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Miscarriage and intrauterine growth restriction (IUGR) are devastating complications in fetal/neonatal alloimmune thrombocytopenia (FNAIT). We previously reported the mechanisms for bleeding diatheses, but it is unknown whether placental, decidual immune cells or other abnormalities at the maternal–fetal interface contribute to FNAIT. Here we show that maternal immune responses to fetal platelet antigens cause miscarriage and IUGR that are associated with vascular and immune pathologies in murine FNAIT models. Uterine natural killer (uNK) cell recruitment and survival beyond mid-gestation lead to elevated NKp46 and CD107 expression, perforin release and trophoblast apoptosis. Depletion of NK cells restores normal spiral artery remodeling and placental function, prevents miscarriage, and rescues hemorrhage in neonates. Blockade of NK activation receptors (NKp46, FcɣRIIIa) also rescues pregnancy loss. These findings shed light on uNK antibody-dependent cell-mediated cytotoxicity of invasive trophoblasts as a pathological mechanism in FNAIT, and suggest that anti-NK cell therapies may prevent immune-mediated pregnancy loss and ameliorate FNAIT., Fetal/neonatal alloimmune thrombocytopenia (FNAIT) is a gestational disease caused by maternal immune responses against fetal platelets. Using a FNAIT mouse model and human trophoblast cell lines, here the authors show that uterine natural killer cell-mediated trophoblast apoptosis contributes to FNAIT pathogenesis.

Availability of universal marker for the diagnosis of platelet apoptosis is an important but currently unresolved goal of platelet physiology investigations. Mitochondrial inner transmembrane potential (▵Ψm) depolarization is frequently used as a marker of apoptosis in nucleated cells and anucleate platelets. Since ▵Ψm depolarization in platelets is also frequently associated with concurrent induction of other apoptotic responses, it may appear that ▵Ψm depolarization is a good universal marker of platelet apoptosis. However, data presented in the current study indicate that this is incorrect. We report here fundamental differences in the effects of potassium ionophore valinomycin and calcium ionophore A23187 on human platelet apoptosis. Although both A23187-triggered and valinomycin-triggered ▵Ψm depolarization are strongly induced, the former is dependent on the opening of mitochondrial permeability transition pore (MPTP) and the latter is MPTP-independent. Furthermore, effects of calcium and potassium ionophores on other apoptotic events are also basically different. A23187 induces caspase-3 activation, proapoptotic Bax and Bak protein expression, phosphatidylserine exposure, and microparticle formation, whereas valinomycin does not induce these apoptotic manifestations. Discovery of targeted ▵Ψm depolarization not associated with apoptosis in valinomycin-treated platelets indicates that this marker should not be used as a single universal marker of platelet apoptosis in unknown experimental and clinical settings as it may lead to a false-positive apoptosis diagnosis.

Platelet αIIbβ3 integrin and its ligands are essential for thrombosis and hemostasis, and play key roles in myocardial infarction and stroke. Here we show that apolipoprotein A-IV (apoA-IV) can be isolated from human blood plasma using platelet β3 integrin-coated beads. Binding of apoA-IV to platelets requires activation of αIIbβ3 integrin, and the direct apoA-IV-αIIbβ3 interaction can be detected using a single-molecule Biomembrane Force Probe. We identify that aspartic acids 5 and 13 at the N-terminus of apoA-IV are required for binding to αIIbβ3 integrin, which is additionally modulated by apoA-IV C-terminus via intra-molecular interactions. ApoA-IV inhibits platelet aggregation and postprandial platelet hyperactivity. Human apoA-IV plasma levels show a circadian rhythm that negatively correlates with platelet aggregation and cardiovascular events. Thus, we identify apoA-IV as a novel ligand of αIIbβ3 integrin and an endogenous inhibitor of thrombosis, establishing a link between lipoprotein metabolism and cardiovascular diseases., Activation of integrin αIIbβ3 at the surface of platelets is required for their aggregation and for thrombus formation. Here Xu et al. identify apolipoprotein A-IV as a novel ligand for platelet αIIbβ3 integrin, and find it inhibits platelet aggregation and thrombosis.

