The Syk‐kinase inhibitor R406 impairs platelet activation and monocyte tissue factor expression triggered by heparin‐PF4 complex directed antibodies

Summary. Background: Heparin‐induced thrombocytopenia (HIT) is a rare but severe complication of heparin therapy in which immunoglobulin G (IgG) antibodies against the platelet factor 4–heparin complex activate platelets through the FcγRIIA receptor. Clustering of FcγRIIA initiates signaling cascades involving tyrosine kinases including the spleen tyrosine kinase (Syk). Moreover, besides the critical role of platelets, the expression of tissue factor (TF) by human monocytes triggered by HIT antibodies has been shown to contribute to the hypercoagulability and the thrombotic complications in HIT patients. Objectives: We investigated the effect of R406, a small molecule inhibitor of Syk developed as a potential treatment of autoimmune diseases, allergic disorders and B‐cell related hematological malignancies, on FcγRIIA‐mediated platelet activation. To further assess the potential activity of Syk inhibitors in HIT treatment, the effect of R406 was also evaluated on HIT antibodies‐induced expression of TF and procoagulant activity of monocytic cells. Results: We show that R406 is a potent inhibitor of platelet signaling and functions initiated by FcγRIIA cross‐linking by specific antibodies or by sera from HIT patients. Syk inhibition efficiently prevents FcγRIIA‐induced LAT phosphorylation and activation of phosphoinositide 3‐kinase, Akt, phospholipase Cγ2 and p38 MAP‐kinase. As a consequence, FcγRIIA‐induced platelet aggregation, granule secretion and microparticles production are strongly inhibited by R406. Moreover, the Syk inhibitor efficiently impairs the expression of TF and the procoagulant activity of human monocytes triggered by HIT antibodies. Conclusion: Syk inhibitors may be of therapeutic interest in the treatment of HIT by reducing HIT antibodies‐mediated platelet activation and monocyte procoagulant activity.     

Platelet immunoreceptor tyrosine‐based activation motif (ITAM) and hemITAM signaling and vascular integrity in inflammation and development

Platelets are essential for maintaining hemostasis following mechanical injury to the vasculature. Besides this established function, novel roles of platelets are becoming increasingly recognized, which are critical in non‐injury settings to maintain vascular barrier integrity. For example, during embryogenesis platelets act to support the proper separation of blood and lymphatic vessels. This role continues beyond birth, where platelets prevent leakage of blood into the lymphatic vessel network. During the course of inflammation, platelets are necessary to prevent local hemorrhage due to neutrophil diapedesis and disruption of endothelial cell‐cell junctions. Surprisingly, platelets also work to secure tumor‐associated blood vessels, inhibiting excessive vessel permeability and intra‐tumor hemorrhaging. Interestingly, many of these novel platelet functions depend on immunoreceptor tyrosine‐based activation motif (ITAM) signaling but not on signaling via G protein‐coupled receptors, which plays a crucial role in platelet plug formation at sites of mechanical injury. Murine platelets express two ITAM‐containing receptors: the Fc receptor γ‐chain (FcRγ), which functionally associates with the collagen receptor GPVI, and the C‐type lectin‐like 2 (CLEC‐2) receptor, a hemITAM receptor for the mucin‐type glycoprotein podoplanin. Human platelets express an additional ITAM receptor, FcγRIIA. These receptors share common downstream effectors, including Syk, SLP‐76 and PLCγ2. Here we will review the recent literature that highlights a critical role for platelet GPVI/FcRγ and CLEC‐2 in vascular integrity during development and inflammation in mice and discuss the relevance to human disease.     

Platelets kill bacteria by bridging innate and adaptive immunity via platelet factor 4 and FcγRIIA

Essentials

• Human platelets specifically interact with IgG opsonized bacteria through FcγRIIA.
• Platelet factor 4 (PF4) binds to polyanions (P) and undergoes a conformational change.
• Anti‐PF4/P IgG opsonizes PF4‐coated Gram‐positive and Gram‐negative bacteria.
• Platelets specifically kill E.coli opsonized with PF4 and human anti‐PF4/P IgG.

