A key role for platelet GPVI in neutrophil recruitment, migration, and NETosis in the early stages of acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are associated with high morbidity and mortality. Excessive neutrophil infiltration into the pulmonary airspace is the main cause for the acute inflammation and lung injury. Platelets have been implicated in the pathogenesis of ALI/ARDS, but the underlying mechanisms are not fully understood. Here, we show that the immunoreceptor tyrosine-based activation motif-coupled immunoglobulin-like platelet receptor, glycoprotein VI (GPVI), plays a key role in the early phase of pulmonary thrombo-inflammation in a model of lipopolysaccharide (LPS)-induced ALI in mice. In wild-type (WT) control mice, intranasal LPS application triggered severe pulmonary and blood neutrophilia, hypothermia, and increased blood lactate levels. In contrast, GPVI-deficient mice as well as anti-GPVI-treated WT mice were markedly protected from pulmonary and systemic compromises and showed no increased pulmonary bleeding. High-resolution multicolor microscopy of lung sections and intravital confocal microcopy of the ventilated lung revealed that anti-GPVI treatment resulted in less stable platelet interactions with neutrophils and overall reduced platelet-neutrophil complex (PNC) formation. Anti-GPVI treatment also reduced neutrophil crawling and adhesion on endothelial cells, resulting in reduced neutrophil transmigration and alveolar infiltrates. Remarkably, neutrophil activation was also diminished in anti-GPVI-treated animals, associated with strongly reduced formation of PNC clusters and neutrophil extracellular traps (NETs) compared with that in control mice. These results establish GPVI as a key mediator of neutrophil recruitment, PNC formation, and NET formation (ie, NETosis) in experimental ALI. Thus, GPVI inhibition might be a promising strategy to reduce the acute pulmonary inflammation that causes ALI/ARDS.

Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to thrombin and accelerates thrombus formation in vivo.

Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

Immunoglobulin G-dependent inhibition of inflammatory bone remodeling requires pattern recognition receptor Dectin-1.

Immunoglobulin G (IgG) antibodies are major drivers of inflammation during infectious and autoimmune diseases. In pooled serum IgG (IVIg), however, antibodies have a potent immunomodulatory and anti-inflammatory activity, but how this is mediated is unclear. We studied IgG-dependent initiation of resolution of inflammation in cytokine- and autoantibody-driven models of rheumatoid arthritis and found IVIg sialylation inhibited joint inflammation, whereas inhibition of osteoclastogenesis was sialic acid independent. Instead, IVIg-dependent inhibition of osteoclastogenesis was abrogated in mice lacking receptors Dectin-1 or FcγRIIb. Atomistic molecular dynamics simulations and super-resolution microscopy revealed that Dectin-1 promoted FcγRIIb membrane conformations that allowed productive IgG binding and enhanced interactions with mouse and human IgG subclasses. IVIg reprogrammed monocytes via FcγRIIb-dependent signaling that required Dectin-1. Our data identify a pathogen-independent function of Dectin-1 as a co-inhibitory checkpoint for IgG-dependent inhibition of mouse and human osteoclastogenesis. These findings may have implications for therapeutic targeting of autoantibody and cytokine-driven inflammation.

G6b-B regulates an essential step in megakaryocyte maturation.

G6b-B is a megakaryocyte lineage-specific immunoreceptor tyrosine-based inhibition motif-containing receptor, essential for platelet homeostasis. Mice with a genomic deletion of the entire Mpig6b locus develop severe macrothrombocytopenia and myelofibrosis, which is reflected in humans with null mutations in MPIG6B. The current model proposes that megakaryocytes lacking G6b-B develop normally, whereas proplatelet release is hampered, but the underlying molecular mechanism remains unclear. We report on a spontaneous recessive single nucleotide mutation in C57BL/6 mice, localized within the intronic region of the Mpig6b locus that abolishes G6b-B expression and reproduces macrothrombocytopenia, myelofibrosis, and osteosclerosis. As the mutation is based on a single-nucleotide exchange, Mpig6bmut mice represent an ideal model to study the role of G6b-B. Megakaryocytes from these mice were smaller, displayed a less-developed demarcation membrane system, and had a reduced expression of receptors. RNA sequencing revealed a striking global reduction in the level of megakaryocyte-specific transcripts, in conjunction with decreased protein levels of the transcription factor GATA-1 and impaired thrombopoietin signaling. The reduced number of mature MKs in the bone marrow was corroborated on a newly developed Mpig6b-null mouse strain. Our findings highlight an unexpected essential role of G6b-B in the early differentiation within the megakaryocytic lineage.