Integrins are a large family of heterodimeric transmembrane receptors differentially expressed on almost all metazoan cells. Integrin β subunits contain a highly conserved plexin-semaphorin-integrin (PSI) domain. The CXXC motif, the active site of the protein-disulfide-isomerase (PDI) family, is expressed twice in this domain of all integrins across species. However, the role of the PSI domain in integrins and whether it contains thiol-isomerase activity have not been explored. Here, recombinant PSI domains of murine β3, and human β1 and β2 integrins were generated and their PDI-like activity was demonstrated by refolding of reduced/denatured RNase. We identified that both CXXC motifs of β3 integrin PSI domain are required to maintain its optimal PDI-like activity. Cysteine substitutions (C13A and C26A) of the CXXC motifs also significantly decreased the PDI-like activity of full-length human recombinant β3 subunit. We further developed mouse anti-mouse β3 PSI domain monoclonal antibodies (mAbs) that cross-react with human and other species. These mAbs inhibited αIIbβ3 PDI-like activity and its fibrinogen binding. Using single-molecular Biomembrane-Force-Probe assays, we demonstrated that inhibition of αIIbβ3 endogenous PDI-like activity reduced αIIbβ3-fibrinogen interaction, and these anti-PSI mAbs inhibited fibrinogen binding via different levels of both PDI-like activity-dependent and -independent mechanisms. Importantly, these mAbs inhibited murine/human platelet aggregation in vitro and ex vivo, and murine thrombus formation in vivo, without significantly affecting bleeding time or platelet count. Thus, the PSI domain is a potential regulator of integrin activation and a novel target for antithrombotic therapies. These findings may have broad implications for all integrin functions, and cell-cell and cell-matrix interactions.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality and can occur with any type of transfusion. TRALI is thought to be primarily mediated by donor antibodies activating recipient neutrophils resulting in pulmonary endothelial damage. Nonetheless, details regarding the interactions between donor antibodies and recipient factors are unknown. A murine antibody-mediated TRALI model was used to elucidate the roles of the F(ab')2 and Fc regions of a TRALI-inducing immunoglobulin G anti-major histocompatibility complex (MHC) class I antibody (34.1.2s). Compared with intact antibody, F(ab')2 fragments significantly increased serum levels of the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2); however, pulmonary neutrophil levels were only moderately increased, and no pulmonary edema or mortality occurred. Fc fragments did not modulate any of these parameters. TRALI induction by intact antibody was completely abrogated by in vivo peripheral blood monocyte depletion by gadolinium chloride (GdCl3) or chemokine blockade with a MIP-2 receptor antagonist but was restored upon repletion with purified monocytes. The results suggest a two-step process for antibody-mediated TRALI induction: the first step involves antibody binding its cognate antigen on blood monocytes, which generates MIP-2 chemokine production that is correlated with pulmonary neutrophil recruitment; the second step occurs when antibody-coated monocytes increase Fc-dependent lung damage.

BACKGROUND: Treatment with human platelet antigen (HPA)-matched platelets (PLTs) is the optimal therapy for bleeding secondary to neonatal alloimmune thrombocytopenia. Recent advances in high-throughput DNA-based blood group and PLT antigen genotyping have made it possible to screen plateletpheresis donors for potential HPA-matched PLT transfusion. STUDY DESIGN AND METHODS: This prospective study evaluated genomic DNA from plateletpheresis donors for single-nucleotide polymorphisms (SNPs) associated with HPA-1, -2, -3, -4, -5, and -15 to determine whether high-throughput multiplex genomic DNA PCR and oligonucleotide extension technology can be used for mass-scale PLT antigen genotyping. Genotyping using SNP technology was confirmed using sequence-specific polymerase chain reaction (SSP-PCR). RESULTS: Of the 748 donors screened, 277 were found to be negative for antigens implicated in alloimmune thrombocytopenia. In addition, two donors were homozygous for HPA-1b/b and -2b/b, six donors for HPA-1b/b and -3b/b, one for HPA-2b/b and -3b/b, one for HPA-1b/b and -5b/b, 10 for HPA-1b/b and -15 b/b, four for HPA-5b/b and -15b/b, and one for HPA-2b/b and -15b/b. Retesting using SSP-PCR was conducted for 60 donors. Discrepant results occurred between SNP and SSP-PCR in less than 20% of samples for HPA-1b/1b/HPA-3b/3b, HPA-5b/5b, and HPA-15b/b. DISCUSSION: High-throughput multiplex PCR SNP and confirmatory molecular genotyping are useful for mass-scale screening of apheresis PLT donors to provide antigen-negative genotypes. Refinements to mass-scale multiplex analysis technology would reduce further the confirmatory testing needed.