Summary

Background

Activated platelets release the chemokine platelet factor 4 (PF4) stored in their granules. PF4 binds to polyanions (P) on bacteria, undergoes a conformational change and exposes neoepitopes. These neoepitopes induce production of anti‐PF4/P antibodies. As PF4 binds to a variety of bacteria, anti‐PF4/P IgG can bind and opsonize several bacterial species.

Objective

Here we investigated whether platelets are able to kill bacteria directly after recognizing anti‐PF4/P IgG opsonized bacteria in the presence of PF4 via their FcγRIIA.

Methods

Using platelet‐bacteria suspension co‐culture experiments and micropatterns with immobilized viable bacteria, in combination with pharmacological inhibitors and human anti‐ PF4/P IgG we analyzed the role of platelet‐mediated killing of bacteria.

Results

In the presence of PF4, human anti‐PF4/P IgG and platelets, E. coli killing (> 50%) with colony forming units (CFU mL^?1) 0.71 × 10⁴ ± 0.19 was observed compared with controls incubated only with anti‐PF4/P IgG (CFU mL^?1 3.4 × 10⁴ ± 0.38). Blocking of platelet FcγRIIA using mAb IV.3 (CFU mL^?1 2.5 × 10⁴ ± 0.45), or integrin αIIbβ3 (CFU mL^?1 2.26 × 10⁴ ± 0.31), or disruption of cytoskeletal functions (CFU mL^?1 2.7 × 10⁴ ± 0.4) markedly reduced E. coli killing by this mechanism. Our observation of E. coli killing by platelets on micropatterned arrays is compatible with the model that platelets kill bacteria by covering them, actively concentrating them into the area under their granulomere and then releasing antimicrobial substances of platelet α‐granules site directed towards bacteria.

Conclusion

These findings collectively indicate that by bridging of innate and adaptive immune mechanisms, platelets and anti‐PF4/polyanion antibodies cooperate in an antibacterial host response.

     

Determinants of donor platelet variability when testing for heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia is a common immune-mediated drug reaction that can be complicated by life-threatening arterial thrombosis. The diagnosis can be confirmed by demonstrating heparin-dependent release of radiolabeled serotonin from washed normal platelets in the presence of patient serum. However, certain serum samples from these patients produce 14C-serotonin release from some but not other normal donor platelets. We investigated this problem of donor platelet variability by studying the reactivities of 10 serum samples from patients with heparin-induced thrombocytopenia with platelets from 10 normal donors (100 serum and platelet combinations). We observed a marked variability in reactivity for patient serum and platelets from normal donors; this initially appeared random. However, closer examination indicated that the reactivities varied hierarchically. Because heparin-induced thrombocytopenia is caused by binding of heparin-dependent IgG to platelet Fc receptors, we examined whether platelet Fc receptor number or function explained the variability in platelet reactivity. We observed that platelet Fc receptor function, as measured by platelet release associated with heat-aggregated IgG, was highly correlated with platelet reactivity to heparin-induced thrombocytopenia serum samples. No significant correlation, however, was found between Fc receptor number and platelet response. Reaction of murine monoclonal antibodies that activate human platelets by means of the platelet Fc receptors was not predictive of platelet reactivity to heparin-induced thrombocytopenia serum.(ABSTRACT TRUNCATED AT 250 WORDS)     

Platelet FcγRIIA HIS131ARG polymorphism and platelet function: antibodies to platelet-bound fibrinogen induce platelet activation

