FcγRIIA Activation Research Articles

Affinity capillary electrophoresis - mass spectrometry permits direct binding assessment of IgG and FcγRIIa in a glycoform-resolved manner.

The impact of antibody glycoforms on FcγRIIa activation and immune responses is poorly understood. Yet, glycoform binding assessment remains one of the major analytical challenges requiring long enrichment or glycoengineering steps. Here, we developed and applied an affinity capillary electrophoresis-mass spectrometry approach to selectively assess the binding of different antibody glycoforms to the FcγIIa receptor without the need of glycoengineering. The approach required only low microgram amounts of antibody and receptor and enables assessing the binding of high and low-abundance glycoforms. The approach indicated clear differences in binging between doubly-, hemi-glycosylated and non-glycosylated antibodies as well as for mutated (Leu234Ala, Leu235Ala – Pro329-Gly (LALA-PG)) IgG1 antibodies silenced for Fcγ binding. The LALA-PG mutated antibody showed no binding to the FcγIIa receptor (excluding potential non-specific binding effects) while the non-glycosylated IgG1 showed a strongly reduced, but still minor binding. The highest binding affinity was for the antibody carrying two complex-type glycans. Man5 glycans resulted in decreased binding compared to complex-type glycans, with the lowest binding for the IgG containing two Man5. For complex-type glycans, galactosylation showed a subtle increase in binding to the FcγIIa receptor, and sialylation showed an increase in binding for lower sialylated species. Fucosylation did not influence binding to the FcγIIa receptor. Finally, the assay was evaluated for the two variants of the FcγRIIa receptor (allotypes H131 and R131) showing highly comparable glycoform selectivity. Overall, the proposed approach allows the direct comparison of binding affinities of different antibody species in mixtures promising a fast establishment of their structure-function relationships.

291 Dual-specific antibodies blocking both PD-L1 and PD-L2 engagement of PD-1 restore anti-tumor immunity

BackgroundInhibition of T cell activation and effector function via engagement of the co-inhibitory receptor PD-1 is a critical mechanism enabling tumors to evade host immunity. The two ligands for PD-1, PD-L1 and PD-L2, can be expressed by a variety of immunosuppressive stromal cells, particularly of the myeloid lineage, endothelial cells, and by tumors themselves. In addition to PD-1, PD-L1 engages B7-1 in an additional co-inhibitory interaction. Blocking only PD-1 or only PD-L1 thus does not relieve all inhibitory components of this pathway. We hypothesized that bispecific antibodies blocking both PD-L1 and PD-L2 could more fully restore tumor-specific T cell activation and potentiate anti-cancer immunotherapy. Furthermore, we speculated that enhancing the cytotoxic effector function of these antibodies might further enhance their efficacy through the depletion of tumor cells and supportive stroma.MethodsWe investigated the capacity of monoclonal antibodies capable of bivalent binding to both PD-L1 and PD-L2 to restore the function of PD-1-suppressed T cells in vitro. To assess the in vivo therapeutic efficiency of bispecific PD-Ligand antibodies with ADCC capacities, mouse IgG2a and modified human IgG1 versions were generated. We assessed their ADCC activity in vitro using a bioluminescent reporter assay, and their therapeutic efficiency in vivo in syngeneic or human-cell derived tumors.ResultsThe bispecific antibodies we generated restore the function of PD-1-suppressed T cells in vitro with equivalent efficiency to the FDA approved PD-1 antibody Pembrolizumab. Moreover, our modified human bispecific antibodies lead to significantly higher FcγRIIa activation than FDA-approved clinical human IgG1 PD-L1 antibodies in vitro. In vivo, ADCC-capable PD-Ligand bispecific antibodies suppress the growth of U2940 lymphoma in immunodeficient mice more efficiently than Rituximab, and in a syngeneic model of PD-L1/PD-L2 double positive colon carcinoma, these antibodies demonstrate superiority to PD-1 blocking antibodies to limit tumor growth and increase survival. Furthermore, treatment with our bispecific antibodies increases T cell proliferation and cytotoxicity and reduces density of immunosuppressive myeloid stroma in vivo.ConclusionsADCC-capable PD-Ligand bispecific antibodies display higher therapeutic potential than existing anti-PD-1 antibodies and represent a new class of PD-1 pathway therapeutics with significant potential for the treatment of a variety of human cancers.AcknowledgementsI would like to thank Anupallavi Srinivasamani, PhD student in Michael Curran’s lab, who performed a considerable amount of the work on this project before I joined.

Effects of the Btk-Inhibitors Remibrutinib (LOU064) and Rilzabrutinib (PRN1008) With Varying Btk Selectivity Over Tec on Platelet Aggregation and in vitro Bleeding Time.