Heterotrimeric G Protein Subunit Gαq Is a Master Switch for Gßγ-Mediated Calcium Mobilization by Gi-Coupled GPCRs.

Mechanisms that control mobilization of cytosolic calcium [Ca2+]i are key for regulation of numerous eukaryotic cell functions. One such paradigmatic mechanism involves activation of phospholipase Cß (PLCß) enzymes by G protein ßγ subunits from activated Gαi-Gßγ heterotrimers. Here, we report identification of a master switch to enable this control for PLCß enzymes in living cells. We find that the Gαi-Gßγ-PLCß-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gßγ can bind to PLCß but does not elicit Ca2+ signals. Removal of an auto-inhibitory linker that occludes the active site of the enzyme is required and sufficient to empower "stand-alone control" of PLCß by Gßγ. This dependence of Gi-Gßγ-Ca2+ on Gαq places an entire signaling branch of G-protein-coupled receptors (GPCRs) under hierarchical control of Gq and changes our understanding of how Gi-GPCRs trigger [Ca2+]i via PLCß enzymes.

BIN2 orchestrates platelet calcium signaling in thrombosis and thrombo-inflammation.

Store-operated Ca2+ entry (SOCE) is the major route of Ca2+ influx in platelets. The Ca2+ sensor stromal interaction molecule 1 (STIM1) triggers SOCE by forming punctate structures with the Ca2+ channel Orai1 and the inositol trisphosphate receptor (IP3R), thereby linking the endo-/sarcoplasmic reticulum to the plasma membrane. Here, we identified the BAR domain superfamily member bridging integrator 2 (BIN2) as an interaction partner of STIM1 and IP3R in platelets. Deletion of platelet BIN2 (Bin2fl/fl,Pf4-Cre mice) resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. This severely defective Ca2+ signaling translated into impaired thrombus formation under flow and a protection of Bin2fl/fl,Pf4-Cre mice in models of arterial thrombosis and stroke. Our results establish BIN2 as a central regulator of platelet activation in thrombosis and thrombo-inflammatory disease settings.

CD84 Links T Cell and Platelet Activity in Cerebral Thrombo-Inflammation in Acute Stroke.

RATIONALE: Ischemic stroke is a leading cause of morbidity and mortality worldwide. Recanalization of the occluded vessel is essential but not sufficient to guarantee brain salvage. Experimental and clinical data suggest that infarcts often develop further due to a thromboinflammatory process critically involving platelets and T cells, but the underlying mechanisms are unknown. OBJECTIVE: We aimed to determine the role of CD (cluster of differentiation)-84 in acute ischemic stroke after recanalization and to dissect the underlying molecular thromboinflammatory mechanisms. METHODS AND RESULTS: Here, we show that mice lacking CD84-a homophilic immunoreceptor of the SLAM (signaling lymphocyte activation molecule) family-on either platelets or T cells displayed reduced cerebral CD4+ T-cell infiltration and thrombotic activity following experimental stroke resulting in reduced neurological damage. In vitro, platelet-derived soluble CD84 enhanced motility of wild-type but not of Cd84-/- CD4+ T cells suggesting homophilic CD84 interactions to drive this process. Clinically, human arterial blood directly sampled from the ischemic cerebral circulation indicated local shedding of platelet CD84. Moreover, high platelet CD84 expression levels were associated with poor outcome in patients with stroke. CONCLUSIONS: These results establish CD84 as a critical pathogenic effector and thus a potential pharmacological target in ischemic stroke.

Platelets in Thrombo-Inflammation: Concepts, Mechanisms, and Therapeutic Strategies for Ischemic Stroke.

