Adaptor Protein SLP-76 Research Articles

T Cell Receptor (TCR)-Induced PLC-γ1 Sumoylation via PIASxβ and PIAS3 SUMO E3 Ligases Regulates the Microcluster Assembly and Physiological Function of PLC-γ1.

The SUMO modification system plays an important role in T cell activation, yet how sumoylation regulates TCR-proximal signaling remains largely unknown. We show here that Phospholipase C-γ1 (PLC-γ1) is conjugated by SUMO1 at K54 and K987 upon TCR stimulation and that K54 sumoylation is pivotal for PLC-γ1-mediated T cell activation. We further demonstrate that TCR-induced K54 sumoylation of PLC-γ1 significantly promotes the formation of PLC-γ1 microclusters and the association of PLC-γ1 with the adaptor proteins SLP76 and Gads, but only slightly affects the phosphorylation of PLC-γ1 on Y783, which determines the enzyme catalytic activity. Moreover, upon TCR stimulation, the SUMO E3 ligases PIASxβ and PIAS3 both interact with PLC-γ1 and cooperate to sumoylate PLC-γ1, facilitating the assembly of PLC-γ1 microclusters. Together, our findings reveal a critical role of PLC-γ1 K54 sumoylation in PLC-γ1 microcluster assembly that controls PLC-γ1-mediated T cell activation, suggesting that sumoylation may have an important role in the microcluster assembly of TCR-proximal signaling proteins.

Glycerol monolaurate induces filopodia formation by disrupting the association between LAT and SLP-76 microclusters.

Glycerol monolaurate (GML) is a monoglyceride with potent antimicrobial properties that suppresses T cell receptor (TCR)-induced signaling and T cell effector function. Actin rearrangement is needed for the interaction of T cells with antigen-presenting cells and for migration to sites of infection. Because of the critical role actin rearrangement plays in T cell effector function, we analyzed the effect of GML on the rearrangement of the actin cytoskeleton after TCR activation. We found that GML-treated human T cells were less adherent than untreated T cells and did not form actin ring structures but instead developed numerous inappropriate actin-mediated filopodia. The formation of these filopodia was not due to disruption of TCR-proximal regulators of actin or microtubule polymerization. Instead, total internal reflection fluorescence microscopy demonstrated mislocalization of actin nucleation protein Arp2 microclusters, but not those containing the adaptor proteins SLP-76 and WASp, or the actin nucleation protein ARPC3, which are necessary for TCR-induced actin rearrangement. Additionally, SLP-76 microclusters colocalized with WASp and WAVE microclusters but not with LAT. Together, our data suggest that GML alters actin cytoskeletal rearrangements and identify diverse functions for GML as a T cell-suppressive agent.

Sensitivity-Enhanced Four-Dimensional Amide-Amide Correlation NMR Experiments for Sequential Assignment of Proline-Rich Disordered Proteins.

Proline is prevalent in intrinsically disordered proteins (IDPs). NMR assignment of proline-rich IDPs is a challenge due to low dispersion of chemical shifts. We propose here new sensitivity-enhanced 4D NMR experiments that correlate two pairs of amide resonances that are either consecutive (NH i-1, NH i) or flanking a proline at position i-1 (NH i-2, NH i). The maximum 2-fold enhancement of sensitivity is achieved by employing two coherence order-selective (COS) transfers incorporated unconventionally into the pulse sequence. Each COS transfer confers an enhancement over amplitude-modulated transfer by a factor of √2 specifically when transverse relaxation is slow. The experiments connect amide resonances over a long fragment of sequence interspersed with proline. When this method was applied to the proline-rich region of B cell adaptor protein SLP-65 (pH 6.0) and α-synuclein (pH 7.4), which contain a total of 52 and 5 prolines, respectively, 99% and 92% of their nonprolyl amide resonances have been successfully assigned, demonstrating its robustness to address the assignment problem in large proline-rich IDPs.

The molecular mechanisms involved in lectin-induced human platelet aggregation.