Summary.  The His131Arg polymorphism of platelet FcγRIIA affects the binding affinity of certain IgG subclasses. The Arg131 allele has been associated with (auto)immune thrombocytopenia and heparin-induced thrombocytopenia in some studies. Because FcγRIIA can transmit platelet activation signals, we studied platelet responsiveness from 73 healthy donors to determine if this polymorphism modulated platelet function. Platelet function was studied by agonist and shear-induced activation, and standard aggregation. FcγRIIA was genotyped by allele-specific PCR. Compared with His131, the Arg131 allele was associated with significantly greater binding of activation-dependent antibodies. This effect was most prominent for the receptor-induced binding site (RIBS) antibodies F26 ( P  < 0.0001) and RIBS1 ( P  = 0.0057), and the ligand-induced binding site antibody LIBS1 ( P  = 0.0367). Unexpectedly, Arg131-positive platelets did not show greater fibrinogen binding, platelet aggregation or shear-induced platelet activation. We considered whether enhanced Fc binding and FcγRIIA cross-linking were responsible for those discrepancies. The increased binding of the two RIBS antibodies to the Arg131 isoform was abolished by blocking FcγRIIA, and the FcγRIIA genotype effect on F26 IgG binding was lost when F26 F(ab')₂ fragments were used. Furthermore, intact F26 and RIBS1 IgG directly and specifically induced P-selectin expression, and this effect was greatest in Arg131-positive platelets. We concluded that (a) the His131Arg polymorphism of FcγRIIA does not affect intrinsic platelet reactivity; (b) RIBS antibodies are able to cross-link FcγRIIA and activate platelets, and this activation has a modest effect on Arg131 platelets; and (c) flow cytometric based platelet assays may need to compensate for this FcγRIIA His131Arg effect on platelet activation.     

Simultaneous engagement of thrombin and Fc gamma RIIA receptors results in platelets expressing high levels of procoagulant proteins.

Collagen and thrombin-activated (COAT) platelets represent a unique subset of activated platelets that exhibit high levels of several adhesive and procoagulant alpha-granule proteins on their surface. In this report we demonstrate that a similar subpopulation of platelets can also be generated by the combined stimulation of Fc gamma RIIA and thrombin receptors. Platelets activated in this manner are referred to as Fc receptor and thrombin-activated (FcRT) platelets, and they share many of the characteristics of the formerly observed COAT platelets, including aminophospholipid exposure, adhesive and procoagulant protein enrichment, increased frequency among young platelets, and sensitivity to transglutaminase inhibitors. Although Fc gamma RIIA receptor activation can be achieved either with anti-CD9 monoclonal antibodies (ALB-6 and ML-13) or with direct Fc receptor cross-linking, FcRT platelet generation occurs only with concurrent or slightly delayed thrombin stimulation. In fact, when thrombin was the second agonist, time delays of up to 120 seconds after Fc gamma RIIA receptor stimulation had little effect on the generation of FcRT platelets; however, a similar delay for convulxin plus thrombin activation results in a 90% diminution in COAT platelet production. FcRT platelet formation in platelet-poor plasma and whole blood was also investigated, and results were similar to those observed with gel-filtered platelets. Previous experiments with COAT platelet formation used physiologic agonists (collagen and thrombin) that might be encountered under either physiologic or pathologic conditions; however, the current experiments with Fc receptor stimulation offer the first example in which these highly prohemostatic platelets are likely to be strictly pathogenic.     

An αIIbβ3 antagonist prevents thrombosis without causing Fc receptor γ‐chain IIa‐mediated thrombocytopenia

Essentials

• FcγRIIa‐mediated thrombocytopenia is associated with drug‐dependent antibodies (DDAbs).
• We investigated the correlation between α_IIbβ₃ binding epitopes and induction of DDAbs.
• An FcγRIIa‐transgenic mouse model was used to evaluate thrombocytopenia among anti‐thrombotics.
• An antithrombotic with binding motif toward α_IIbβ‐propeller domain has less bleeding tendency.

Summary

Background

Thrombocytopenia, a common side effect of Arg‐Gly‐Asp‐mimetic antiplatelet drugs, is associated with drug‐dependent antibodies (DDAbs) that recognize conformation‐altered integrin α_IIbβ₃.

Objective

To explore the correlation between α_IIbβ₃ binding epitopes and induction of DDAb binding to conformation‐altered α_IIbβ₃, we examined whether two purified disintegrins, TMV‐2 and TMV‐7, with distinct binding motifs have different effects on induction of α_IIbβ₃ conformational change and platelet aggregation in the presence of AP2, an IgG₁ inhibitory mAb raised against α_IIbβ₃.

Methods

We investigated the possible mechanisms of intrinsic platelet activation of TMV‐2 and TMV‐7 in the presence of AP2 by examining the signal cascade, tail bleeding time and immune thrombocytopenia in Fc receptor γ‐chain IIa (FcγRIIa) transgenic mice.