Platelets are anucleate cells known for their essential function in hemostasis and formation of thrombi under pathologic conditions. In recent years, strong evidence emerged demonstrating the critical involvement of platelets in inflammatory processes including acute ischemic stroke (AIS), which is one of the leading causes of death and disability worldwide. Recanalization of the occluded brain artery to reconstitute cerebral blood flow is the primary goal in the treatment of stroke patients. However, despite successful reperfusion many patients show progression of infarct sizes, a phenomenon referred to as ischemia/reperfusion injury (I/RI). Cerebral I/RI involves both thrombotic as well as inflammatory pathways acting in concert to cause tissue damage, defining AIS as a prototypic thrombo-inflammatory disease. Currently used antiplatelet drugs applied to AIS patients eventually increase the risk of partially life-threatening hemorrhages, making more targeted pharmacological intervention necessary. Experimental evidence indicates that inhibition of platelet surface receptors that regulate initial platelet adhesion and activation might be suitable targets in thrombo-inflammatory settings, while inhibitors of platelet aggregation are not. In this review, we will summarize the recent developments in elucidating the role of the main platelet receptors in AIS and discuss their potential as pharmaceutical targets. Furthermore, we will also briefly discuss the important platelet-triggered intrinsic coagulation pathway with the pro-inflammatory kallikrein-kinin system in the context of ischemic stroke.

Critical redundant functions of the adapters Grb2 and Gads in platelet (hem)ITAM signaling in mice.

Platelets are essential for normal hemostasis; however, pathological conditions can also trigger unwanted platelet activation precipitating thrombosis and ischemic damage of vital organs such as the heart or brain. Glycoprotein (GP)VI- and C-type lectin-like receptor 2 (CLEC-2)-mediated (hem)immunoreceptor tyrosine-based activation motif (ITAM) signaling represents a major pathway for platelet activation. The two members of the Growth-factor receptor-bound protein 2 (Grb2) family of adapter proteins expressed in platelets - Grb2 and Grb2-related adapter protein downstream of Shc (Gads) - are part of the hem(ITAM) signaling cascade by forming an adapter protein complex with linker for activation of T cells (LAT). To date, a possible functional redundancy between these two adapters in platelet activation has not been investigated. We here generated megakaryocyte- and platelet-specific Grb2/Gads double knockout (DKO) mice and analyzed their platelet function in vitro and in vivo. The DKO platelets exhibited virtually abolished (hem)ITAM signaling whereas only partial defects were seen in Grb2 or Gads single-deficient platelets. This was based on impaired phosphorylation of key molecules in the (hem)ITAM signaling cascade and translated into impaired hemostasis and partially defective arterial thrombosis, thereby exceeding the defects in either Grb2 KO or Gads KO mice. Despite this severe (hem)ITAM signaling defect, CLEC-2 dependent regulation of blood-lymphatic vessel separation was not affected in the DKO animals. These results provide direct evidence for critically redundant roles of Grb2 and Gads for platelet function in hemostasis and thrombosis, but not development.

Heparan sulfates are critical regulators of the inhibitory megakaryocyte-platelet receptor G6b-B.

The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.

The Gp1ba-Cre transgenic mouse: a new model to delineate platelet and leukocyte functions.

Conditional knockout (KO) mouse models are invaluable for elucidating the physiological roles of platelets. The Platelet factor 4-Cre recombinase (Pf4-Cre) transgenic mouse is the current model of choice for generating megakaryocyte/platelet-specific KO mice. Platelets and leukocytes work closely together in a wide range of disease settings, yet the specific contribution of platelets to these processes remains unclear. This is partially a result of the Pf4-Cre transgene being expressed in a variety of leukocyte populations. To overcome this issue, we developed a Gp1ba-Cre transgenic mouse strain in which Cre expression is driven by the endogenous Gp1ba locus. By crossing Gp1ba-Cre and Pf4-Cre mice to the mT/mG dual-fluorescence reporter mouse and performing a head-to-head comparison, we demonstrate more stringent megakaryocyte lineage-specific expression of the Gp1ba-Cre transgene. Broader tissue expression was observed with the Pf4-Cre transgene, leading to recombination in many hematopoietic lineages, including monocytes, macrophages, granulocytes, and dendritic and B and T cells. Direct comparison of phenotypes of Csk, Shp1, or CD148 conditional KO mice generated using either the Gp1ba-Cre or Pf4-Cre strains revealed similar platelet phenotypes. However, additional inflammatory and immunological anomalies were observed in Pf4-Cre-generated KO mice as a result of nonspecific deletion in other hematopoietic lineages. By excluding leukocyte contributions to phenotypes, the Gp1ba-Cre mouse will advance our understanding of the role of platelets in inflammation and other pathophysiological processes in which platelet-leukocyte interactions are involved.