We have compared the effect of three legume lectins, wheat germ agglutinin (WGA), Phaseolus vulgaris agglutinin (PHA) and Lens culinaris agglutinin (LCA), on the function of human platelets. We have found that WGA is more active than PHA in stimulating platelet activation/aggregation, while LCA has no effect. Studies on the mechanisms involved show that WGA and PHA induce phosphorylation/activation of PLCγ2 and increase [Ca2+]i. For the first time, it has been shown that Src/Syk pathway, the adapter protein SLP-76 and the exchange protein VAV, participate in the PLCγ2 activation by these lectins. Moreover WGA and PHA stimulate the PI3K/AKT pathway. PI3K, through its product phosphatidylinositol-3,4,5-trisphosphate activates Bruton's tyrosine kinase (BTK) and contributes to PLCγ2 activation. In conclusion, our findings suggest that PLCγ2 activation induced by WGA and PHA is regulated by Src/Syk and by PI3K/BTK pathways through their concerted action.

T Cell Costimulation by CD6 Is Dependent on Bivalent Binding of a GADS/SLP-76 Complex.

ABSTRACTThe cell surface receptor CD6 regulates T cell activation in both activating and inhibitory manners. The adaptor protein SLP-76 is recruited to the phosphorylated CD6 cytoplasmic Y662 residue during T cell activation, providing an activating signal to T cells. In this study, a biochemical approach identified the SH2 domain-containing adaptor protein GADS as the dominant interaction partner for the CD6 cytoplasmic Y629 residue. Functional experiments in human Jurkat and primary T cells showed that both mutations Y629F and Y662F abolished costimulation by CD6. In addition, a restraint on T cell activation by CD6 was revealed in primary T cells expressing CD6 mutated at Y629 and Y662. These data are consistent with a model in which bivalent recruitment of a GADS/SLP-76 complex is required for costimulation by CD6.

In Vitro Formation of a Multi-Protein Complex Involved in TCR Signaling

The T cell antigen receptor (TCR) binds a ligand consisting of antigenic peptide bound to a cell surface molecule encoded by the major histocompatibility complex (pMHC) to begin the process of T cell activation. TCR engagement leads to protein tyrosine kinase (PTK) recruitment to the TCR and PTK activation, and activated PTKs phosphorylate the membrane-bound adapter protein LAT and the cytosolic adapter protein SLP-76. Phosphorylated LAT and SLP-76 recruit a number of other adapters and enzymes including GADS, PLCγ, GRB2, NCK, VAV and ITK. The protein complexes nucleating at LAT are highly heterogeneous, and the formation of these complexes is highly co-operative. Different LAT complexes activate different effector function. For example, the LAT/GADS/SLP-76 complex recruits the PTK ITK to activate PLCγ, leading to Ca2+ flux and MAPK activation. Here we study the LAT-GADS-SLP-76 tri-molecular complex by generating it in vitro from purified component proteins and a LAT phospho-peptide. Isolation of this complex and others should enable generation of structural information, which would be required to design specific inhibitors for modulation of signaling.

Serine Phosphorylation of SLP76 Is Dispensable for T Cell Development but Modulates Helper T Cell Function.

The adapter protein SLP76 is a key orchestrator of T cell receptor (TCR) signal transduction. We previously identified a negative feedback loop that modulates T cell activation, involving phosphorylation of Ser376 of SLP76 by the hematopoietic progenitor kinase 1 (HPK1). However, the physiological relevance of this regulatory mechanism was still unknown. To address this question, we generated a SLP76-S376A-expressing knock-in mouse strain and investigated the effects of Ser376 mutation on T cell development and function. We report here that SLP76-S376A-expressing mice exhibit normal thymocyte development and no detectable phenotypic alterations in mature T cell subsets or other lymphoid and myeloid cell lineages. Biochemical analyses revealed that mutant T cells were hypersensitive to TCR stimulation. Indeed, phosphorylation of several signaling proteins, including SLP76 itself, phospholipase Cγ1 and the protein kinases AKT and ERK1/2, was increased. These modifications correlated with increased Th1-type and decreased Th2-type cytokine production by SLP76-S376A T cells, but did not result in significant changes of proliferative capacity nor activation-induced cell death susceptibility. Hence, our results reveal that SLP76-Ser376 phosphorylation does not mediate all HPK1-dependent regulatory effects in T cells but it fine-tunes helper T cell responses.

A mutation within the SH2 domain of slp-76 regulates the tissue distribution and cytokine production of iNKT cells in mice.