Integrin αIIbβ3 plays key roles in thrombosis and hemostasis primarily through mediating platelet adhesion and aggregation. We recently reported that the active site of thiol-isomerase enzymes, CXXC motif, is expressed twice within the plexin-semaphorin-integrin (PSI) domain across all integrins and species, and the PSI domain of β3 integrin possesses endogenous thiol-isomerase activity, which may be a novel target for anti-thrombotic therapy (Blood, 2017). We developed four mouse anti-mouse β3 integrin PSI domain monoclonal antibodies (mAbs). These mAbs cross-react with β3 PSI domains of human, mouse, pig, rat, and rabbit tested but not other regions of β3 integrin, other integrins or other thiol-isomerase enzymes. They inhibit the thiol-isomerase activity of β3 PSI domain, decrease platelet adhesion/aggregation and thrombosis without increasing bleeding. Interestingly, the inhibitory effect of these mAbs on thrombosis in vivo (no anti-coagulant) was 10-20 times greater than their inhibitory effect on platelet aggregation in anti-coagulated platelet-rich plasma in vitro. This motivated us to explore whether this PSI domain contributes to blood coagulation. To asses blood clot formation and retraction, blood was incubated in non-stick tubes for two hours at 37°C in clot retraction assays. These assays showed less clot retraction and significantly lower dry clot weight in human and mouse whole blood treated with these anti-PSI mAbs compared to controls (p Disulfide bond exchanges are required for blood coagulation and thiol-isomerase enzymes play important roles in thrombosis. Given that blood coagulation is rapidly amplified on the platelet surface (i.e. cell-based coagulation), many coagulation factors are in close proximity to β3 integrin PSI domains (with thiol-isomerase activity). The study of interaction between β3 PSI domain and coagulation factors is currently being exploring. This is the first evidence of an allosteric therapeutic target of β3 integrin. This work suggests that a novel interaction exists between platelets and the coagulation cascade that requires the β3 integrin PSI domain and its thiol-isomerase activity. Importantly, the PSI domain can be targeted to partially block both platelet aggregation and coagulation, resulting in less bleeding while still providing a strong overall anti-thrombotic effect. Disclosures No relevant conflicts of interest to declare.

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. Although it has been reported that von Willebrand factor (VWF) affects P-selectin expression on endothelial cells, little information is available regarding regulation of platelet P-selectin expression. Here, we first observed that P-selectin expression was significantly decreased on platelets of fibrinogen and VWF double-deficient mice. Subsequently, we identified this was due to fibrinogen deficiency. Impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in human hypofibrinogenemic patients. We demonstrated that this impairment is unlikely due to excessive P-selectin shedding, deficient fibrinogen-mediated cell surface P-selectin binding, or impaired platelet granule release, but rather is due to decreased platelet P-selectin content. Fibrinogen transfusion completely recovered this impairment in fibrinogen-deficient (Fg−/−) mice, and engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process. Furthermore, Fg−/− platelets significantly increased P-selectin expression following transfusion into β3 integrin–deficient mice and when cultured with fibrinogen. These data suggest fibrinogen may play important roles in inflammation, thrombosis, and hemostasis via enhancement of platelet P-selectin expression. Since human fibrinogen levels vary significantly in normal and diseased populations, P-selectin as an activation marker on platelets should be used with caution.

