Circulating lymphocytes continuously enter lymph nodes (LNs) for immune surveillance through specialised blood vessels named high endothelial venules (HEVs)1–5, a process that increases dramatically during immune responses. How HEVs permit lymphocyte transmigration while maintaining vascular integrity is unknown. Here, we report a role for the transmembrane O-glycoprotein podoplanin (PDPN, also known as gp38 and T1α)6–8 in maintaining HEV barrier function. Mice with postnatal deletion of PDPN lost HEV integrity and exhibited spontaneous bleeding in mucosal LNs, and bleeding in the draining peripheral LN after immunisation. Blocking lymphocyte homing rescued bleeding, indicating that PDPN is required to protect the barrier function of HEVs during lymphocyte trafficking. Further analyses demonstrated that PDPN expressed on fibroblastic reticular cells (FRCs)7, which surround HEVs, functions as an activating ligand for platelet C-type lectin-like receptor 2 (CLEC-2)9,10. Mice lacking FRC PDPN or platelet CLEC-2 exhibited significantly reduced levels of VE-cadherin (VE-cad), which is essential for overall vascular integrity11,12, on HEVs. Infusion of wild-type (WT) platelets restored HEV integrity in CLEC-2-deficient mice. Activation of CLEC-2 induced release of sphingosine-1-phosphate (S1P)13,14 from platelets, which promoted expression of VE-cad on HEVs ex vivo. Furthermore, draining peripheral LNs of immunised mice lacking S1P had impaired HEV integrity similar to PDPN- and CLEC-2-deficient mice. These data demonstrate that local S1P release after PDPN-CLEC-2-mediated platelet activation is critical for HEV integrity during immune responses.

Damage to the integrity of the vessel wall leads to exposure of the subendothelial extracellular matrix (ECM), triggering platelet activation and aggregation. This process is essential for primary hemosta-sis but it may also lead to arterial thrombosis. Although the mechanisms underlying platelet activation on the ECM are well explored, it is less clear which receptors mediate cellular activation in a growing thrombus. Here we studied the role of the recently identified C-type lectin-like receptor 2 (CLEC-2) in this process. We show that anti-CLEC-2 antibody treatment of mice leads to complete and highly specific loss of CLEC-2 in circulating plate-lets for several days. CLEC-2-deficient platelets displayed normal adhesion under flow, but subsequent aggregate formation was severely defective in vitro and in vivo. As a consequence, CLEC-2 deficiency was associated with increased bleeding times and profound protection from occlusive arterial thrombus formation. These results reveal an essential function of CLEC-2 in hemostasis and thrombosis. (Blood. 2009;114:3464-3472)

Vascular injury initiates rapid platelet activation that is critical for hemostasis, but it also may cause thrombotic diseases, such as myocardial infarction or ischemic stroke. Reorganizations of the platelet cytoskeleton are crucial for platelet shape change and secretion and are thought to involve activation of the small GTPase RhoA. In this study, we analyzed the in vitro and in vivo consequences of megakaryocyte-and platelet-specific RhoA gene deletion in mice. We found a pronounced macrothrombocytopenia in RhoA-deficient mice, with platelet counts of approximately half that of wild-type controls. The mutant cells displayed an altered shape but only a moderately reduced life span. Shape change of RhoAdeficient platelets in response to G 13 -coupled agonists was abolished, and it was impaired in response to G q stimulation. Similarly, RhoA was required for efficient secretion of ␣ and dense granules downstream of G 13 and G q . Furthermore, RhoA was essential for integrinmediated clot retraction but not for actomyosin rearrangements and spreading of activated platelets on fibrinogen. In vivo, RhoA deficiency resulted in markedly prolonged tail bleeding times but also significant protection in different models of arterial thrombosis and in a model of ischemic stroke. Together, these results establish RhoA as an important regulator of platelet function in thrombosis and hemostasis. (Blood. 2012;119(4): 1054-1063) IntroductionPlatelet activation on the injured vessel wall is induced by multiple signaling pathways and leads to extensive cytoskeletal rearrangements that are crucial for conversion from discoid to spheric shape, granule secretion, spreading, and clot retraction. Many of these processes are thought to involve proteins of the Rho family of small GTPases, such as RhoA, Rac1, and Cdc42. 1 Like most Rho GTPases, RhoA (ϳ 23 kDa) is active when bound to GTP, whereas the protein is inactive in the GDP-bound state. The active state enables RhoA to bind effector molecules such as Rho-associated, coiled-coil-containing protein kinase (ROCK) and mammalian diaphanous homologue (mDia). RhoA plays a central role in the organization of the actin cytoskeleton in various cell types, mainly through its ability to form stress fibers and to regulate actomyosin contractility. 2 In addition, RhoA has been implicated in numerous further cellular processes, such as cytokinesis, regulation of microtubule dynamics, and the formation of focal adhesions. 3,4 Platelet activation by soluble agonists, such as adenosine diphosphate (ADP), thromboxane A 2 (TxA 2 ), or thrombin, induces signaling through receptors coupled to heterotrimeric G proteins (G q , G i , and G 13 ). 5 Studies on mice deficient in the G protein subunits G␣ 13 6 and G␣ q , 7-9 as well as inhibitor studies using human platelets 10-14 demonstrated a critical involvement of RhoA in the induction of platelet shape change downstream of G␣ 13 after stimulation with low doses of the TxA 2 analog U46619 or thrombin. In detail, RhoA-mediated activation of ROCK and sub...