Results

TMV‐7 has a binding motif that recognizes the α_IIb β‐propeller domain of α_IIbβ₃, unlike that of TMV‐2. TMV‐7 neither primed the platelets to bind ligand, nor caused a conformational change of α_IIbβ₃ as identified with the ligand‐induced binding site mAb AP5. In contrast to eptifibatide and TMV‐2, cotreatment of TMV‐7 with AP2 did not induce FcγRIIa‐mediated platelet aggregation and the downstream activation cascade. Both TMV‐2 and TMV‐7 efficaciously prevented occlusive thrombosis in vivo. Notably, both eptifibatide and TMV‐2 caused severe thrombocytopenia mediated by FcγRIIa, prolonged tail bleeding time in vivo, and repressed human whole blood coagulation indexes, whereas TMV‐7 did not impair hemostatic capacity.

Conclusions

TMV‐7 shows antiplatelet and antithrombotic activities resulting from a mechanism different from that of all other tested α_IIbβ₃ antagonists, and may offer advantages as a therapeutic agent with a better safety profile.     

Allelic polymorphisms of human Fc gamma receptor IIA and Fc gamma receptor IIIB. Independent mechanisms for differences in human phagocyte function.

Two different allelic polymorphisms among the isoforms of human Fc gamma receptors have been defined: the low-responder (LR)-high-responder (HR) polymorphism of huFc gamma RIIA expressed on both PMN and monocytes and the NA1-NA2 polymorphism of the neutrophil Fc gamma RIII (huFc gamma RIIIB). To address the issues of whether the LR-HR polymorphism has a significant impact on Fc gamma R-mediated functions in human blood cells and whether any differences in LR-HR might be related to higher Fc gamma R-mediated phagocytosis in NA1 donors, we examined Fc gamma R-specific binding and internalization by donors homozygous for the two huFc gamma RIIA alleles. PMN from LR homozygotes showed consistently higher levels of internalization of erythrocytes opsonized with pooled human IgG (E-hIgG). The absence of an LR-HR phagocytic difference with erythrocytes opsonized with either anti-Fc gamma RIIA MAb IV.3 or rabbit IgG, as opposed to E-hIgG, suggested that the Fc piece of the opsonin might be important for this LR-HR difference. Accordingly, we studied HR and LR homozygotes with human IgG subclass-specific probes. Both PMN (independent of huFc gamma RIIIB phenotype) and monocytes from LR donors bound and internalized erythrocytes coated with human IgG2 (E-hIgG2) efficiently, whereas phagocytes from HR donors did so poorly. E-hIgG2 internalization was completely abrogated by blockade of the ligand binding site of huFc gamma RIIA with IV.3 Fab, indicating that huFc gamma RIIA is essential for the handling of hIgG2 and that the mechanism of the LR-HR phagocytic difference is at the level of ligand binding to huFc gamma RIIA. In contrast, the difference in internalization of E-hIgG between NA1 and NA2 homozygous donors was independent of the huFc gamma RIIA phenotype and did not manifest differences in ligand binding. Thus, the two known allelic polymorphisms of human Fc gamma R have distinct and independent mechanisms for altering receptor function, which may influence host defense and immune complex handling.     

Therapeutic plasma exchange (TPE) as a plausible rescue therapy in severe vaccine-induced immune thrombotic thrombocytopenia

Vaccine-induced immune thrombotic thrombocytopenia (VITT) is associated with high titers of immunoglobulin G class antibodies directed against the cationic platelet chemokine platelet factor 4 (PF4). These antibodies activate platelets via FcγIIa receptors. VITT closely resembles heparin-induced thrombocytopenia. Inflammation and tissue trauma substantially increase the risk for forming pathogenic PF4 antibodies. We therefore propose the use of therapeutic plasma exchange as rescue therapy in VITT to deplete antibodies plus factors promoting inflammation such as excess cytokines in the circulation as well as extracellular vesicles derived from activated platelets.     