The role of the mitochondrial permeability transition pore (MPTP) in apoptosis of nucleated cells is well documented. In contrast, the role of MPTP in apoptosis of anucleated platelets is largely unknown. The aim of this study was to elucidate the contribution of MPTP in the control of different manifestations of platelet apoptosis by analyzing the effect of cyclosporin A (CsA), a potent inhibitor of MPTP formation. Using flow cytometry, we studied the effect of pretreatment of platelets with CsA on apoptotic responses in human platelets stimulated with calcium ionophore A23187. We found that CsA inhibited A23187-stimulated platelet apoptosis, completely preventing (i) depolarization of mitochondrial inner membrane potential (DeltaPsim), (ii) activation of cytosolic apoptosis executioner caspase-3, (iii) platelet shrinkage, and (iv) fragmentation of platelets to microparticles, but (v) only partially (approximately 25%), inhibiting phosphatidylserine (PS) exposure on the platelet surface. This study shows that MPTP formation is upstream of DeltaPsim depolarization, caspase-3 activation, platelet shrinkage and microparticle formation, and stringently controls these apoptotic events in A23187-stimulated platelets but is less involved in PS externalization. These data also indicate that CsA may rescue platelets from apoptosis, preventing caspase-3 activation and inhibiting the terminal cellular manifestations of platelet apoptosis, such as platelet shrinkage and degradation to microparticles. Furthermore, the results suggest a novel potentially useful application of CsA as an inhibitor of platelet demise through apoptosis in thrombocytopenias associated with enhanced platelet apoptosis.

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+, Fnflox/flox, Fg/VWF−/−) mice and littermate control (Cre−, Fnflox/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 β3 integrin antibodies but was not affected by anti–mouse GPIbα 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 FeCl3-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.

Transfusion-related immunomodulation (TRIM) has been correlated with the presence of white cells (WBCs) in blood transfusions, but the role of components such as platelets (PLTs) in mediating TRIM has not been extensively examined. We designed a murine PLT transfusion model to study whether leukoreduced PLTs mediate TRIM effects.CBA recipient mice were administered four weekly transfusions of either fresh (4 hr) or aged (24 and 72 hr) donor leukoreduced PLTs from allogeneic BALB/c mice and then transplanted with skin grafts from donor-matched mice. TRIM was measured by comparing the times to graft rejection and these were correlated with immunoglobulin G (IgG) antibody development measured by flow cytometry.Compared with nontransfused control recipients, four transfusions of fresh, extremely leukoreduced (0.05 WBCs/mL), allogeneic PLTs significantly (p0.002) reduced the recipient's ability to reject donor-matched skin grafts (survival49 days compared with14 days in nontransfused controls) despite the presence of high-titered serum IgG donor antibodies. In contrast, however, aged PLTs or fresh PLTs devoid of MHC Class I molecules were unable to affect skin graft survival nor stimulate antibody production. The PLT age-related inability to induce TRIM was shown to be due to loss of PLT-associated MHC Class I molecules; soluble supernatant MHC molecules that were transfused were unable to induce TRIM.These results suggest that fresh PLTs can induce TRIM independently of WBCs due to their MHC antigen expression whereas aging results in loss of MHC and ability to mediate TRIM. The findings support the concept that either active MHC removal from fresh PLTs or passive removal by, for example, storage, may reduce any deleterious effects of TRIM in transfusion recipients.

