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)

Platelet aggregation is essential for hemostasis, but can also cause myocardial infarction and stroke. A key but poorly understood step in platelet activation is increased function of the major adhesive receptor, αIIbβ3 integrin, which enables adhesion and aggregation. Phospholipases (PL), in response to agonist receptor stimulation, cleave membrane phospholipids to generate lipid second messengers. An essential role in platelet activation has been established for PLC, but not for PLD and its product phosphatidic acid. Here, we report the generation of Pld1−/− mice and show that their platelets display impaired αIIbβ3 integrin activation in response to classic agonists, and defective glycoprotein Ib-dependent aggregate formation under high shear flow conditions. This defect resulted in protection from thrombosis and ischemic brain infarction, without affecting tail bleeding times. These results indicate that PLD1 may be a critical regulator of platelet activity in the setting of ischemic cardiovascular and cerebrovascular events.

IntroductionPlatelet adhesion, activation, and aggregation are essential for primary hemostasis at sites of vascular injury but are also critically important for the development of acute thrombotic occlusion at regions of atherosclerotic plaque rupture, the major pathophysiologic mechanism underlying myocardial infarction and ischemic stroke. 1 Platelet activation is triggered by various agonists, including subendothelial collagen, ADP released from activated platelets, thrombin generated by the coagulation cascade, or the collagen receptor glycoprotein VI (GPVI)-specific agonists convulxin (CVX) and collagen-related peptide (CRP). 2 The agonists lead to platelet granule release, integrin ␣ IIb ␤ 3 activation, phosphatidylserine exposure, aggregation, and thrombus formation. 2 All those platelet responses depend on an increase of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ), 3,4 which is accomplished by inositol-1,4,5-triphosphatemediated Ca 2ϩ release from intracellular stores triggering subsequent stimulation of store-operated Ca 2ϩ entry (SOCE) across the plasma membrane. 5 Two key players in platelet SOCE have recently been identified: The 4-transmembrane-spanning poreforming calcium release-activated channel moiety Orai1, which mediates entry of extracellular Ca 2ϩ , and stromal interaction molecule 1 (STIM1), an Orai1 regulating Ca 2ϩ sensor expressed predominantly in the endoplasmic reticulum. [6][7][8] Regulators of Orai1 in other cell types include receptor for activated protein kinase C-1, 9 reactive oxygen species, 10 and lipid rafts. 11 However, regulation of Orai1 in platelets is poorly understood. Platelet activation has been shown to be regulated in vitro and in vivo by the PI3K/Akt signaling cascade. 12,13 Interference with PI3K signaling has previously been shown to compromise Ca 2ϩ influx into platelets. 14,15 Signaling molecules regulated by PI3K signaling include the serum-and glucocorticoid-inducible kinase 1 (SGK1), a kinase belonging to the AGC family of serine/threonine protein kinases. 16,17 SGK1 has originally been cloned as a glucocorticoidsensitive gene but later shown to be regulated by a variety of hormones and other triggers, including thrombin, growth factors IGF-1 and TGF-␤, oxidative stress, and ischemia. 17 SGK1 has previously been reported to regulate a wide variety of carriers and ion channels, including the epithelial Ca 2ϩ channels TRPV5 and TRPV6. 17 Most recently, SGK1 has been shown to be critically important for the Ca 2ϩ entry into mast cells after activation of the IgE receptor, 18 an effect mediated by regulation of Orai1. 19 Furthermore, SGK1 participates in the regulation of renal tubular Na ϩ reabsorption, salt appetite, and thus blood pressure. 17 A gain-of-function SGK1 gene variant, the combined presence of single nucleotide polymorphism in intron 6 (rs1743966) and in exon 8 (rs1057293), is associated with enhanced blood pressure. 20 Submitted June 9, 2011; accepted August 28, 2011. Prepublished online as Blood First Edition paper, October 26, 2011; DOI 10.1182...

