Guangheng Zhu

Crosstalk between Platelets and the Immune System: Old Systems with New Discoveries

Platelets are small anucleate cells circulating in the blood. It has been recognized for more than 100 years that platelet adhesion and aggregation at the site of vascular injury are critical events in hemostasis and thrombosis; however, recent studies demonstrated that, in addition to these classic roles, platelets also have important functions in inflammation and the immune response. Platelets contain many proinflammatory molecules and cytokines (e.g., P-selectin, CD40L, IL-1β, etc.), which support leukocyte trafficking, modulate immunoglobulin class switch, and germinal center formation. Platelets express several functional Toll-like receptors (TLRs), such as TLR-2, TLR-4, and TLR-9, which may potentially link innate immunity with thrombosis. Interestingly, platelets also contain multiple anti-inflammatory molecules and cytokines (e.g., transforming growth factor-β and thrombospondin-1). Emerging evidence also suggests that platelets are involved in lymphatic vessel development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2. Besides the active contributions of platelets to the immune system, platelets are passively targeted in several immune-mediated diseases, such as autoimmune thrombocytopenia, infection-associated thrombocytopenia, and fetal and neonatal alloimmune thrombocytopenia. These data suggest that platelets are important immune cells and may contribute to innate and adaptive immunity under both physiological and pathological conditions.

Plasma fibronectin depletion enhances platelet aggregation and thrombus formation in mice lacking fibrinogen and von Willebrand factor

We previously showed that platelet aggregation and thrombus formation occurred in mice lacking both fibrinogen (Fg) and von Willebrand factor (VWF) and that plasma fibronectin (pFn) promoted thrombus growth and stability in injured arterioles in wild-type mice. To examine whether pFn is required for Fg/VWF-independent thrombosis, we generated Fg/VWF/conditional pFn triple-deficient (TKO; Cre(+), Fn(flox/flox), Fg/VWF(-/-)) mice and littermate control (Cre(-), Fn(flox/flox), Fg/VWF(-/-)) mice. Surprisingly, TKO platelet aggregation was not abolished, but instead was enhanced in both heparinized platelet-rich plasma and gel-filtered platelets. This enhancement was diminished when TKO platelets were aggregated in pFn-positive control platelet-poor plasma (PPP), whereas aggregation was enhanced when control platelets were aggregated in pFn-depleted TKO PPP. The TKO platelet aggregation can be completely inhibited by our newly developed mouse anti-mouse beta(3) integrin antibodies but was not affected by anti-mouse GPIbalpha antibodies. Enhanced platelet aggregation was also observed when heparinized TKO blood was perfused in collagen-coated perfusion chambers. Using intravital microscopy, we further showed that thrombogenesis in TKO mice was enhanced in both FeCl(3)-injured mesenteric arterioles and laser-injured cremaster arterioles. Our data indicate that pFn is not essential for Fg/VWF-independent thrombosis and that soluble pFn is probably an important inhibitory factor for platelet aggregation.

Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests that antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc–FcγR interactions. However, we and others have demonstrated that anti-GPIbα (but not GPIIbIIIa)-mediated ITP is often refractory to therapies targeting FcγR pathways. Here, we generate mouse anti-mouse monoclonal antibodies (mAbs) that recognize GPIbα and GPIIbIIIa of different species. Utilizing these unique mAbs and human ITP plasma, we find that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induces Fc-independent platelet activation, sialidase neuraminidase-1 translocation and desialylation. This leads to platelet clearance in the liver via hepatocyte Ashwell–Morell receptors, which is fundamentally different from the classical Fc–FcγR-dependent macrophage phagocytosis. Importantly, sialidase inhibitors ameliorate anti-GPIbα-mediated thrombocytopenia in mice. These findings shed light on Fc-independent cytopenias, designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of refractory ITP.

Fibrinogen and von Willebrand factor-independent platelet aggregation in vitro and in vivo

