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 α2β1 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, TxA2, 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 αIIbβ3, increasing their ligand affinity. Binding of αIIbβ3 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 αIIbβ3, 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.Key words: platelet, receptors, thrombus formation.Citation: Rivera J, Lozano ML, Navarro-Núñez L, Vicente V. Platelet receptors and signaling in the dynamics of thrombus formation. Haematologica 2009; 94:700-711. doi:10.3324/haematol.2008 This is an open-access paper. ABSTRACT Platelet receptors and signaling in the dynamics of thrombus formationJosé Rivera, María Luisa Lozano, Leyre Navarro-Núñez, and Vicente Vicente Unit of Hematology and Clinical Oncology, Centro Regional de Hemodonación, University of Murcia, Spain © F e r r a t a S t o r t i F o u n d a t i o nand metastasis, or immunological host defense. 1Internally, platelets contain a cytoskeleton, a dense tubular system, few mitochondrias, glycogen granules, dense (δ) and α storage granules and peroxisomes. The α-granules retain relevant proteins for the hemostatic function of platelets, such as von Willebrand factor (VWF), fibrinogen, P-selectin,...

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. (Blood. 2012;119(7):1747-1756) IntroductionRecently, several mutant mouse models have shown a defect in the separation of the lymphatic vasculature from the blood vasculature typically resulting in the appearance of blood-filled lymphatic vessels in the skin at embryonic day (E) 14.5 (review in Tammela and Alitalo 1 ). Mice deficient in the tyrosine kinase Syk show this phenotype during gestation and die around the time of birth. 2-4 A similar defect is found in mice deficient in the adapter protein SLP76 (Lcp2) 4 or in PLC␥2, 5 which play vital roles downstream of Syk in immunoreceptor tyrosine-based activation motif (ITAM) and integrin signaling cascades, providing circumstantial evidence that the Syk-SLP76-PLC␥2 pathway is required for normal lymphatic development.The C-type lectin-like protein type 2 (CLEC-2, encoded by the Clec1b gene) is highly expressed on platelets and at lower levels on other hematopoietic cells [6][7][8][9] and signals through a cytosolic YxxL sequence known as a hemITAM. 10,11 These receptors signal through a similar pathway used by ITAM receptors which have a dual YxxL/I sequence. HemITAM receptors activate Syk, initiating a signaling cascade partially dependent on SLP76 that leads to activation of PLC␥2. 6,12,13 The role of CLEC-2 in hemostasis and thrombosis is debatable because some lines of evidence suggest that it is required 14,15 and others show that it has no significant involvement in these processes. 16 CLEC-2 has been recognized as a receptor for the transmembrane protein podoplanin. 17,18 Podoplanin is expressed on lymphatic endothelial cells (LECs), lung type-1 alveolar cells, and kidney podocytes but not in blood endothelial cells (BECs). Podoplanin-deficient mice die shortly after birth because of an inability to inflate their lungs and, like Syk-deficient mice, show dilated, tortuous blood-filled lymphatics in mid-gestation. 19,20 A similar phenotype is seen in mice lacking megakaryocytes/...

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Background: The interaction of platelet CLEC-2 with Podoplanin is critical for development of the lymphatics.Results: CLEC-2 forms a central cluster upon engagement with Podoplanin, which clusters Podoplanin. Clustering is dependent on Syk and is critical for adhesion.Conclusion: Clustering regulates the interaction of platelets with lymphatic endothelial cells.Significance: These findings account for the similar lymphatic phenotype of CLEC-2- and Syk-deficient mice.

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What is already known about this subject • Flavonoids are largely recognized as potential inhibitors of platelet function, through nonspecific mechanisms such as antioxidant activity and/or inhibition of several enzymes and signalling proteins.• In addition, we, and few others, have shown that certain antiaggregant flavonoids may behave as specific TXA2 receptor (TP) ligands in platelets.• Whether flavonoids interact with TP isoforms in other cell types is not known, and direct evidence that flavonoid-TP interaction inhibits signalling downstream TP has not been shown. What this study adds• This study first demonstrates that certain flavonoids behave as ligands for both TP isoforms, not only in platelets, but also in human myometrium and in TP-transfected HEK 293T cells.• Differences in the effect of certain flavonoids in platelet signalling, induced by either U46619 or thrombin, suggest that abrogation of downstream TP signalling is related to their specific blockage of the TP, rather than to a nonspecific effect on tyrosine kinases or other signalling proteins. AimsFlavonoids may affect platelet function by several mechanisms, including antagonism of TxA2 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. MethodsPlatelets 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 TPaand TPb-transfected HEK 293T cells was explored using binding assays and the TP antagonist 3 H-SQ29548. ResultsApigenin, genistein, luteolin and quercetin impaired U46619-induced calcium mobilization in a concentration-dependent manner (IC50 10-30 mm). 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. ConclusionsThese 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.

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