Platelet Fc Receptor: INCREASED EXPRESSION IN MYELOPROLIFERATIVE DISEASE

The platelet Fc receptor, a membrane receptor for immune complexes or aggregated immunoglobulin (Ig)G, was compared in normal and myeloproliferative platelets. Washed platelets from 11 normal donors and 27 patients were incubated with fluorescein-conjugated ovalbumin-anti-ovalbumin complexes and examined by phase and fluorescence microscopy. Only 3.2±1% of the normal platelets stained, whereas 76±16% of the myeloproliferative platelets stained with the immune complex. The fluorescent staining was mediated by a platelet Fc receptor, as shown by the absence of platelet staining with immune complex containing antibody preincubated with Staphylococcal protein A to block the Fc region. In addition, no staining occurred with antigen or antibody alone or after preincubation of platelets with aggregated IgG. Platelets from normal or myeloproliferative donors did not stain with the immune complexes when the incubation was performed in plasma. The increased expression of Fc receptors on myeloproliferative platelets was corroborated by studies of [¹⁴C]serotonin release by immune complexes or aggregated IgG in 8 patients and 17 normal donors. Serotonin uptake was similar in both groups. Myeloproliferative platelets released significantly more serotonin than normal platelets at each concentration of immune complex or aggregated IgG; in addition, myeloproliferative platelets released serotonin in response to much smaller concentrations of immune complex or aggregated IgG. [¹⁴C]Serotonin release by myeloproliferative platelets was not increased above that of normal platelets when thrombin was used as the stimulus. The results were independent of patient age, sex, therapy, hematocrit, or platelet size. Interaction of circulating immune complexes with platelets bearing increased Fc receptors may contribute to the abnormal hemostasis associated with the myeloproliferative syndromes.     

Bevacizumab immune complexes activate platelets and induce thrombosis in FCGR2A transgenic mice

Summary.  Background:  Treatment with Bevacizumab has been associated with arterial thromboembolism in colorectal cancer patients. However, the mechanism of this remains poorly understood, and preclinical testing in mice failed to predict thrombosis. Objective:  We investigated whether thrombosis might be the result of platelet activation mediated via the FcγRIIa (IgG) receptor – which is not present on mouse platelets – and aimed to identify the functional roles of heparin and platelet surface localization in Bev-induced FcγRIIa activation. Methods and results:  We found that Bev immune complexes (IC) activate platelets via FcγRIIa, and therefore attempted to reproduce this finding in vivo using FcγRIIa (hFcR) transgenic mice. Bev IC were shown to be thrombotic in hFcR mice in the presence of heparin. This activity required the heparin-binding domain of Bev's target, vascular endothelial growth factor (VEGF). Heparin promoted Bev IC deposition on to platelets in a mechanism similar to that observed with antibodies from patients with heparin-induced thrombocytopenia. When sub-active amounts of ADP or thrombin were used to prime platelets (simulating hypercoagulability in patients), Bev IC-induced dense granule release was significantly potentiated, and much lower (sub-therapeutic) heparin concentrations were sufficient for Bev IC-induced platelet aggregation. Conclusions:  The prevailing rationale for thrombosis in Bev therapy is that VEGF blockade leads to vascular inflammation and clotting. However, we conclude that Bev can induce platelet aggregation, degranulation and thrombosis through complex formation with VEGF and activation of the platelet FcγRIIa receptor, and that this provides a better explanation for the thrombotic events observed in vivo .     

Platelet Receptor Expression and Shedding: Glycoprotein Ib-IX-V and Glycoprotein VI

Quantity, quality, and lifespan are 3 important factors in the physiology, pathology, and transfusion of human blood platelets. The aim of this review is to discuss the proteolytic regulation of key platelet-specific receptors, glycoprotein(GP)Ib and GPVI, involved in the function of platelets in hemostasis and thrombosis, and nonimmune or immune thrombocytopenia. The scope of the review encompasses the basic science of platelet receptor shedding, practical aspects related to laboratory analysis of platelet receptor expression/shedding, and clinical implications of using the proteolytic fragments as platelet-specific biomarkers in vivo in terms of platelet function and clearance. These topics can be relevant to platelet transfusion regarding both changes in platelet receptor expression occurring ex vivo during platelet storage and/or clinical use of platelets for transfusion. In this regard, quantitative analysis of platelet receptor profiles on blood samples from individuals could ultimately enable stratification of bleeding risk, discrimination between causes of thrombocytopenia due to impaired production vs enhanced clearance, and monitoring of response to treatment prior to change in platelet count.     