Summary.  Background: Fibrinogen (Fg) has been considered essential for platelet aggregation. However, we recently demonstrated formation of occlusive thrombi in Fg‐deficient mice and in mice doubly deficient for Fg and von Willebrand factor (Fg/VWF−/−). Methods and results: Here we studied Fg/VWF‐independent platelet aggregation in vitro and found no aggregation in citrated platelet‐rich plasma of Fg/VWF−/− mice. Surprisingly, in Fg/VWF−/− plasma without anticoagulant, adenosine diphosphate induced robust aggregation of Fg/VWF−/− platelets but not of β3‐integrin‐deficient (β3−/−) platelets. In addition, β3−/− platelets did not significantly incorporate into thrombi in Fg/VWF−/− mice. This Fg/VWF‐independent aggregation was blocked by thrombin inhibitors (heparin, hirudin, PPACK), and thrombin or thrombin receptor activation peptide (AYPGKF‐NH2) induced aggregation of gel‐filtered Fg/VWF−/− platelets in 1 mm Ca2+ PIPES buffer. Notably, aggregation in PIPES buffer was only 50–60% of that observed in Fg/VWF−/− plasma. Consistent with the requirement for thrombin in vitro, hirudin completely inhibited thrombus formation in Fg/VWF−/− mice. These data define a novel pathway of platelet aggregation independent of both Fg and VWF. Although this pathway was not detected in the presence of anticoagulants, it was observed under physiological conditions in vivo and in the presence of Ca2+in vitro. Conclusions: β3 integrin, thrombin, and Ca2+ play critical roles in this Fg/VWF‐independent aggregation, and both plasma and platelet granule proteins contribute to this process.

Animal model of fetal and neonatal immune thrombocytopenia: role of neonatal Fc receptor in the pathogenesis and therapy

Fetal and neonatal immune thrombocytopenia (FNIT) is a severe bleeding disorder in which maternal antibodies cross the placenta and destroy fetal/neonatal platelets. It has been demonstrated that the neonatal Fc receptor (FcRn) regulates immunoglobulin G (IgG) homeostasis and plays an important role in transplacental IgG transport. However, the role of FcRn in the pathogenesis and therapy of FNIT has not been studied. Here, we developed an animal model of FNIT using combined β3 integrin-deficient and FcRn-deficient (β3(-/-)FcRn(-/-)) mice. We found that β3(-/-)FcRn(-/-) mice are immunoresponsive to β3(+/+)FcRn(-/-) platelets. The generated antibodies were β3 integrin specific and were maintained at levels that efficiently induced thrombocytopenia in adult β3(+/+)FcRn(-/-) mice. FNIT was observed when immunized β3(-/-)FcRn(+/+) females were bred with β3(+/+)FcRn(+/+) males, while no FNIT occurred in β3(-/-)FcRn(-/-) females bred with β3(+/+)FcRn(-/-) males, suggesting that FcRn is indispensable for the induction of FNIT. We further demonstrated that fetal FcRn was responsible for the transplacental transport of various IgG isotypes. We found that anti-FcRn antibody and intravenous IgG prevented FNIT, and that intravenous IgG ameliorated FNIT through both FcRn-dependent and -independent pathways. Our data suggest that targeting FcRn may be a potential therapy for human FNIT as well as other maternal pathogenic antibody-mediated diseases.

Maternal anti-platelet β3 integrins impair angiogenesis and cause intracranial hemorrhage

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening disease in which intracranial hemorrhage (ICH) is the major risk. Although thrombocytopenia, which is caused by maternal antibodies against β3 integrin and occasionally by maternal antibodies against other platelet antigens, such as glycoprotein GPIbα, has long been assumed to be the cause of bleeding, the mechanism of ICH has not been adequately explored. Utilizing murine models of FNAIT and a high-frequency ultrasound imaging system, we found that ICH only occurred in fetuses and neonates with anti-β3 integrin-mediated, but not anti-GPIbα-mediated, FNAIT, despite similar thrombocytopenia in both groups. Only anti-β3 integrin-mediated FNAIT reduced brain and retina vessel density, impaired angiogenic signaling, and increased endothelial cell apoptosis, all of which were abrogated by maternal administration of intravenous immunoglobulin (IVIG). ICH and impairment of retinal angiogenesis were further reproduced in neonates by injection of anti-β3 integrin, but not anti-GPIbα antisera. Utilizing cultured human endothelial cells, we found that cell proliferation, network formation, and AKT phosphorylation were inhibited only by murine anti-β3 integrin antisera and human anti-HPA-1a IgG purified from mothers with FNAIT children. Our data suggest that fetal hemostasis is distinct and that impairment of angiogenesis rather than thrombocytopenia likely causes FNAIT-associated ICH. Additionally, our results indicate that maternal IVIG therapy can effectively prevent this devastating disorder.

Severe platelet desialylation in a patient with glycoprotein Ib/IX antibody-mediated immune thrombocytopenia and fatal pulmonary hemorrhage

Immune thrombocytopenia (ITP) is a common bleeding disorder characterized by autoantibody-mediated platelet destruction. Antibodies predominantly target platelet glycoproteins GPIIb/IIIa (70–80%) and GPIb/IX (20–40%), with some patients having both. In animal models, anti-GPIb/IX but not anti-

Fc-independent phagocytosis: implications for IVIG and other therapies in immune-mediated thrombocytopenia.

Phagocytes were first described by Dr. Metchnikoff in 1873. The roles of phagocytes in innate and adaptive immunity have been well established to date, although the molecular mechanisms involved in initiating phagocytosis (through Fc or other receptors) remain to be further explored. Phagocytes in the reticuloendothelial system, particularly macrophages, have been implicated in the clearance of senescent blood cells. The destruction of these cells may be primarily mediated via an Fc-independent pathway. Fc-independent phagocytosis may also play an important role in platelet clearance, including in autoimmune thrombocytopenia (ITP), and in clearance of platelet-rich emboli detached from sites of vascular injury. In ITP, the two major platelet auto-antigens have been located on glycoprotein (GP)IIbIIIa and the GPIb complex. It has been demonstrated that anti-GPIb antibodies, in contrast to anti-GPIIbIIIa, can induce thrombocytopenia in an Fc-independent manner. We further demonstrated in an animal model that intravenous IgG (IVIG) is unable to ameliorate thrombocytopenia caused by most anti-GPIb antibodies, despite its efficacy in anti- GPIIbIIIa-mediated thrombocytopenia. Our data was supported by subsequent retrospective studies with ITP patients by several independent groups. Most recently, we found that anti-GPIb-mediated ITP was also resistant to steroid therapy and that platelet activation and apoptosis induced by anti-GPIb antibodies may be involved in the Fc-independent platelet clearance. Therefore, identification of antibody specificity in patients, e.g. anti-GPIIbIIIa (Fc-dependent) versus anti-GPIb (Fc-independent), may be important for therapies against ITP, as well as other immune-mediated thrombocytopenias.

CD8 + T cells are predominantly protective and required for effective steroid therapy in murine models of immune thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune bleeding disorder characterized by autoantibodies targeting platelet surface proteins, most commonly GPIIbIIIa (αIIbβ3 integrin), leading to platelet destruction. Recently, CD8(+) cytotoxic T-lymphocytes (CTLs) targeting platelets and megakaryocytes have also been implicated in thrombocytopenia. Because steroids are the most commonly administered therapy for ITP worldwide, we established both active (immunized splenocyte engraftment) and passive (antibody injection) murine models of steroid treatment. Surprisingly, we found that, in both models, CD8(+) T cells limited the severity of the thrombocytopenia and were required for an efficacious response to steroid therapy. Conversely, CD8(+) T-cell depletion led to more severe thrombocytopenia, whereas CD8(+) T-cell transfusion ameliorated thrombocytopenia. CD8(+) T-regulatory cell (Treg) subsets were detected, and interestingly, dexamethasone (DEX) treatment selectively expanded CD8(+) Tregs while decreasing CTLs. In vitro coculture studies revealed CD8(+) Tregs suppressed CD4(+) and CD19(+) proliferation, platelet-associated immunoglobulin G generation, CTL cytotoxicity, platelet apoptosis, and clearance. Furthermore, we found increased production of anti-inflammatory interleukin-10 in coculture studies and in vivo after steroid treatment. Thus, we uncovered subsets of CD8(+) Tregs and demonstrated their potent immunosuppressive and protective roles in experimentally induced thrombocytopenia. The data further elucidate mechanisms of steroid treatment and suggest therapeutic potential for CD8(+) Tregs in immune thrombocytopenia.

Fc-independent phagocytosis: implications for intravenous IgG therapy in immune thrombocytopenia.

Phagocytes were first described 120 years ago. Although the molecular mechanisms involved in initiating phagocytosis (through Fc or other receptors) are still not fully understood, the roles of phagocytes in innate and adaptive immunity have been well studied. Phagocytes in the reticuloendothelial system, particularly macrophages, have been implicated in the clearance of senescent blood cells. The destruction of these cells may be primarily mediated through an Fc-independent pathway. Fc-independent phagocytosis may also play an important role in platelet clearance, including immune thrombocytopenia (ITP). The two major platelet antigens targeted in ITP are GPIIbIIIa and the GPIb complex. It has been demonstrated that anti-GPIbalpha antibodies, in contrast to anti-GPIIbIIIa, can induce thrombocytopenia in an Fc-independent manner. We further demonstrated in an animal model that intravenous IgG is not able to ameliorate thrombocytopenia caused by most anti-GPIbalpha antibodies, though it is effective in anti-GPIIbIIIa mediated thrombocytopenia. Our data was supported by a recent retrospective study of ITP patients. Therefore, identification of antibody specificity (e.g. anti-GPIIbIIIa (Fc-dependent) versus anti-GPIbalpha (Fc-independent)) in patients may be important for ITP therapy.