Background: Bruton tyrosine kinase inhibitors (BTKi) are used in B-cell malignancies and in development against various autoimmune diseases. Since Btk is also involved in specific pathways of platelet activation, BTKi might be considered to target platelet GPVI/GPIb-mediated atherothrombosis and platelet FcγRIIA-dependent immune disorders. However, BTKi treatment of patients with B-cell malignancies is frequently associated with mild bleeding events caused possibly by off-target inhibition of Tec. Here, we compared the platelet effects of two novel BTKi that exhibit a high (remibrutinib) or low (rilzabrutinib) selectivity for Btk over Tec.Methods and Results: Remibrutinib and rilzabrutinib were pre-incubated with anticoagulated blood. Platelet aggregation and in vitro bleeding time (closure time) were studied by multiple electrode aggregometry (MEA) and platelet-function analyzer-200 (PFA-200), respectively. Both BTKi inhibited atherosclerotic plaque-stimulated GPVI-mediated platelet aggregation, remibrutinib being more potent (IC50 = 0.03 μM) than rilzabrutinib (IC50 = 0.16 μM). Concentrations of remibrutinib (0.1 μM) and rilzabrutinib (0.5 μM), >80% inhibitory for plaque-induced aggregation, also significantly suppressed (>90%) the Btk-dependent pathways of platelet aggregation upon GPVI, von Willebrand factor/GPIb and FcγRIIA activation stimulated by low collagen concentrations, ristocetin and antibody cross-linking, respectively. Both BTKi did not inhibit aggregation stimulated by ADP, TRAP-6 or arachidonic acid. Remibrutinib (0.1 μM) only slightly prolonged closure time and significantly less than rilzabrutinib (0.5 μM).Conclusion: Remibrutinib and rilzabrutinib inhibit Btk-dependent pathways of platelet aggregation upon GPVI, VWF/GPIb, and FcγRIIA activation. Remibrutinib being more potent and showing a better profile of inhibition of Btk-dependent platelet activation vs. hemostatic impairment than rilzabrutinib may be considered for further development as an antiplatelet drug.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes

AbstractHeparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex–mediated thrombotic disorders.

Apoptosis Signal-Regulating Kinase 1 (ASK1) Is a Novel Regulator of Heparin-Induced Thrombocytopenia and Thrombosis in Mice

Heparin-induced thrombocytopenia and thrombosis (HITT) is a life-threatening (20-30% mortality risk) disease in which IgG antibodies against the heparin-PF4 complex bind and activate platelets via FcγRIIA. Our laboratory has identified that the Apoptosis Signal-Regulating Kinase (ASK1), a MAP3K, is present in both human and murine platelets and potentiates many platelet functions. Given that ASK1 regulates platelet function, and that platelets are known to play a major role in the pathogenesis of HITT, we hypothesized that ASK1 is a novel regulator of HITT.To establish if ASK1 is activated downstream of FcγRIIA, we first stimulated washed human platelets with anti-CD9 (700ng/mL). Anti-CD9 induces the activation of FcγRIIA. We found that anti-CD9 induced a robust activation of ASK1, as measured by phosphorylation of ASK1 Thr845 (a marker of ASK1's kinase activity). ASK1 exclusively activates p38 in platelets; therefore we also measured phosphorylation of p38 a marker of ASK1's signaling activity. We found that anti-CD9 also induced a robust phosphorylation of p38.To determine the role ASK1 plays in platelet-FcγRIIA signaling and HITT, we crossed Ask1-/- mice to FcγRIIA+/+(hFcR) mice. We found that genetic ablation of Ask1 did not have any effect on anti-CD9-induced activation of PLCγ2 or Syk in hFcR/ Ask1-/- (KO) platelets compared to hFcR/ Ask1+/+ (WT). However, loss of Ask1 did result in the complete absence of p38 activation in KO platelets following activation of FcγRIIA by anti-CD9 (500ng/mL). Further, we observed that anti-CD9-induced integrin αIIbβ3 activation, α-granule secretion, and platelet aggregation were all significantly attenuated in KO platelets (P<0.01). These in vitro results strongly suggested that ASK1 plays a prominent role in FcγRIIA-mediated platelet activation.To further investigate the role of ASK1 in HITT pathogenesis, WT and KO mice were subjected to an in vivo model of HITT by injecting anti-CD9 IgG. Platelet counts were measured in samples of whole blood collected at various time points post-injection, and compared to resting platelet counts taken before HITT was initiated. We found that KO mice were significantly (P<0.01) protected from thrombocytopenia compared to WT mice when injected with 250μg/kg anti-CD9. However, when injected with 500μg/kg anti-CD9, both WT and KO mice displayed the same level of thrombocytopenia. Despite not protecting from thrombocytopenia, when observed for signs of shock, we found that KO mice were significantly (P<0.01) protected from anti-CD9 induced shock compared to WT mice regardless of the dose.Taken together these in vitro and in vivo data strongly suggest that ASK1 regulates FcγRIIA-mediated platelet activation, and that ASK1 plays a key role in the pathogenesis of HITT. Furthermore, these data highlight the much-overlooked ASK1/p38 signaling pathway as key mediator of platelet activation during HITT pathogenesis. ASK1 may be a potential target for the development of novel drugs to treat HITT. DisclosuresNo relevant conflicts of interest to declare.