Antibodies from patients with heparin-induced thrombocytopenia/thrombosis are specific for platelet factor 4 complexed with heparin or bound to endothelial cells.

Heparin-induced thrombocytopenia/thrombosis (HITP) is thought to be mediated by immunoglobulins that activate platelets in the presence of pharmacologic concentrations of heparin, but the molecular basis for this relatively common and often serious complication of heparin therapy has not been established. We found that plasma from each of 12 patients with HITP contained high titer (> or = 1:200) antibodies that reacted with immobilized complexes of heparin and platelet factor 4 (PF4), a heparin-binding protein contained in platelet alpha-granules. Recombinant human PF4 behaved similarly to PF4 isolated from platelets in this assay system. Complexes formed at an apparent heparin/PF4 molecular ratio of approximately 1:2 (fresh heparin) and approximately 1:12 (outdated heparin) were most effective in binding antibody. Immune complexes consisting of PF4, heparin, and antibody reacted with resting platelets; this interaction was inhibited by a monoclonal antibody specific for the Fc gamma RII receptor and by excess heparin. Human umbilical vein endothelial cells, known to express heparin-like glycosaminoglycan molecules on their surface, were recognized by antibody in the presence of PF4 alone; this reaction was inhibited by excess heparin, but not by anti-Fc gamma RII. Antibodies reactive with heparin/PF4 were not found in normal plasma, but IgG and IgM antibodies were detected at dilutions of 1:10 (IgG) and 1:50 (IgM) in 3 of 50 patients (6%) with other types of immune thrombocytopenia. These findings indicate that antibodies associated with HITP react with PF4 complexed with heparin in solution or with glycosaminoglycan molecules on the surface of endothelial cells and provide the basis for a new hypothesis to explain the development of thrombocytopenia with thrombosis or disseminated intravascular coagulation in patients sensitive to heparin.     

Large and small platelets—(When) do they differ?

Platelets are most important in providing cellular hemostasis but also take part in inflammation and immune processes. Increased platelet size has been regarded as a feature describing a young and more reactive subpopulation until studies were published which questioned this concept. Moreover, changes of platelet size given by the mean platelet volume (MPV) were described for immune thrombocytopenia, cardiovascular disease, atherosclerosis, venous thromboembolism, chronic lung disease, sepsis, cancer‐associated thrombosis, autoimmune disorders, and others. This review summarizes the literature on what is known about platelets with different size and describes controversies of studies with large and small platelets putting a focus on their thrombogenicity, age, and on the association of MPV with the mentioned diseases.     

Reduction of disulfide bonds within anti-D results in enhanced Fcγ receptor blockade

BACKGROUND: We have investigated whether chemicals known to disrupt disulfide bonds are capable of altering immunoglobulin anti‐D structure resulting in an increased efficacy of the chemically modified anti‐D to inhibit Fcγ receptor (FcγR)‐mediated phagocytosis. If successful, this would provide a rationale to explore this mechanism of enhancing FcγR blockade for future use in immunoglobulin therapies for immune cytopenias. STUDY DESIGN AND METHODS: Anti‐D that was shown to block 50 percent of the FcγR‐mediated phagocytosis of opsonized red blood cells (RBCs) using a monocyte monolayer assay (MMA) was combined with two different thiol‐containing compounds, dithiothreitol (DTT) or p‐toluenesulfonylmethyl mercaptan, with or without treatment with iodoacetamide, and allowed to react. Excess chemical was removed by extensive dialysis. FcγR blockade was assessed by MMA with dialyzed, untreated, or chemically treated anti‐D using both D+ and D? opsonized target RBCs. Toxicity was determined by fluorescence‐activated cell sorting. Aggregates and oligomerization of chemically treated anti‐D were examined using gel filtration–high pressure liquid chromatography. RESULTS: Using disulfide‐reducing compounds to chemically modify anti‐D significantly increases the efficacy of the anti‐D to induce an FcγR blockade and decrease phagocytosis in vitro of opsonized D+ or D? RBCs. This effect was shown not due to unbound residual chemical, toxicity, or formation of immunoglobulin G oligomers. S‐alkylation was required when using low concentrations of reducing compound. CONCLUSION: Our results demonstrate that irreversible reduction of interchain disulfide bonds within the immunoglobulin anti‐D results in a significantly increased efficacy to inhibit FcγR‐mediated phagocytosis regardless of opsonized target cell. With the use of this strategy, more effective and less expensive immunoglobulin treatment for immune cytopenias such as immune thrombocytopenic purpura or autoimmune hemolytic anemia may be developed.     

Evidence that a 210,000-molecular-weight glycoprotein (GP 210) serves as a platelet Fc receptor.

We previously identified a 210,000-mol-wt platelet glycoprotein (GP 210) that is missing from Bernard-Soulier platelets, and found that an antibody against GP 210 inhibits ristocetin-induced platelet agglutination. We now show by immunoblotting that GP 210 binds heat-aggregated rabbit and human IgG, as well as keyhole limpet hemocyanin (KLH)-anti-KLH and ovalbumin (OA)-anti-OA immune complexes. Immune complex binding to GP 210 was preserved on chymotrypsin-treated platelets that lacked glycoprotein Ib (GP Ib). In contrast, ristocetin-induced platelet agglutination resulted in disappearance of immunologically detectable GP 210 and loss of immune complex binding, even though GP Ib remained intact. Purified Fc fragments inhibited binding of anti-GP 210 antibody to intact platelets and to GP 210 on immunoblots. The Fc fragments also blocked immune complex binding to GP 210. Conversely, anti-GP 210 antiserum and F(ab)2 fragments inhibited binding of fluorescein-labeled Fc fragments to intact platelets. We conclude that GP 210 functions as a platelet Fc receptor.     

Human Fc gamma RII, in the absence of other Fc gamma receptors, mediates a phagocytic signal.

Fc gamma receptors are important components in the binding and phagocytosis of IgG-sensitized cells. Studies on the role of these receptors have been limited by the fact that most hematopoietic cells express more than one Fc gamma receptor. We studied the role of Fc gamma RIIA in isolation on a human erythroleukemia cell line (HEL) which expresses Fc gamma RIIA as its only Fc gamma receptor. HEL cells were observed to bind and phagocytose IgG-sensitized red blood cells (RBCs) in a dose-dependent manner. We then examined the role of Fc gamma RI and Fc gamma RII in isolation and in combination, in transfected COS-1 cells. Fc gamma RIIA-transfected COS cells also mediated both the binding and phagocytosis of IgG-sensitized RBCs. In contrast, phagocytosis was not observed in Fc gamma RI-transfected cells, although these cells avidly bound IgG-sensitized RBCs. Furthermore, coexpression of both receptors by doubly transfected cells did not affect the phagocytic efficiency of Fc gamma RIIA. These studies establish that Fc gamma RIIA can mediate phagocytosis and suggest that transfected COS-1 cells provide a model for examining this process. Since HEL cells exhibit characteristics of cells of the megakaryocyte-platelet lineage, including expression of Fc gamma RII as the only Fc gamma receptor, Fc gamma RIIA on megakaryocytes and platelets may be involved in the ingestion of IgG-containing immune complexes. Furthermore, these studies indicate that Fc gamma RI and Fc gamma RIIA differ in their requirements for transduction of a phagocytic signal.     

Non‐genomic effects of PPARγ ligands: inhibition of GPVI‐stimulated platelet activation

Summary. Background: Peroxisome proliferator‐activated receptor‐_γ (PPAR_γ) is expressed in human platelets although in the absence of genomic regulation in these cells, its functions are unclear. Objective: In the present study, we aimed to demonstrate the ability of PPAR_γ ligands to modulate collagen‐stimulated platelet function and suppress activation of the glycoprotein VI (GPVI) signaling pathway. Methods: Washed platelets were stimulated with PPAR_γ ligands in the presence and absence of PPAR_γ antagonist GW9662 and collagen‐induced aggregation was measured using optical aggregometry. Calcium levels were measured by spectrofluorimetry in Fura‐2AM‐loaded platelets and tyrosine phosphorylation levels of receptor‐proximal components of the GPVI signaling pathway were measured using immunoblot analysis. The role of PPAR_γ agonists in thrombus formation was assessed using an in vitro model of thrombus formation under arterial flow conditions. Results: PPAR_γ ligands inhibited collagen‐stimulated platelet aggregation that was accompanied by a reduction in intracellular calcium mobilization and P‐selectin exposure. PPAR_γ ligands inhibited thrombus formation under arterial flow conditions. The incorporation of GW9662 reversed the inhibitory actions of PPAR_γ agonists, implicating PPAR_γ in the effects observed. Furthermore, PPAR_γ ligands were found to inhibit tyrosine phosphorylation levels of multiple components of the GPVI signaling pathway. PPAR_γ was found to associate with Syk and LAT after platelet activation. This association was prevented by PPAR_γ agonists, indicating a potential mechanism for PPAR_γ function in collagen‐stimulated platelet activation. Conclusions: PPAR_γ agonists inhibit the activation of collagen‐stimulation of platelet function through modulation of early GPVI signalling.     