Congenital macrothrombocytopenia with focal myelofibrosis due to mutations in human G6b-B is rescued in humanized mice.

Unlike primary myelofibrosis (PMF) in adults, myelofibrosis in children is rare. Congenital (inherited) forms of myelofibrosis (cMF) have been described, but the underlying genetic mechanisms remain elusive. Here we describe 4 families with autosomal recessive inherited macrothrombocytopenia with focal myelofibrosis due to germ line loss-of-function mutations in the megakaryocyte-specific immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B (G6b, C6orf25, or MPIG6B). Patients presented with a mild-to-moderate bleeding diathesis, macrothrombocytopenia, anemia, leukocytosis and atypical megakaryocytes associated with a distinctive, focal, perimegakaryocytic pattern of bone marrow fibrosis. In addition to identifying the responsible gene, the description of G6b-B as the mutated protein potentially implicates aberrant G6b-B megakaryocytic signaling and activation in the pathogenesis of myelofibrosis. Targeted insertion of human G6b in mice rescued the knockout phenotype and a copy number effect of human G6b-B expression was observed. Homozygous knockin mice expressed 25% of human G6b-B and exhibited a marginal reduction in platelet count and mild alterations in platelet function; these phenotypes were more severe in heterozygous mice that expressed only 12% of human G6b-B. This study establishes G6b-B as a critical regulator of platelet homeostasis in humans and mice. In addition, the humanized G6b mouse will provide an invaluable tool for further investigating the physiological functions of human G6b-B as well as testing the efficacy of drugs targeting this receptor.

Uncoupling ITIM receptor G6b-B from tyrosine phosphatases Shp1 and Shp2 disrupts murine platelet homeostasis.

The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation (G6b-B diY/F) were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in G6b-deficient mice and patients. Platelets from G6b-B diY/F mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from G6b-B diY/F, G6b KO, and megakaryocyte-specific Shp2 KO mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis.

Cutting Edge: Imbalanced Cation Homeostasis in MAGT1-Deficient B Cells Dysregulates B Cell Development and Signaling in Mice.

Cation homeostasis, in relation to various immune-suppressive diseases, is a novel field of investigation. Recently, patients with a loss-of-function mutation in magnesium transporter 1 (MAGT1) were reported to present a dysregulated Mg2+ homeostasis in T lymphocytes. Using Magt1-knockout mice (Magt1-/y ), we show that Mg2+ homeostasis was impaired in Magt1-/y B cells and Ca2+ influx was increased after BCR stimulation, whereas T and NK cell function was unaffected. Consequently, mutant B cells displayed an increased phosphorylation of BCR-related proteins differentially affecting protein kinase C activation. These in vitro findings translated into increased frequencies of CD19+ B cells and marginal zone B cells and decreased frequencies of plasma cells among CD45+ splenocytes in vivo. Altogether, our study demonstrates for the first time, to our knowledge, that abolished MAGT1 function causes imbalanced cation homeostasis and developmental responses in B cells. Therefore, this study might contribute to a further understanding of B cell-related pathologies.

Twinfilin 2a regulates platelet reactivity and turnover in mice.

Regulated reorganization of the actin cytoskeleton is a prerequisite for proper platelet production and function. Consequently, defects in proteins controlling actin dynamics have been associated with platelet disorders in humans and mice. Twinfilin 2a (Twf2a) is a small actin-binding protein that inhibits actin filament assembly by sequestering actin monomers and capping filament barbed ends. Moreover, Twf2a binds heterodimeric capping proteins, but the role of this interaction in cytoskeletal dynamics has remained elusive. Even though Twf2a has pronounced effects on actin dynamics in vitro, only little is known about its function in vivo. Here, we report that constitutive Twf2a-deficient mice (Twf2a-/-) display mild macrothrombocytopenia due to a markedly accelerated platelet clearance in the spleen. Twf2a-/- platelets showed enhanced integrin activation and α-granule release in response to stimulation of (hem) immunoreceptor tyrosine-based activation motif (ITAM) and G-protein-coupled receptors, increased adhesion and aggregate formation on collagen I under flow, and accelerated clot retraction and spreading on fibrinogen. In vivo, Twf2a deficiency resulted in shortened tail bleeding times and faster occlusive arterial thrombus formation. The hyperreactivity of Twf2a-/- platelets was attributed to enhanced actin dynamics, characterized by an increased activity of n-cofilin and profilin 1, leading to a thickened cortical cytoskeleton and hence sustained integrin activation by limiting calpain-mediated integrin inactivation. In summary, our results reveal the first in vivo functions of mammalian Twf2a and demonstrate that Twf2a-controlled actin rearrangements dampen platelet activation responses in a n-cofilin- and profilin 1-dependent manner, thereby indirectly regulating platelet reactivity and half-life in mice.