Another receptor that mediates strong platelet activation is CLEC-2, a C-type lectin-like type II transmembrane receptor, that was identified as the receptor for the platelet activating snake venom, rhodocytin.13 Interestingly, CLEC-2 is a so-called hemITAM receptor containing only a single © 2013 American Heart Association, Inc. Objective-Platelet inhibition is a major strategy to prevent acute ischemic cardiovascular and cerebrovascular events, which may, however, be associated with an increased bleeding risk. The (hem)immunoreceptor tyrosine activation motifbearing platelet receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2), might be promising antithrombotic targets because they can be depleted from circulating platelets by antibody treatment, leading to sustained antithrombotic protection, but only moderately increased bleeding times in mice. Approach and Results-We investigated whether both (hem)immunoreceptor tyrosine activation motif-bearing receptors can be targeted simultaneously and what the in vivo consequences of such a combined therapeutic GPVI/CLEC-2 deficiency are. We demonstrate that isolated targeting of either GPVI or CLEC-2 in vivo does not affect expression or function of the respective other receptor. Moreover, simultaneous treatment with both antibodies resulted in the sustained loss of both GPVI and CLEC-2, while leaving other activation pathways intact. However, GPVI/CLEC-2-depleted mice displayed a dramatic hemostatic defect and profound impairment of arterial thrombus formation. Furthermore, a strongly diminished hemostatic response could also be reproduced in mice genetically lacking GPVI and CLEC-2. Conclusions-These

Platelet activation at sites of vascular injury is triggered through different signaling pathways leading to activation of phospholipase (PL) Cbeta or PLCgamma2. Active PLCs trigger Ca(2+) mobilization and entry, which is a prerequisite for adhesion, secretion, and thrombus formation. PLCbeta isoenzymes are activated downstream of G protein-coupled receptors (GPCRs), whereas PLCgamma2 is activated downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors, such as the major platelet collagen receptor glycoprotein (GP) VI or CLEC-2. The mechanisms underlying PLC regulation are not fully understood. An involvement of small GTPases of the Rho family (Rho, Rac, Cdc42) in PLC activation has been proposed but this has not been investigated in platelets. We here show that murine platelets lacking Rac1 display severely impaired GPVI- or CLEC-2-dependent activation and aggregation. This defect was associated with impaired production of inositol 1,4,5-trisphosphate (IP(3)) and intracellular calcium mobilization suggesting inappropriate activation of PLCgamma2 despite normal tyrosine phosphorylation of the enzyme. Rac1 ( -/- ) platelets displayed defective thrombus formation on collagen under flow conditions which could be fully restored by co-infusion of ADP and the TxA(2) analog U46619, indicating that impaired GPVI-, but not G-protein signaling, was responsible for the observed defect. In line with this, Rac1 ( -/- ) mice were protected in two collagen-dependent arterial thrombosis models. Together, these results demonstrate that Rac1 is essential for ITAM-dependent PLCgamma2 activation in platelets and that this is critical for thrombus formation in vivo.