TCR ligation is critical for the selection, activation, and integrin expression of T lymphocytes. Here, we explored the role of the TCR adaptor protein slp-76 on iNKT-cell biology. Compared to B6 controls, slp-76(ace/ace) mice carrying a missense mutation (Thr428Ile) within the SH2-domain of slp-76 showed an increase in iNKT cells in the thymus and lymph nodes, but a decrease in iNKT cells in spleens and livers, along with reduced ADAP expression and cytokine response. A comparable reduction in iNKT cells was observed in the livers and spleens of ADAP-deficient mice. Like ADAP(-/-) iNKT cells, slp-76(ace/ace) iNKT cells were characterized by enhanced CD11b expression, correlating with an impaired induction of the TCR immediate-early gene Nur77 and a decreased adhesion to ICAM-1. Furthermore, CD11b-intrinsic effects inhibited cytokine release, concanavalin A-mediated inflammation, and iNKT-cell accumulation in the liver. Unlike B6 and ADAP(-/-) mice, the expression of the transcription factors Id3 and PLZF was reduced, whereas NP-1-expression was enhanced in slp-76(ace/ace) mice. Blockade of NP-1 decreased the recovery of iNKT cells from peripheral lymph nodes, identifying NP-1 as an iNKT-cell-specific adhesion factor. Thus, slp-76 contributes to the regulation of the tissue distribution, PLZF, and cytokine expression of iNKT cells via ADAP-dependent and -independent mechanisms.

Adapting to the nucleus

In addition to mediating T cell receptor signaling at the plasma membrane, the adaptor protein SLP-76 is required for optimal nuclear entry of transcription factors.

Quantitative reduction of the TCR adapter protein SLP-76 unbalances immunity and immune regulation.

Gene variants that disrupt TCR signaling can cause severe immune deficiency, yet less disruptive variants are sometimes associated with immune pathology. Null mutations of the gene encoding the scaffold protein Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76), for example, cause an arrest of T cell positive selection, whereas a synthetic membrane-targeted allele allows limited positive selection but is associated with proinflammatory cytokine production and autoantibodies. Whether these and other enigmatic outcomes are due to a biochemical uncoupling of tolerogenic signaling, or simply a quantitative reduction of protein activity, remains to be determined. In this study we describe a splice variant of Lcp2 that reduced the amount of wild-type SLP-76 protein by ~90%, disrupting immunogenic and tolerogenic pathways to different degrees. Mutant mice produced excessive amounts of proinflammatory cytokines, autoantibodies, and IgE, revealing that simple quantitative reductions of SLP-76 were sufficient to trigger immune dysregulation. This allele reveals a dose-sensitive threshold for SLP-76 in the balance of immunity and immune dysregulation, a common disturbance of atypical clinical immune deficiencies.

3P195 免疫シグナルアダプター分子SLP-76 の複合体の形成と成長 : 1 分子追跡による解明(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

3P195 免疫シグナルアダプター分子SLP-76 の複合体の形成と成長 : 1 分子追跡による解明(細胞生物的課題,ポスター,第52回日本生物物理学会年会(2014年度))

Strength of TCR signal from self‐peptide modulates autoreactive thymocyte deletion and Foxp3+Treg‐cell formation

Autoreactive CD4(+) CD8(-) (CD4SP) thymocytes can be subjected to deletion when they encounter self-peptide during their development, but they can also undergo selection to become CD4SPFoxp3(+) Treg cells. We have analyzed the relationship between these distinct developmental fates using mice in which signals transmitted by the TCR have been attenuated by mutation of a critical tyrosine residue of the adapter protein SLP-76. In mice containing polyclonal TCR repertoires, the mutation caused increased frequencies of CD4SPFoxp3(+) thymocytes. CD4SP thymocytes expressing TCR Vβ-chains that are subjected to deletion by endogenous retroviral superantigens were also present at increased frequencies, particularly among Foxp3(+) thymocytes. In transgenic mice in which CD4SP thymocytes expressing an autoreactive TCR undergo both deletion and Treg-cell formation in response to a defined self-peptide, SLP-76 mutation abrogated deletion of autoreactive CD4SP thymocytes. Notably, Foxp3(+) Treg-cell formation still occurred, albeit with a reduced efficiency, and the mutation was also associated with decreased Nur77 expression by the autoreactive CD4SP thymocytes. These studies provide evidence that the strength of the TCR signal can play a direct role in directing the extent of both thymocyte deletion and Treg-cell differentiation, and suggest that distinct TCR signaling thresholds and/or pathways can promote CD4SP thymocyte deletion versus Treg-cell formation.

The Chemokine CXCL12 Generates Costimulatory Signals in T Cells to Enhance Phosphorylation and Clustering of the Adaptor Protein SLP-76

The CXC chemokine CXCL12 mediates the chemoattraction of T cells and enhances the stimulation of T cells through the T cell receptor (TCR). The adaptor SLP-76 [Src homology 2 (SH2) domain-containing leukocyte protein of 76 kD] has two key tyrosine residues, Tyr(113) and Tyr(128), that mediate signaling downstream of the TCR. We investigated the effect of CXCL12 on SLP-76 phosphorylation and the TCR-dependent formation of SLP-76 microclusters. Although CXCL12 alone failed to induce SLP-76 cluster formation, it enhanced the number, stability, and phosphorylation of SLP-76 microclusters formed in response to stimulation of the TCR by an activating antibody against CD3, a component of the TCR complex. Addition of CXCL12 to anti-CD3-stimulated cells resulted in F-actin polymerization that stabilized SLP-76 microclusters in the cells' periphery at the interface with antibody-coated coverslips and increased the interaction between SLP-76 clusters and those containing ZAP-70, the TCR-associated kinase that phosphorylates SLP-76, as well as increased TCR-dependent gene expression. Costimulation with CXCL12 and anti-CD3 increased the extent of phosphorylation of SLP-76 at Tyr(113) and Tyr(128), but not that of other TCR-proximal components, and mutation of either one of these residues impaired the CXCL12-dependent effect on SLP-76 microcluster formation, F-actin polymerization, and TCR-dependent gene expression. The effects of CXCL12 on SLP-76 microcluster formation were dependent on the coupling of its receptor CXCR4 to G(i)-family G proteins (heterotrimeric guanine nucleotide-binding proteins). Thus, we identified a costimulatory mechanism by which CXCL12 and antigen converge at SLP-76 microcluster formation to enhance T cell responses.

Abstract 486: Platelet ITAM Signaling is Critical for Maintenance of Vascular Integrity in Inflammation

Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in in[[Unable to Display Character: fl]]ammation seems to be independent of their ability to form a clot. However, the underlying molecular mechanism remains unknown. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling but not G protein-coupled receptor (GPCR) signaling is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a novel protocol for the adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from wild-type platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of CLEC-2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into thrombocytopenic mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel anti-thrombotic agents directed towards the ITAM signaling pathway.

Platelet ITAM signaling is critical for vascular integrity in inflammation

Platelets play a critical role in maintaining vascular integrity during inflammation, but little is known about the underlying molecular mechanisms. Here we report that platelet immunoreceptor tyrosine activation motif (ITAM) signaling, but not GPCR signaling, is critical for the prevention of inflammation-induced hemorrhage. To generate mice with partial or complete defects in these signaling pathways, we developed a protocol for adoptive transfer of genetically and/or chemically inhibited platelets into thrombocytopenic (TP) mice. Unexpectedly, platelets with impaired GPCR signaling, a crucial component of platelet plug formation and hemostasis, were indistinguishable from WT platelets in their ability to prevent hemorrhage at sites of inflammation. In contrast, inhibition of GPVI or genetic deletion of Clec2, the only ITAM receptors expressed on mouse platelets, significantly reduced the ability of platelets to prevent inflammation-induced hemorrhage. Moreover, transfusion of platelets without ITAM receptor function or platelets lacking the adapter protein SLP-76 into TP mice had no significant effect on vascular integrity during inflammation. These results indicate that the control of vascular integrity is a major function of immune-type receptors in platelets, highlighting a potential clinical complication of novel antithrombotic agents directed toward the ITAM signaling pathway.

The PAK Signaling System Links Rho Gtpase Activation to Platelet Lamellopodia Formation, Aggregation and Aggregate Stability Under Shear

Abstract 1060Platelets serve as the primary mediators of hemostasis and thrombosis, circulating as surveyors for gaps in vascular integrity. As platelets encounter exposed extracellular matrix proteins, receptors on the platelet surface trigger intracellular signaling events that result in rapid platelet activation and a complex rearrangement of platelet morphology to form filopodia and lamellipodia. Rac1, a member of the Rho GTPase family, has emerged as a key regulator in platelet actin dynamics. However, the specific downstream events following Rac1 activation that mediate platelet actin cytoskeleton reorganization remain ill-defined.The Rho GTPase, Rac, supports the autocatalytic activation of the p21 activated kinases, or PAKs, to mediate actin reorganization processes in focal adhesion formation and cell migration. Upon activation by GTP-bound Rac, the PAKs phosphorylate a number of substrates to coordinate actin dynamics. Platelets express a number of PAK isoforms, and like Rac, PAK has been shown to be activated as platelets spread on collagen in a Src and PI3K dependent manner. Furthermore, the adaptor protein SLP-76 has been proposed to potentiate PAK activity downstream of Rac activation to mediate platelet lamellipodia formation. However, the specific roles of PAK in platelet function have yet to be characterized. Thus we set out to elucidate the role of PAK in platelet function and to define the connection between Rac activation, PAK, and platelet cytoskeletal reorganization.Our initial experiments with mass spectrometry revealed that following platelet activation, Rac1 associates with a set of PAK effectors, GIT1, GEFH1, LIMK1, and Merlin. We next demonstrated a co-localization of Rac1 and PAK with actin at the leading edge of spread platelets on fibrinogen. In addition, inhibition of PAK signaling by two different pharmacologic inhibitors blocked platelet focal adhesion and lamellopodia formation on both fibrinogen and collagen. Inhibition of PAK signaling abrogated intracellular calcium mobilization in platelets, prevented platelet aggregation to the GPVI-agonist, CRP, and destabilized platelet lamellipodia, resulting in the retraction of lamellipodia in spread platelets. Finally, inhibition of PAK resulted in the disaggregation of platelet aggregates formed under shear flow conditions.Together, these results demonstrate that the PAK signaling system is a key orchestrator of platelet actin dynamics, linking Rho GTPase activation to PAK effector function and platelet lamellopodia formation, thus filling an important gap in the understanding of platelet actin cytoskeletal organization. In addition, these data characterize the integral role of PAK in platelet spreading, aggregation, and aggregate stability. Elucidating the mechanisms that mediate platelet spreading and aggregate formation may highlight important steps in the platelet activation cascade at which to pharmacologically intervene in order to inhibit or treat pathologic thrombi formation. Disclosures:No relevant conflicts of interest to declare.

SLP‐76 is required for optimal CXCR4‐stimulated T lymphocyte firm arrest to ICAM‐1 under shear flow

Rapid arrest of T cells at target sites upon engagement of chemokine receptors is crucial to the proper functioning of the immune system. Although T-cell arrest always occurs under hydrodynamic forces in vivo, most studies investigating the molecular mechanisms of arrest have been performed under static conditions. While the requirement of the adapter protein SLP-76 (Src homology 2-domain containing leukocyte-specific phosphoprotein of 76 kDa) in TCR-induced integrin activation has been demonstrated, its role in chemokine-triggered T-cell adhesion is unknown. Using a flow chamber system, we show that SLP-76 plays an important role in regulating the transition from tethering and rolling to firm adhesion of T cells under physiological shear flow in response to CXCL12α (stromal cell-derived factor-1α); SLP-76-deficient primary T cells exhibited defective adhesion with a significant decrease in the number of firmly arrested cells. We further demonstrate the N-terminal phosphotyrosines of SLP-76 play a critical role in T-cell adhesion under flow. These findings reveal a novel role for SLP-76 in CXCR4-mediated T lymphocyte trafficking.

Complementary Phosphorylation Sites in the Adaptor Protein SLP-76 Promote Synergistic Activation of Natural Killer Cells

The cytotoxic effects of natural killer (NK) cells and their ability to secrete cytokines require synergistic signals from specific pairs of co-activation receptors, such as CD314 (also known as NKG2D) and CD244 (2B4), which bind to distinct ligands present on target cells. These signals are required to overcome inhibition mediated by the E3 ubiquitin ligase c-Cbl of the guanine nucleotide exchange factor Vav1, which promotes activation of NK cells. Here, we showed that the adaptor protein SLP-76 (Src homology 2 domain-containing leukocyte phosphoprotein of 76 kilodaltons) was required for this synergy and that distinct tyrosine residues in SLP-76 were phosphorylated by each member of a pair of synergistic receptors. Selective phosphorylation of tyrosine 113 or tyrosine 128 in SLP-76 enabled binding of SLP-76 to Vav1. Selective phosphorylation of SLP-76 at these residues was restricted to receptors that stimulated ligand-dependent target cell killing; antibody-dependent stimulation of the Fc receptor CD16 promoted phosphorylation at both sites. Knockdown and reconstitution experiments with SLP-76 mutant proteins showed the distinct role of each tyrosine in the synergistic mobilization of Ca2+, revealing an unexpected degree of selectivity in the phosphorylation of SLP-76 by NK cell co-activation receptors. Together, these data suggest that combined phosphorylation of separate tyrosine residues in SLP-76 forms the basis of synergistic NK cell activation.

The SKAP55 dimer stabilizes TCR-induced SLP-76 microclusters (121.27)

Abstract The adapter molecule SKAP55 enables the formation of T-cell:APC conjugates, facilitates T-cell cytokine production, and is required for optimal T cell proliferation in response to antigen. However, the molecular basis for the involvement of SKAP55 in these events remains unclear. Here, we demonstrate that both endogenous SKAP55 and exogenous SKAP55 chimeras enter T cell receptor (TCR)-induced signaling microclusters containing the adapter protein SLP-76. Using a SKAP55-deficient Jurkat T cell line, we demonstrate that SKAP55 is required for both the persistence and the movement of SLP-76 microclusters. Furthermore, the recruitment of SKAP55 into microclusters requires the presence of both SLP-76 and ADAP, an intermediary adapter protein capable of interacting with both SLP-76 and SKAP55. In addition, we show that SKAP55 homodimerizes via an N-terminal dimerization motif (DM). Our structure-function analyses indicate that the dimerization domain and the ADAP-interacting Src-homology domain 3 (SH3) domain of SKAP55 are both required for the recruitment of SKAP55 into SLP-76 microclusters, and for the stabilization of SLP-76 microclusters. Importantly, an artificial tandem dimer composed of two functional SH3 domains joined by a flexible linker was sufficient for entry into, and stabilization of these signaling complexes. This data indicates that SKAP55 could contribute to T-cell activation by increasing the overall avidity and stability of TCR-induced SLP-76 microclusters.

Abstract 53: Immunoreceptor Tyrosine Activation Motif Signaling in Platelets Is Critical for the Maintenance of Vascular Integrity During Inflammation

Introduction: Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in inflammation seems to be independent of their ability to form a clot. Goal: To identify signaling molecules critical for the contribution of platelets to vascular integrity in inflammation. Methods: Transgenic mice with platelet-specific expression of the interleukin (IL)4 receptor were rendered thrombocytopenic by infusion of antibodies to IL4R. Platelets from various knockout mice or inhibitor-treated wild-type (WT) platelets were transfused into thrombocytopenic (TP) mice and tested for their ability to support vascular integrity in a reverse passive Arthus reaction (rpA) model. Tissue hemoglobin (Hb) was used to quantify red blood cells in the inflamed tissue, a marker of disrupted vascular integrity. Results: Impaired vascular integrity at the site of inflammation was markedly higher in TP mice compared to controls (Hb: 1448±176 vs. 100±8 μg/cm2). Transfusion of WT platelets into TP mice strongly reduced tissue Hb (150±46 μg/ cm2). Transfused platelets from mice lacking the thrombin receptor PAR4 and/or functional P2Y12 (ADP receptor) also prevented disruption of vascular integrity (Hb: 210±66, 136±16 μg and 360±120 μg/cm2), suggesting that signaling by G protein-coupled receptors is not important for this response. Furthermore, infusion of heparin into WT mice did not lead to increased tissue Hb. Immunoreceptor tyrosine activation motif (ITAM) signaling, however, was critical for platelet-dependent maintenance of vascular integrity, as tissue Hb was markedly increased in TP mice transfused with platelets lacking the signal trandsucing adapter protein SLP-76 (1212±160 μg/cm2) or platelets lacking functional ITAM signaling receptors GPVI (852±196 μg/cm2), CLEC-2 (1062±188 μg/cm2), or both (1498±128 μg/cm2). Data are mean±SEM, n=4-5 mice per group. Conclusions: We have established a novel mouse model for the adoptive transfer of platelets into TP mice. Using this model we can show that the ITAM but not GPCR signaling in platelets is critical for the maintenance of vascular integrity during inflammation. Our studies highlight a potential clinical complication of future anti-platelet agents targeted towards ITAM signaling receptors such as GPVI.