BACKGROUND: Anti-D treatment is effective in increasing platelet (PLT) counts in patients with autoimmune thrombocytopenic purpura (AITP); however, the exact mechanism of action is unknown. Previous results have suggested that anti-D-coated red blood cells (RBCs) affect reticuloendothelial system phagocytosis by stimulating agents (e.g., reactive oxygen species) that alter signaling pathways within the phagocyte. To address this, a flow cytometric assay was used to compare the kinetics and signaling pathways responsible for opsonized PLT and RBC phagocytosis. STUDY DESIGN AND METHODS: Human RBCs or PLTs were labeled with the fluorescent dye CM-Green, opsonized with Rh immune globulin or anti-MHC, respectively, and incubated with THP-1 monocytes with or without signal transduction inhibitors and intracellular fluorescence was analyzed. RESULTS: Compared with opsonized PLTs, phagocytosis of opsonized RBCs was significantly slower (p < 0.0001) and, within 2 hours, induced a state of phagocytic refractoriness; resting the mononuclear cells (MNCs) for up to 24 hours did not rescue their ability to further mediate PLT phagocytosis. Inhibitors of phosphatidylinositol 3-kinase (wortmannin, LY294002, myricetin, and quercetin), protein kinase C (staurosporine), and Syk kinase (piceatannol) inhibited both opsonized RBC and opsonized PLT phagocytosis. In contrast, opsonized RBC phagocytosis was significantly (p < 0.0001) enhanced by the tyrosine phosphatase inhibitor phenyl arsine oxide, whereas PLT phagocytosis was significantly reduced (p < 0.0001). Of interest, phosphatase inhibition during opsonized RBC phagocytosis induced a longer (48 hr) phagocytic refractoriness period in the MNCs. CONCLUSION: These results suggest that the early kinetics and signaling events related to phosphatase activity regulate how mononuclear phagocytes engulf opsonized RBCs and induce phagocytic refractoriness for further PLT phagocytosis.

Haemolytic disease of the newborn (HDN) can be prevented by the passive administration of anti-D to the mother. The most accepted theory to describe this activity of anti-D is based upon its ability to clear opsonized erythrocytes before their recognition by the maternal immune system. We examined this hypothesis using a murine model of immunity to foreign erythrocytes. Whereas transfusion of foreign erythrocytes into mice induced immunoglobulin (Ig)M and IgG antibodies specific for the erythrocytes, these humoral immune responses were inhibited when the erythrocytes were opsonized with IgG. To specifically determine if immunological evasion occurs with these opsonized erythrocytes, we examined T-cell responses from these mice. An erythrocyte-specific T-cell response was clearly detected. We then tested whether phagocytosis of opsonized erythrocytes is sufficient to prevent the antibody response. We exposed mononuclear phagocytic cells to sheep red blood cells (SRBC) in vitro and then adoptively transferred the phagocytic cells to recipient mice; opsonized SRBC unexpectedly increased, rather than decreased, the antibody response. These data indicate that removal of opsonized erythrocytes by phagocytic cells does not prevent their immunological recognition and suggest that antigen clearance may not be the predominant mechanism of anti-erythrocyte action in downregulating the humoral immune response.

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., Immune thrombocytopenia (ITP) is caused by autoantibody-mediated platelet clearance, but refractoriness to current immunomodulatory therapies is common. Here the authors show that desialylated platelets can be cleared via hepatic Ashwell–Morell receptor, a process that can be attenuated by sialidase inhibitors, suggesting a new therapy for ITP.

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.

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.

The exact mechanism of action of IVIg in the amelioration of immune thrombocytopenic purpura (ITP) is still unclear. Studies have suggested that IVIg may function through the regulation of cytokines, including interleukin-1 receptor antagonist (IL-1Ra), an inhibitor of phagocytosis. Using a mouse model relevant to ITP, we confirm an increase in mouse serum levels of IL-1Ra after exposure to IVIg, yet a recombinant IL-1Ra did not ameliorate thrombocytopenia. IVIg has also been shown to affect the expression of other regulatory cytokines. We have also recently established that IVIg specifically targets activating FcγRs on CD11c+ dendritic cells (DCs) as its primary mechanism of action in the amelioration of murine ITP. Herein, we show that IVIg functions therapeutically in mice lacking specific cytokines or their receptors that can potentially affect DC/macrophage function (IL-1 receptor, IL-4, IL-10, IL-12β, TNF-α, IFN-γ receptor, MIP-1α). This suggests that while IVIg may mediate the release of a variety of cytokines, the cytokines tested do not directly participate in the mechanism of IVIg action.

