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

Abstract

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/...

Platelets mediate lymphovenous hemostasis to maintain blood-lymphatic separation throughout life

“…Some groups have reported that platelet activation by CLEC2 signaling may regulate LEC growth in vitro through secretion of platelet granule contents (6,10). However, we have not observed such effects on LEC growth in vitro or detected major defects in the pattern or rate of lymphatic vascular development in vivo (8,11), and how such an angiogenic mechanism would explain an ongoing requirement in mature, presumably nonangiogenic, animals is not clear.…”

“…Mice deficient in PDPN, CLEC2, or the CLEC2 signaling proteins SYK and SLP-76 exhibit blood-filled lymphatics during fetal life and die shortly after birth due to defective lymphatic function (3)(4)(5)(6). Tissue-specific deletion experiments reveal that CLEC2 and its intracellular signaling effectors are required in platelets, while PDPN is required in endothelial cells to prevent this phenotype (4)(5)(6)(7).…”

“…Mice deficient in PDPN, CLEC2, or the CLEC2 signaling proteins SYK and SLP-76 exhibit blood-filled lymphatics during fetal life and die shortly after birth due to defective lymphatic function (3)(4)(5)(6). Tissue-specific deletion experiments reveal that CLEC2 and its intracellular signaling effectors are required in platelets, while PDPN is required in endothelial cells to prevent this phenotype (4)(5)(6)(7). In addition, studies of mature mice reconstituted with hematopoietic cells deficient in Clec2, Syk, or SLP-76, as well as mature mice in which PDPN expression is ablated through conditional deletion of the O-glycan synthase enzyme required for its expression, revealed that this pathway is also required in mature animals to prevent blood-filled lymphatics and death due to lymphatic dysfunction (3,4,8,9).…”

Mutation of Threonine 34 in Mouse Podoplanin-Fc Reduces CLEC-2 Binding and Toxicity in Vivo While Retaining Anti-lymphangiogenic Activity

“…So it appears that CLEC-2-dependent functions of Pdpn are involved wherever there is a connection between blood and lymphatic vessels. It was reported that platelets might inhibit LEC function (39,40). Whether this is a direct effect via Pdpn-mediated changes in LECs, or an indirect effect due to a soluble factor (i.e.…”

Fucoidan Is a Novel Platelet Agonist for the C-type Lectin-like Receptor 2 (CLEC-2)

“…Mice-Platelet specific CLEC-2 knock-out mice were produced as described previously (50). Briefly, the CLEC-2 gene (CLEC1b) was flanked with LoxP sites, and this mouse was cross-bred with a PF4-Cre expressing mouse to drive excision of the gene in only the megakaryocyte/platelet lineage.…”