Shifting FcγRIIA-ITAM from activation to inhibitory configuration ameliorates arthritis

Rheumatoid arthritis–associated (RA-associated) inflammation is mediated through the interaction between RA IgG immune complexes and IgG Fc receptors on immune cells. Polymorphisms within the gene encoding the human IgG Fc receptor IIA (hFcγRIIA) are associated with an increased risk of developing RA. Within the hFcγRIIA intracytoplasmic domain, there are 2 conserved tyrosine residues arranged in a noncanonical immunoreceptor tyrosine–based activation motif (ITAM). Here, we reveal that inhibitory engagement of the hFcγRIIA ITAM either with anti-hFcγRII F(ab′)₂ fragments or intravenous hIgG (IVIg) ameliorates RA-associated inflammation, and this effect was characteristic of previously described inhibitory ITAM (ITAMi) signaling for hFcαRI and hFcγRIIIA, but only involves a single tyrosine. In hFcγRIIA-expressing mice, arthritis induction was inhibited following hFcγRIIA engagement. Moreover, hFcγRIIA ITAMi-signaling reduced ROS and inflammatory cytokine production through inhibition of guanine nucleotide exchange factor VAV-1 and IL-1 receptor–associated kinase 1 (IRAK-1), respectively. ITAMi signaling was mediated by tyrosine 304 (Y304) within the hFcγRIIA ITAM, which was required for recruitment of tyrosine kinase SYK and tyrosine phosphatase SHP-1. Anti-hFcγRII F(ab′)₂ treatment of inflammatory synovial cells from RA patients inhibited ROS production through induction of ITAMi signaling. These data suggest that shifting constitutive hFcγRIIA-mediated activation to ITAMi signaling could ameliorate RA-associated inflammation.     

Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans.

Allelic variants of Fc gamma R confer distinct phagocytic capacities providing a mechanism for heritable susceptibility to immune complex disease. Human Fc gamma RIIa has two codominantly expressed alleles, R131 and H131, which differ substantially in their ability to ligate human IgG2. The Fc gamma RIIa-H131 is the only human Fc gamma R which recognizes IgG2 efficiently and optimal IgG2 handling occurs only in the homozygous state. Therefore, since immune complex clearance is essential in SLE, we hypothesized that Fc gamma RIIA genes are important disease susceptibility factors for SLE, particularly lupus nephritis. In a two-stage cross-sectional study, we compared the distribution of Fc gamma RIIA alleles in African Americans with SLE to that in African American non-SLE controls. A pilot study of 43 SLE patients and 39 controls demonstrated a skewed distribution of Fc gamma RIIA alleles, with only 9% of SLE patients homozygous for Fc gamma RIIa-H131 compared with 36% of controls (odds ratio, 0.18; 95% CI, 0.05-0.69, P = 0.009). This was confirmed with a multicenter study of 214 SLE patients and 100 non-SLE controls. The altered distribution of Fc gamma RIIA alleles was most striking in lupus nephritis. Trend analysis of the genotype distribution showed a highly significant decrease in Fc gamma RIIA-H131 as the likelihood for lupus nephritis increased (P = 0.0004) consistent with a protective effect of the Fc gamma RIIA-H131 gene. The skewing in the distribution of Fc gamma RIIA alleles identifies this gene as a risk factor with pathophysiologic importance for the SLE diathesis in African Americans.