Cooperative and alternate functions for STIM1 and STIM2 in macrophage activation and in the context of inflammation.

Calcium (Ca(2+)) signaling in immune cells, including macrophages, controls a wide range of effector functions that are critical for host defense and contribute to inflammation and autoimmune diseases. However, receptor-mediated Ca(2+) responses consist of complex mechanisms that make it difficult to identify the pathogenesis and develop therapy. Previous studies have revealed the importance of the Ca(2+) sensor STIM1 and store-operated Ca(2+)-entry (SOCE) for Fcγ-receptor activation and IgG-induced inflammation. Here, we identify the closely related STIM2 as mediator of cell migration and cytokine production downstream of GPCR and TLR4 activation in macrophages and show that mice lacking STIM2 are partially resistant to inflammatory responses in peritonitis and LPS-induced inflammation. Interestingly, STIM2 modulates the migratory behavior of macrophages independent from STIM1 and without a strict requirement for Ca(2+) influx. While STIM2 also contributes in part to FcγR activation, the C5a-induced amplification of IgG-mediated phagocytosis is mainly dependent on STIM1. Blockade of STIM-related functions limits mortality in experimental models of AIHA and LPS-sepsis in normal mice. These results suggest benefits of Ca(2+)-inhibition for suppression of exacerbated immune reactions and illustrate the significance of alternate functions of STIM proteins in macrophage activation and in the context of innate immune inflammation.

Orai1 controls C5a-induced neutrophil recruitment in inflammation.

Stromal interaction molecule 1 (STIM1)-dependent store operated calcium-entry (SOCE) through Orai1-mediated calcium (Ca(2+) ) influx is considered a major pathway of Ca(2+) signaling, serving T-cell, mast cell, and platelet responses. Here, we show that Orai1 is critical for neutrophil function. Orai1-deficient neutrophils present defects in fMLP and complement C5a-induced Ca(2+) influx and migration, although they respond normally to another chemoattractant, CXCL2. Up until now, no specific contribution of Orai1 independent from STIM1 or SOCE has been recognized in immune cells. Here, we observe that Orai1-deficient neutrophils exhibit normal STIM1-dependent SOCE and STIM1-deficient neutrophils respond to fMLP and C5a efficiently. Despite substantial cytokine production, Orai1(-/-) chimeric mice show impaired neutrophil recruitment in LPS-induced peritonitis. Moreover, Orai1 deficiency results in profoundly defective C5a-triggered neutrophil lung recruitment in hypersensitivity pneumonitis. Comparative evaluation of inflammation in Stim1(-/-) chimeras reveals a distinct pathogenic contribution of STIM1, including its involvement in IgG-induced C5a production. Our data establish Orai1 as key signal mediator of C5aR activation, contributing to inflammation by a STIM1-independent pathway of Ca(2+) -influx in neutrophils.

Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization.

Platelet aggregation at sites of vascular injury is not only essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemi-immunoreceptor tyrosine-based activation motif-containing C-type lectinlike receptor 2 (CLEC-2) mediates powerful platelet activation through a Src- and spleen tyrosine kinase (Syk)-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 not only contributes to thrombus formation and stabilization but also plays a central role in blood-lymphatic vessel development, tumor metastasis, and prevention of inflammatory bleeding, making it a potential pharmacologic target to modulate these processes. We have previously shown that injection of the anti-CLEC-2 antibody, INU1, results in virtually complete immunodepletion of platelet CLEC-2 in mice, which is, however, preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. The mechanisms underlying this targeted CLEC-2 downregulation have remained elusive. Here, we show that INU1-induced CLEC-2 immunodepletion occurs through Src-family kinase-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with platelet-specific Syk deficiency, INU1-induced CLEC-2 internalization/degradation was fully preserved whereas the associated thrombocytopenia was largely prevented. These results show for the first time that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia.