irrelevant in the pathogenesis of bleeding disorders.

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. Interactions between these elements are largely influenced by flow and trigger signaling events that reinforce adhesion and promote platelet activation. Thereafter, soluble agonists, ADP, thrombin, TxA(2), produced/released at the site of vascular injury act in autocrine and paracrine mode to amplify platelet activation and to recruit circulating platelets to the developing thrombus. Specific interactions of these agonists with their G-protein coupled receptors generate inside-out signaling leading to conformational activation of integrins, in particular alpha(IIb)beta(3), increasing their ligand affinity. Binding of alpha(IIb)beta(3) to its ligands, mainly fibrinogen, supports processes such as clot retraction and platelet aggregation. Stabilization of thrombi is supported by the late wave of signaling events promoted by close contact between aggregated platelets. The best known contact-dependent signaling is outside-in signaling through alphaIb beta(3), but new ones are being clarified such as those mediated by interaction of Eph receptors with ephrins, or by Sema 4D and Gas-6 binding to their receptors. Finally, newly identified mechanisms appear to control thrombus growth, including back-shifting of activated integrins and actuation of compensatory molecules such as ESAM or PECAM-1. The expanding knowledge of thrombotic disease is expected to translate into the development of new drugs to help management and prevention of thrombosis.

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. In this study, the ability of 20 naturally occurring flavonoids belonging to different structural subtypes to inhibit [3H]-SQ29548 binding to platelet-rich plasma was compared to establish the structural basis explaining their TP antagonistic activity. The results show a key contribution of C7 and C8 carbons in the A ring, gamma-pyrone structure conjugated with a double bond between C2 and C3 carbons in the C ring, and C2', C3', and C4' carbons in the B ring as the structural determinants that create the active flavonoid skeleton in TP blockade. These data might help in the design of new TP antagonists with potential antithrombotic effects and provide additional evidence for the correlation between biological properties of flavonoids and their structure.

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. Predonation venous hemoglobin was also determined in all donors using a Coulter Max-M analyzer. RESULTS: The three photometers provided acceptable reproducibility (CV below 5%), and displayed a significant correlation between the capillary blood samples and the venous hemoglobin (R2 0.5-0.8). HemoCue showed the best agreement with venous hemoglobin determination, followed by STAT-Site MHgb, and the CompoLab HB system. The copper sulphate test provided the highest rate of donors acceptance (83%) despite unacceptable hemoglobin levels, and the lowest rate for donor deferral (1%) despite acceptable hemoglobin levels. The percentage of donors correctly categorized for blood donation by the portable hemoglobinometers was 85%, 82%, and 76% for CompoLab HB system, HemoCue and STAT-Site, respectively. CONCLUSION: Our data suggest that hemoglobin determination remains a conflictive issue in donor selection in the mobile setting. Without appropriate performance control, capillary hemoglobin screening by either the copper sulphate method or by the novel portable hemoglobinometers could be inaccurate, thus potentially affecting both donor safety and the blood supply.

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. DESIGN AND METHODS: We evaluated the role of the TUBB1 Q43P polymorphism and its synergism with other polymorphisms in the risk of developing subarachnoid (SAH) and intracerebral hemorrhage (ICH). We performed the study in 109 patients with SAH, 259 patients with ICH, and 449 subjects from the general population from southern Spain. RESULTS: No relationship was found between the TUBB1 Q43P polymorphism and SAH. In contrast, this polymorphism significantly increased the risk of ICH in men (OR, 2.78; 95% CI, 1.16-6.63; p=0.021) and was associated with an earlier age of occurrence of an ICH event (p=0.011). Carriers of the TUBB1 Q43P polymorphism displayed lower platelet reactivity towards collagen. A potent synergistic effect was observed in ICH patients carrying the TUBB1 Q43P polymorphism combined with either FVII -323 Del/Ins of a decanucleotide (OR 20.76; 95% CI, 3.57-120.71; p<0.001) or FXIII V34L (OR 7.19; 95% CI, 1.99-25.95; p=0.003). INTERPRETATION AND CONCLUSIONS: This is the first evidence linking the TUBB1 Q43P platelet polymorphism with hemorrhagic stroke in humans. The TUBB1 Q43P polymorphism, by causing a lower reactivity in platelets carrying the variant form of b1-tubulin, protects against thrombotic disorders but increases the risk of ICH in men.

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. METHODS: Platelets were treated with flavonoids, or other selected inhibitors, and then stimulated with U46619. Similar assays were performed in aspirinized platelets activated with thrombin. Effects on calcium release were analysed by fluorometry and changes in whole protein tyrosine phosphorylation and activation of ERK 1/2 by Western blot analysis. The binding of flavonoids to TP in platelets, human myometrium and TPalpha- and TPbeta-transfected HEK 293T cells was explored using binding assays and the TP antagonist (3)H-SQ29548. RESULTS: Apigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC(50) 10-30 microm). These flavonoids caused a significant impairment of U46619-induced platelet tyrosine phosphorylation and of ERK 1/2 activation. By contrast, in aspirin-treated platelets all these flavonoids, except quercetin, displayed minor effects on thrombin-induced calcium mobilization, ERK 1/2 and total tyrosine phosphorylation. Finally, apigenin, genistein and luteolin inhibited by >50% (3)H-SQ29548 binding to different cell types. CONCLUSIONS: These data further suggest that flavonoids may inhibit platelet function by binding to TP and by subsequent abrogation of downstream signalling. Binding of these compounds to TP occurs in human myometrium and in TP-transfected HEK 293T cells and suggests that antagonism of TP might mediate the effects of flavonoids in different tissues.

Latent and polymeric antithrombin: clearance and potential thrombotic risk.

Antithrombin, the most potent anticoagulant in vivo, displays a significant conformational flexibility. The native five-stranded anticoagulant form transforms under different conditions or mutations to inactive six-stranded conformations: latent or polymer. However, the function, potential deleterious effects, and clearance of these forms are not completely known. The dimerization of latent antithrombin with a native molecule has been suggested to have thrombotic potential. We have assessed the potential thrombogenicity of high amounts of latent and polymeric antithrombin by experiments performed in mice and human plasma. Moreover, we have analyzed the clearance of (125)I-labeled native, latent, polymer, and thrombin-complexed antithrombins in rat, as well as the clearance of latent antithrombin from plasma of patients treated with commercial concentrates. Our results show that high plasma levels of latent or polymeric antithrombin do not interfere with the anticoagulant function of native antithrombin. Moreover, we confirm that all monomeric forms of antithrombin have similar turnover. Finally, we show that polymers have the longest half-life of all conformers, being in circulation for prolonged periods of time. In conclusion, our data support that latent and polymeric antithrombin would not likely have a thrombotic effect, thus dispelling doubts about the potential harmful effect of latent antithrombin present in commercial concentrates for therapeutic use. Moreover, the suggested antiangiogenic role of latent antithrombin, together with its stability in plasma and its negligible thrombogenicity raises the possibility of its use as a new antiangiogenic drug.