Platelet activation at sites of vascular injury is crucial for hemostasis, but it may also cause myocardial infarction or stroke. Cytoskeletal reorganization is essential for platelet activation and secretion. The small GTPase Cdc42 has been implicated as an important mediator of filopodia formation and exocytosis in various cell types, but its exact function in platelets is not established. Here, we show that the megakaryocyte/platelet-specific loss of Cdc42 leads to mild thrombocytopenia and a small increase in platelet size in mice. Unexpectedly, Cdc42-deficient platelets were able to form normally shaped filopodia and spread fully on fibrinogen upon activation, whereas filopodia formation upon selective induction of GPIb signaling was reduced compared with wild-type platelets. Furthermore, Cdc42-deficient platelets showed enhanced secretion of ␣ granules, a higher adenosine diphosphate (ADP)/adenosine triphosphate (ATP) content, increased aggregation at low agonist concentrations, and enhanced aggregate formation on collagen under flow. In vivo, lack of Cdc42 resulted in faster occlusion of ferric chloride-injured arterioles. The life span of Cdc42-deficient platelets was markedly reduced, suggesting increased clearing of the cells under physiologic conditions. These data point to novel multiple functions of Cdc42 in the regulation of platelet activation, granule organization, degranulation, and a specific role in GPIb signaling. (Blood. 2010;115(16): 3364-3373) IntroductionAt sites of tissue trauma, platelets become activated and rapidly aggregate to form a plug that seals the wound and limits blood loss. On the other hand, platelet activation in pathologic situations can lead to thrombosis, causing myocardial infarction or stroke. Platelet activation by multiple signaling pathways leads to shape change, release of intracellularly stored granules, and spreading on immobilized ligands. Small GTPases of the Rho family, namely RhoA, Cdc42, and Rac1, are thought to play important roles in the cytoskeletal rearrangements occurring during platelet activation by facilitating the formation of stress fibers, filopodia and lamellipodia, respectively. 1 In platelets, signaling from G protein-coupled receptors, such as the thromboxane or thrombin receptors, as well as immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors (GPVI, CLEC-2) was shown to induce activation of Rho GTPases. 2,3 Cdc42 is a small (ϳ 23 kDa) protein that cycles between a GDP-bound inactive and a GTP-bound active state. 4 Cdc42 is an important mediator of filopodia formation in various cell types. According to the "convergent elongation model," active Cdc42 induces activation of Wiskott-Aldrich Syndrome protein (WASP). WASP subsequently activates the ARP2/3 complex, thereby increasing actin turnover and initiating the formation of parallel actin bundles. [5][6][7] Furthermore, Cdc42 can also bind to and activate IRSp53, which recruits the Ena/vasodilator-stimulated phosphoprotein (VASP) family protein Mena, thus promoting filopodi...

Summary. Heterodimeric receptors of the b1 and b3 integrin families mediate platelet adhesion and aggregation in hemostasis and thrombosis. In resting platelets, integrins are expressed in a low-affinity state but they shift to a high-affinity state and efficiently bind their ligands in response to cellular activation. This review summarizes recent advances in understanding the functional regulation and (patho-) physiological significance of individual platelet integrins with a special focus on studies in genetically modified mice. It is now recognized that b1 and b3 integrins have partially redundant roles in the adhesion process and that their activation is regulated by similar mechanisms, involving Ca 2+ -dependent and -independent signaling events and essential functions of talin-1 and kindlin-3 in the terminal activation step.

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.

Cerebral amyloid angiopathy (CAA) is a vascular dysfunction disorder characterized by deposits of amyloid-β (Aβ) in the walls of cerebral vessels. CAA and Aβ deposition in the brain parenchyma contribute to dementia and Alzheimer's disease (AD). We investigated the contribution of platelets, which accumulate at vascular Aβ deposits, to CAA. We found that synthetic monomeric Aβ40 bound through its RHDS (Arg-His-Asp-Ser) sequence to integrin αIIbβ3, which is the receptor for the extracellular matrix protein fibrinogen, and stimulated the secretion of adenosine diphosphate (ADP) and the chaperone protein clusterin from platelets. Clusterin promoted the formation of fibrillar Aβ aggregates, and ADP acted through its receptors P2Y1 and P2Y12 on platelets to enhance integrin αIIbβ3 activation, further increasing the secretion of clusterin and Aβ40 binding to platelets. Platelets from patients with Glanzmann's thrombasthenia, a bleeding disorder in which platelets have little or dysfunctional αIIbβ3, indicated that the abundance of this integrin dictated Aβ-induced clusterin release and platelet-induced Aβ aggregation. The antiplatelet agent clopidogrel, which irreversibly inhibits P2Y12, inhibited Aβ aggregation in platelet cultures; in transgenic AD model mice, this drug reduced the amount of clusterin in the circulation and the incidence of CAA. Our findings indicate that activated platelets directly contribute to CAA by promoting the formation of Aβ aggregates and that Aβ, in turn, activates platelets, creating a feed-forward loop. Thus, antiplatelet therapy may alleviate fibril formation in cerebral vessels of AD patients.

Alzheimer’s disease (AD) is characterized by neurotoxic amyloid-ß plaque formation in brain parenchyma and cerebral blood vessels known as cerebral amyloid angiopathy (CAA). Besides CAA, AD is strongly related to vascular diseases such as stroke and atherosclerosis. Cerebrovascular dysfunction occurs in AD patients leading to alterations in blood flow that might play an important role in AD pathology with neuronal loss and memory deficits. Platelets are the major players in hemostasis and thrombosis, but are also involved in neuroinflammatory diseases like AD. For many years, platelets were accepted as peripheral model to study the pathophysiology of AD because platelets display the enzymatic activities to generate amyloid-ß (Aß) peptides. In addition, platelets are considered to be a biomarker for early diagnosis of AD. Effects of Aß peptides on platelets and the impact of platelets in the progression of AD remained, however, ill-defined. The present study explored the cellular mechanisms triggered by Aß in platelets. Treatment of platelets with Aß led to platelet activation and enhanced generation of reactive oxygen species (ROS) and membrane scrambling, suggesting enhanced platelet apoptosis. More important, platelets modulate soluble Aß into fibrillar structures that were absorbed by apoptotic but not vital platelets. This together with enhanced platelet adhesion under flow ex vivo and in vivo and platelet accumulation at amyloid deposits of cerebral vessels of AD transgenic mice suggested that platelets are major contributors of CAA inducing platelet thrombus formation at vascular amyloid plaques leading to vessel occlusion critical for cerebrovascular events like stroke.