Bruton’s Tyrosine Kinase Inhibitors Impair Fcγriia-Mediated Responses of Healthy Donor Platelets and Chronic Lymphocytic Leukaemia Derived Platelets to Bacteria

Platelets play a key role in innate immunity and can interact with bacteria through multiple molecular mechanisms. One key receptor for platelet-bacteria interactions is FcγRIIa, a low affinity IgG immune receptor found on the surface of platelets, which is associated with platelet aggregation, phagocytosis and the release of bactericidal substances and cytokines from alpha and dense granules. The signalling pathways that regulate FcγRIIa mediated platelet responses to bacteria are not fully understood. Downstream of the FcγRIIa receptor is the protein Bruton's tyrosine kinase (Btk) and potentially other Tec family kinases, yet the role of these kinases in platelet-bacteria interactions is unknown. Btk is a therapeutic target in chronic lymphocytic leukaemia (CLL) with inhibitors of Btk (iBtks) including ibrutinib and acalabrutinib. However, iBtks have off-target effects on platelets, inhibiting aggregation with associated haemorrhagic side effects. iBtk treatment is also associated with a higher incidence of infection in CLL patients. The effect of iBtks on platelet immune function has not been evaluated. We hypothesise that Btk has a role in platelet FcγRIIa signalling in response to bacterial agonists, and that iBtks inhibit such responses, contributing towards the increased risk of infection seen with such therapies in CLL. We show that ibrutinib and acalabrutinib inhibit healthy donor FcγRIIa-mediated platelet aggregation, alpha and dense granule release in response to incubation with Staphylococcus aureus and Escherichia coli, and also in response to FcγRIIa crosslinking with the monoclonal antibody IV.3 (anti-FcγRIIa). The observed lack of granule secretion will reduce the bactericidal substances released by the platelet, as well as the amount of cytokine and chemokine secretion limiting cross-talk with other immune cells. Phosphorylation of Btk at tyrosine 223 (a marker of Btk activation) was detected in response to FcγRIIa agonists, and was inhibited by both ibrutinib and acalabrutinib. Little is known about the effect of iBtk treatment on CLL-platelet responses to bacteria. CLL patients tend to be on concurrent medications for comorbid conditions that could alter platelet responses. We show that treatment-naïve and ibrutinib-treated CLL platelets have aggregation and alpha granule release to thrombin receptor activator peptide 6 and ADP comparable to healthy controls, indicating that CLL platelet responses to these FcγRIIa-independent agonists are normal. Moreover, platelets derived from iBtk naïve CLL patients aggregate normally to bacteria in the presence of autologous plasma. However, platelets from ibrutinib-treated CLL patients have significantly inhibited aggregation and alpha granule release in response to S.aureus, E.coli, and IV.3 crosslinking. Moreover, Btk is phosphorylated at Y223 in response to bacterial agonists in treatment-naïve, but not in ibrutinib-treated CLL platelets, highlighting the lack of Btk activation in iBtk-treated patients. An X-linked agammaglobulinaemia (XLA) patient with a known Btk loss of function mutation was examined to investigate if Btk is vital for the FcγRIIa pathway. XLA-derived platelets aggregated normally in response to multiple bacterial species, suggesting Btk is redundant in mediating platelet aggregatory responses to bacteria. These results suggest that other Tec family kinases might have a role in platelet FcγRIIa activation to bacteria, which could be affected by iBtk treatment too. In conclusion, we propose that iBtks impair the FcγRIIa pathway in platelets, reducing platelet-bacteria responses, possibly contributing to the increased risk of infections in observed in CLL. Disclosures No relevant conflicts of interest to declare.

Fibrillar amyloid peptides promote platelet aggregation through the coordinated action of ITAM‐ and ROS‐dependent pathways

BackgroundAmyloid peptides Aβ40 and Aβ42, whose deposition in brain correlates with Alzheimer disease, are also present in platelets and have prothrombotic activities. ObjectiveIn this study, we analyze the ability of Aβ peptides to form fibrils and to induce platelet activation and aggregation. MethodsAβ40, Aβ42, and their scrambled peptides were diluted in phosphate buffered saline and fibrillogenesis was investigated by ThioflavinT and Congo Red. Aggregation, protein phosphorylation, and reactive oxygen species (ROS) production were analyzed. ResultsAβ40 and Aβ42, but not scrambled peptides, were able to form fibrils when diluted in phosphate buffered saline. Fibrillogenesis of Aβ42 was very rapid, whereas fibril formation by Aβ40 was completed only after 48 hours of incubation. Fibrillar Aβ40 and Aβ42 promoted dose‐dependent aggregation of washed platelets in the presence of extracellular CaCl2. Cleavage of GPIbα by mocarhagin or blockade of the ITAM‐containing FcγRIIA prevented platelet aggregation induced by fibrillary Aβ40 and Aβ42. Fibrillar Aβ peptides stimulated the phosphorylation of FcγRIIA, resulting in the downstream stimulation of PLC, protein kinase C, and phosphoinositide 3‐kinases, whose activity was necessary for full aggregation of platelets. Fibrillar Aβ peptides also induced ROS generation, and NOX inhibitors, as well as ROS scavengers, prevented platelet aggregation. However, Aβ peptide‐induced ROS production did not require binding to GPIbα or activation of FcγRIIA, but was initiated by CD36, which provided an important contribution to full platelet aggregation. ConclusionThese results suggest that fibrillar amyloid Aβ40 and Aβ42 induce platelet aggregation through the recruitment of GPIb‐IX‐V and CD36, which requires the convergence of ITAM‐ and ROS‐dependent pathways.

Oral Bruton tyrosine kinase inhibitors block activation of the platelet Fc receptor CD32a (FcγRIIA): a new option in HIT?

AbstractActivation of the platelet Fc-receptor CD32a (FcγRIIA) is an early and crucial step in the pathogenesis of heparin-induced thrombocytopenia type II (HIT) that has not been therapeutically targeted. Downstream FcγRIIA Bruton tyrosine kinase (BTK) is activated; however, its role in Fc receptor–induced platelet activation is unknown. We explored the potential to prevent FcγRIIA-induced platelet activation by BTK inhibitors (BTKi's) approved (ibrutinib, acalabrutinib) or in clinical trials (zanubrutinib [BGB-3111] and tirabrutinib [ONO/GS-4059]) for B-cell malignancies, or in trials for autoimmune diseases (evobrutinib, fenebrutinib [GDC-0853]). We found that all BTKi's blocked platelet activation in blood after FcγRIIA stimulation by antibody-mediated cross-linking (inducing platelet aggregation and secretion) or anti-CD9 antibody (inducing platelet aggregation only). The concentrations that inhibit 50% (IC50) of FcγRIIA cross-linking–induced platelet aggregation were for the irreversible BTKi's ibrutinib 0.08 µM, zanubrutinib 0.11 µM, acalabrutinib 0.38 µM, tirabrutinib 0.42 µM, evobrutinib 1.13 µM, and for the reversible BTKi fenebrutinib 0.011 µM. IC50 values for ibrutinib and acalabrutinib were four- to fivefold lower than the drug plasma concentrations in patients treated for B-cell malignancies. The BTKi's also suppressed adenosine triphosphate secretion, P-selectin expression, and platelet-neutrophil complex formation after FcγRIIA cross-linking. Moreover, platelet aggregation in donor blood stimulated by sera from HIT patients was blocked by BTKi's. A single oral intake of ibrutinib (280 mg) was sufficient for a rapid and sustained suppression of platelet FcγRIIA activation. Platelet aggregation by adenosine 5′-diphosphate, arachidonic acid, or thrombin receptor-activating peptide was not inhibited. Thus, irreversible and reversible BTKi's potently inhibit platelet activation by FcγRIIA in blood. This new rationale deserves testing in patients with HIT.

IPLA2β Contributes to Activation of Human and Mouse Platelets Via ITAM Receptors Fcγ RIIA and GPVI

Platelet activation by ITAM receptors contributes to hemostasis, thrombosis, vascular integrity and host defense. In the course of our studies of FcγRIIA-mediated platelet activation, we became interested in those mechanisms that require neither full Syk activation nor changes in intracellular calcium. Calcium-independent phospholipase family member iPLA2β has been observed to modulate phospholipid remodeling and second messenger generation in human platelets (Beckett, Thromb Res 2007; Duvernay, Biochem 2015), while iPLA2γ has been studied in knockout mouse platelets (Yoda, JTH 2014) with modest effects noted on thrombosis and hemostasis. These enzymes do not require increased cytoplasmic calcium for their activity in cleaving the acyl group from the sn2 position of phospholipids to yield a free fatty acid and a lysophospholipid. However, the precise role of iPLA2β in human and mouse platelet activation has not been elucidated. Neither has the contribution of iPLA2β in the response to FcγRIIA-mediated activation been reported. We identified the presence of iPLA2β protein in western blots of human and FcγRIIA transgenic mouse platelets. Of interest, multiple isoforms arising from proteolytic cleavage were detected. We treated washed human and FcγRIIA transgenic mouse platelets with agonists to FcγRIIA (IV.3 + GAM) and to GPVI (collagen or collagen-related peptide) in the absence and presence of pharmacologic inhibitors of iPLA2β. At a range of agonist doses up to 3X threshold, we observed significant inhibition of aggregation, dense granule secretion and alpha granule secretion (p&lt;0.05 vs. vehicle only, n = 3 to 4 each). Inhibition occurred with either S-BEL (bromo-enolactone) or with FKGK18 (a fluoroketone), two chemically distinct iPLA2β inhibitor molecules with different modes of action. The IC50 for S-BEL was found to be 1.02 uM for human FcγRIIA, 2.04 uM for human GPVI, and 2.76 uM for transgenic mouse FcγRIIA activated platelets. FKGK18 was less potent, with IC50s at 7.88 uM for human FcγRIIA. In contrast, iPLA2γ inhibitor R-BEL was able to inhibit FcγRIIA -mediated activation, but at an IC50 of 2.62 uM. Notably, iPLA2β inhibition could eliminate ATP secretion from dense granules downstream of FcγRIIA and GPVI activation. When we added ADP to FcγRIIA stimulation in the presence of inhibitory doses of S-BEL, we overcame the inhibition. We have identified for the first time that iPLA2β contributes to aggregation and secretion of both human and FcγRIIA transgenic mouse platelets. The platelets were slightly more sensitive to FcγRIIA than to GPVI inhibition. In other activatable secretory cells, iPLA2β plays both a homeostatic and signaling role. The mechanisms of iPLA2β action in platelets merit further study. Studies are in progress with genetic knockdown and knockout of the enzyme, to complement the findings with inhibitors. Disclosures No relevant conflicts of interest to declare.

Humanised effector-null FcγRIIA antibody inhibits immune complex-mediated proinflammatory responses

ObjectiveImmune complexes (ICs) play a critical role in the pathology of autoimmune diseases. The aim of this study was to generate and characterise a first-in-class anti-FcγRIIA antibody (Ab) VIB9600 (previously...

P2X1 Receptors Amplify FcγRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets.

Platelets express key receptors of the innate immune system such as FcγRIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP)VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent FcγRIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of FcγRIIa in human platelets, following stimulation by cross-linking of an anti-FcγRIIa monoclonal antibody (mAb) IV.3, or bacterial stimulation with Streptococcus sanguinis . Activation was assessed in washed platelet suspensions via measurement of intracellular Ca 2+ ([Ca 2+ ] i ) increases, ATP release and aggregation. P2X1 activity was abolished by pre-addition of α,β-meATP, exclusion of apyrase or the antagonist NF449. FcγRIIa activation evoked a robust increase in [Ca 2+ ] i (441 ± 33 nM at 30 μg/mL mAb), which was reduced to a similar extent (to 66–70% of control) by NF449, pre-exposure to α,β-meATP or apyrase omission, demonstrating a significant P2X1 receptor contribution. FcγRIIa activation-dependent P2X1 responses were partially resistant to nitric oxide (NO), but abrogated by 500 nM prostacyclin (PGI 2 ). Aggregation responses to bacteria and FcγRIIa activation were also inhibited by P2X1 receptor desensitization (to 66 and 42% of control, respectively). However, FcγRIIa-mediated tyrosine phosphorylation and ATP release were not significantly altered by the loss of P2X1 activity. In conclusion, we show that P2X1 receptors enhance platelet FcγRIIa receptor-evoked aggregation through an increase in [Ca 2+ ] i downstream of the initial tyrosine phosphorylation events and early dense granule release. This represents a further route whereby ATP-gated cation channels can contribute to platelet-dependent immune responses in vivo.

TULA-2 Inhibits the Platelet FcγRIIA-Syk Pathway and Heparin-Induced Thrombocytopenia in Mice

Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies against the heparin-PF4 complex activate platelets via FcγRIIA. We previously reported that TULA-2 serves as a negative regulator of FcγRIIA pathway by dephosphorylating Syk in HEL cells. To further investigate the effect of TULA-2 on the FcγRIIA pathway and HIT, we crossed TULA-2-/- with FcγRIIA+/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of Syk, LAT, and PLCγ2 in platelets after FcγRIIA activation. Integrin activation, calcium mobilization, and P-selectin exposure were also enhanced in TULA-2-/- murine platelets compared to TULA-2+/+. Further, anti-GPIX antibody-induced HIT-like thrombocytopenia and thrombin generation were also augmented in TULA-2-/- mice (Figure A). We also found that decreased TULA-2 level shortened tail-bleeding time in mice (Figure B), suggesting a role of TULA-2 in physiological hemostasis.Additionally, TULA-2 KO and WT platelets did not show significant differences in platelet spreading and clot retraction, indicating that outside-in signaling is not affected by the absence of TULA-2. At the protein level, TULA-2 heterozygous knockout (TULA-2+/-) platelets express 50% as much protein as their wildtype counterparts. Interestingly, TULA-2+/- mice showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together the data indicate that not only the absence of TULA-2, but also the relative level of TULA-2 expression modulate FcγRIIA-mediated platelet reactivity, HIT pathogenesis, and hemostasis. Considering that TULA-2 is also a key regulator in platelet GPVI pathway, measuring TULA-2 expression may be a valuable predictor for HIT susceptibility and platelet reactivity in general. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Human platelet activation by Escherichia coli: roles for FcγRIIA and integrin αIIbβ3

Gram-negative Escherichia coli cause diseases such as sepsis and hemolytic uremic syndrome in which thrombotic disorders can be found. Direct platelet–bacterium interactions might contribute to some of these conditions; however, mechanisms of human platelet activation by E. coli leading to thrombus formation are poorly understood. While the IgG receptor FcγRIIA has a key role in platelet response to various Gram-positive species, its role in activation to Gram-negative bacteria is poorly defined. This study aimed to investigate the molecular mechanisms of human platelet activation by E. coli, including the potential role of FcγRIIA. Using light-transmission aggregometry, measurements of ATP release and tyrosine-phosphorylation, we investigated the ability of two E. coli clinical isolates to activate platelets in plasma, in the presence or absence of specific receptors and signaling inhibitors. Aggregation assays with washed platelets supplemented with IgGs were performed to evaluate the requirement of this plasma component in activation. We found a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation in response to E. coli. IgG and αIIbβ3 engagement was required for FcγRIIA activation. Moreover, feedback mediators adenosine 5’-diphosphate (ADP) and thromboxane A2 (TxA2) were essential for platelet aggregation. These findings suggest that human platelet responses to E. coli isolates are similar to those induced by Gram-positive organisms. Our observations support the existence of a central FcγRIIA-mediated pathway by which human platelets respond to both Gram-negative and Gram-positive bacteria.

Anti–miR-148a regulates platelet FcγRIIA signaling and decreases thrombosis in vivo in mice

AbstractFc receptor for IgG IIA (FcγRIIA)–mediated platelet activation is essential in heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. There is considerable interindividual variation in platelet FcγRIIA activation, the reasons for which remain unclear. We hypothesized that genetic variations between FcγRIIA hyper- and hyporesponders regulate FcγRIIA-mediated platelet reactivity and influence HIT susceptibility. Using unbiased genome-wide expression profiling, we observed that human hyporesponders to FcγRIIA activation showed higher platelet T-cell ubiquitin ligand-2 (TULA-2) mRNA expression than hyperresponders. Silent interfering RNA-mediated knockdown of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase following FcγRIIA activation in HEL cells. Significantly, we found miR-148a-3p targeted and inhibited both human and mouse TULA-2 mRNA. Inhibition of miR-148a in FcγRIIA transgenic mice upregulated the TULA-2 level and reduced FcγRIIA- and glycoprotein VI–mediated platelet αIIbβ3 activation and calcium mobilization. Anti–miR-148a also reduced thrombus formation following intravascular platelet activation via FcγRIIA. These results show that TULA-2 is a target of miR-148a-3p, and TULA-2 serves as a negative regulator of FcγRIIA-mediated platelet activation. This is also the first study to show the effects of in vivo miRNA inhibition on platelet reactivity. Our work suggests that modulating miR-148a expression is a potential therapeutic approach for thrombosis.

Mir-148a and Mir-25 Target TULA-2 and Regulate Human Platelet FcγRIIA Signaling

FcγRIIA-mediated platelet activation is important in the pathogenesis of heparin-induced thrombocytopenia (HIT) and other immune-mediated thrombocytopenia and thrombosis disorders. FcgRIIa is also the major transmembrane signaling adapter for the human platelet fibrinogen receptor, integrin aIIbb3. In HIT, antibody to PF4/heparin is necessary but not sufficient for the disease. There is considerable inter-individual variation in platelet FcγRIIA activation; the reasons remain unclear. To identify potential regulators in the FcγRIIA pathway, we determined the platelet RNA expression profile in 154 healthy human donors who were phenotyped for platelet aggregation via FcgRIIA. 87 mRNAs and 4 DE miRNAs were identified as differentially expressed (DE) between hyper- and hypo-responsive donors by microarray and Nanostring analysis, respectively. Among them, hyper-responders show significantly lower T-cell ubiquitin ligand-2 (TULA-2) mRNA level and higher miR-148a-3p and miR-25-3p levels than hyporesponders. TULA-2, also known as UBASH3B, is a known protein tyrosine phosphatase for Syk, and thus a biologically plausible regulator of platelet FcgRIIA.Both miR-148a-3p and miR-25-3p are predicted to target the 3’UTR region on TULA-2 mRNA (Fig., A). Overexpression of miR-148a-3p or miR-25-3p results in down-regulation of the TULA-2 protein level in HCT116 cells and HEL cells (Fig., B). Inhibition of miR-148a-3p by anti-miR (LNA) leads to elevated TULA-2 expression. A luciferase construct with the 3’UTR miR binding sites was generated. A decrease in luciferase activity was observed in assays with co-transfection of the construct and a miR, supporting the binding between miR-148a-3p and miR-25-3p with TULA-2 3’UTR (Fig., C). Finally, we show that knockdown of TULA-2 caused increased Syk-pY (Y323) level in HEL megakaryocytic cell line in response to FcγRIIA crosslinking by IV.3 (mAb against FcγRIIA) + GAM (Goat anti-mouse F(ab)’2)(Fig., D). These results for the first time show that TULA-2 serves as a negative regulator of the FcγRIIA pathway and that miR-25 and miR-148a may contribute to HIT susceptibility. Studies are in progress for the translation of these findings in vivo. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Amplification of bacteria-induced platelet activation is triggered by FcγRIIA, integrin αIIbβ3, and platelet factor 4

AbstractBacterial adhesion to platelets is mediated via a range of strain-specific bacterial surface proteins that bind to a variety of platelet receptors. It is unclear how these interactions lead to platelet activation. We demonstrate a critical role for the immune receptor FcγRIIA, αIIbβ3, and Src and Syk tyrosine kinases in platelet activation by Staphylococcus aureus, Streptococcus sanguinis, Streptococcus gordonii, Streptococcus oralis, and Streptococcus pneumoniae. FcγRIIA activation is dependent on immunoglobulin G (IgG) and αIIbβ3 engagement. Moreover, feedback agonists adenosine 5′-diphosphate and thromboxane A2 are mandatory for platelet aggregation. Additionally, platelet factor 4 (PF4) binds to bacteria and reduces the lag time for aggregation, and gray platelet syndrome α-granule–deficient platelets do not aggregate to 4 of 5 bacterial strains. We propose that FcγRIIA-mediated activation is a common response mechanism used against a wide range of bacteria, and that release of secondary mediators and PF4 serve as a positive feedback mechanism for activation through an IgG-dependent pathway.

Human cytomegalovirus Fcγ binding proteins gp34 and gp68 antagonize Fcγ receptors I, II and III.

Human cytomegalovirus (HCMV) establishes lifelong infection with recurrent episodes of virus production and shedding despite the presence of adaptive immunological memory responses including HCMV immune immunoglobulin G (IgG). Very little is known how HCMV evades from humoral and cellular IgG-dependent immune responses, the latter being executed by cells expressing surface receptors for the Fc domain of IgG (FcγRs). Remarkably, HCMV expresses the RL11-encoded gp34 and UL119-118-encoded gp68 type I transmembrane glycoproteins which bind Fcγ with nanomolar affinity. Using a newly developed FcγR activation assay, we tested if the HCMV-encoded Fcγ binding proteins (HCMV FcγRs) interfere with individual host FcγRs. In absence of gp34 or/and gp68, HCMV elicited a much stronger activation of FcγRIIIA/CD16, FcγRIIA/CD32A and FcγRI/CD64 by polyclonal HCMV-immune IgG as compared to wildtype HCMV. gp34 and gp68 co-expression culminates in the late phase of HCMV replication coinciding with the emergence of surface HCMV antigens triggering FcγRIII/CD16 responses by polyclonal HCMV-immune IgG. The gp34- and gp68-dependent inhibition of HCMV immune IgG was fully reproduced when testing the activation of primary human NK cells. Their broad antagonistic function towards FcγRIIIA, FcγRIIA and FcγRI activation was also recapitulated in a gain-of-function approach based on humanized monoclonal antibodies (trastuzumab, rituximab) and isotypes of different IgG subclasses. Surface immune-precipitation showed that both HCMV-encoded Fcγ binding proteins have the capacity to bind trastuzumab antibody-HER2 antigen complexes demonstrating simultaneous linkage of immune IgG with antigen and the HCMV inhibitors on the plasma membrane. Our studies reveal a novel strategy by which viral FcγRs can compete for immune complexes against various Fc receptors on immune cells, dampening their activation and antiviral immunity.

Abstract 57: 12-Lipoxygenase is a Critical Enzyme Regulating FcyRIIa-Mediated Platelet Activation

Introduction: FcγRIIa plays a key role in platelet activation mediated by immune complexes containing IgG and platelet factor 4 (PF4) bound to heparin. One of the primary mechanisms by which FcγRIIa mediates it effects in the platelet is through its ITAM motif. 12-lipoxygenase (12-LOX) has recently been shown to play an important role in regulation of platelet function through GPVI, which also signals in part through an ITAM motif. We therefore hypothesize that 12-LOX can play an important role in regulation of FcγRIIa-mediated platelet activation resulting in aggregation and thrombosis. Identifying the importance of this regulatory interaction is essential for understanding the role of 12-LOX in regulation of hemostasis and thrombosis, and identifying a possible target for prevention of FcγRIIa -induced platelet activation. Objectives: Determine the requirement of 12-LOX activation in FcγRIIa -mediated platelet activation and identify the underlying mechanism by which 12-LOX activation regulates FcγRIIa. Methods: FcγRIIa-mediated Platelet aggregation was measured in washed human platelets and mouse platelets in the presence of a 12-LOX inhibitor or in FcγRIIa +/+ mice crossed with 12-LOX -/- mice. PLCγ and PKC activation were measured in the absence of 12-LOX activity. Additionally, Rap1, calcium, and αIIbβ3 activity was measured in the presence or absence of 12-LOX activity following stimulation of FcγRIIa with CD9 or goat anti-mouse IV.3 antibody (IV.3). Results: Both human and FcγRIIa +/+ mouse platelets were able to fully aggregate following stimulation with CD9 or IV.3. Pharmacological inhibition of 12-LOX or absence of 12-LOX significantly delayed and attenuated platelet aggregation. Additionally, PLCγ, calcium, Rap1 and activation of αIIbβ3 were inhibited. Conclusions: These observations support our hypothesis that 12-LOX is an essential enzyme in the regulation of FcγRIIa-mediated platelet activation. Further investigation of this regulatory pathway will help to confirm 12-LOX as a viable target for inhibition of FcγRIIa and other ITAM-mediated platelet activation.

Development of a cell-based assay measuring the activation of FcγRIIa for the characterization of therapeutic monoclonal antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) is one of the important mechanisms of action of the targeting of tumor cells by therapeutic monoclonal antibodies (mAbs). Among the human Fcγ receptors (FcγRs), FcγRIIIa is well known as the only receptor expressed in natural killer (NK) cells, and it plays a pivotal role in ADCC by IgG1-subclass mAbs. In addition, the contributions of FcγRIIa to mAb-mediated cytotoxicity have been reported. FcγRIIa is expressed in myeloid effector cells including neutrophils and macrophages, and it is involved in the activation of these effector cells. However, the measurement of the cytotoxicity via FcγRIIa-expressing effector cells is complicated and inconvenient for the characterization of therapeutic mAbs. Here we report the development of a cell-based assay using a human FcγRIIa-expressing reporter cell line. The FcγRIIa reporter cell assay was able to estimate the activation of FcγRIIa by antigen-bound mAbs by a very simple method in vitro. The usefulness of this assay for evaluating the activity of mAbs with different abilities to activate FcγRIIa was confirmed by the examples including the comparison of the activity of the anti-CD20 mAb rituximab and its Fc-engineered variants, and two anti-EGFR mAbs with different IgG subclasses, cetuximab (IgG1) and panitumumab (IgG2). We also applied this assay to the characterization of a force-oxidized mAb, and we observed that oxidation significantly decreased the FcγRIIa activation by EGFR-bound cetuximab. These results suggest that our FcγRIIa reporter assay is a promising tool for the characterization of therapeutic mAbs, including Fc-engineered mAbs, IgG2-subclass mAbs, and their product-related variants.