Intravenous immunoglobulin G (IVIG) is used to treat idiopathic thrombocytopenic purpura (ITP). Although many patients benefit from IVIG, some are refractory to this therapy. ITP is characterized by platelet clearance mediated primarily by antiplatelet antibodies against GPIIbIIIa and/or the GPIbα complex. These 2 groups of antibodies may induce ITP through different mechanisms. We tested the hypothesis that IVIG may not be equally effective in preventing ITP caused by anti-GPIIbIIIa versus anti-GPIbα antibodies in mice. Thrombocytopenia was induced in BALB/c mice using monoclonal antibodies against either mouse GPIIbIIIa (JON1, JON2, and JON3) or GPIbα (p0p3, p0p4, p0p5, p0p9, and p0p11). Pretreatment with IVIG significantly ameliorated ITP in all anti-GPIIbIIIa–injected animals. Conversely, IVIG failed to prevent ITP in all anti-GPIbα–treated mice, except for p0p4. These results were repeated in C57BL/6 mice, and with different IVIG preparations. These data in mice suggest that patients with ITP mediated by anti-GPIbα antibodies may be less responsive to IVIG treatment.

Indirect platelet radioactive antiglobulin tests using anti-IgG and anti-C3 were performed in 483 individuals. The subjects' diagnoses and clinical states were correlated with sensitizing protein (IgG and/or C3). Sensitivity of the technique was increased by increasing the serum to cell ratio and there was good correlation between direct and indirect antiglobulin tests in 56 patients. Seven percent of normal healthy non-thrombocytopenic untransfused males and 88% of patients in whom autoimmune thrombocytopenia was clinically suspected showed a positive test result. In the indirect antiglobulin test, mean +/- SD fg anti-C3d/platelet was 1.75 +/- 0.28 (N = 58) with serum from normal subjects, 3.91 +/- 1.86 (N = 123) with serum from patients with idiopathic thrombocytopenic purpura and systemic lupus erythematosus, and 2.78 +/- 0.91 (N = 52) with serum from patients with lymphoproliferative disease. The differences were significant with P less than 0.001. Levels of bound anti-IgG were in general accord with those of other reports; there was no significant relationship with serum immunoglobulin levels. When the indirect antiglobulin tests were positive for both IgG and C3, there was a significant correlation between IgG and C3 bound to normal platelets (r = 0.671; P less than 0.01). Although sera from 39% of patients whose thrombocytopenia was not initially suspected to be on an autoimmune basis gave a positive test result, indirect platelet radioactive antiglobulin tests using anti-IgG and anti-C3 may be convenient tools in the evaluation and follow-up of immune thrombocytopenias.

Fetal and neonatal alloimmune thrombo cytopenia (FNAITP) is a life-threatening bleeding disorder caused by maternal antibodies directed against fetal platelet antigens. The immunoreactive epitopes in FNAITP are primarily located in the extracellular regions of the platelet glycoprotein IIIa (β3 integrin). Here we have established a novel animal model of FNAITP using β3 integrin–deficient (β3-/-) mice. We demonstrated first that these mice are immunoresponsive to β3 integrin; β3-/- mice transfused with wild-type platelets generated specific anti–β3 antibodies which were able to induce thrombocytopenia in wild-type mice. Subsequently, β3-/- female mice (both naive and immunized) were bred with wild-type male mice to recapitulate the features of FNAITP. The titer of generated maternal antibodies correlated with the severity of FNAITP. High titer maternal anti–β3 anti-bodies caused severe fetal thrombocytopenia, intracranial hemorrhage, and even miscarriage. Furthermore, maternal administration of intravenous immunoglobulin G (IgG) ameliorated FNAITP and down-regulated pathogenic antibodies in both the maternal and fetal circulations.

Toll-like receptors (TLRs) play a critical role in stimulating innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) on invading microorganisms. Platelets also play a role in innate immunity, and we studied whether they express TLR. Results show that human and murine platelets variably expressed TLR2, TLR4, and TLR9 by flow cytometry and Western blotting. TLR4 expression was confirmed by demonstrating murine platelet binding to lipopolysaccharide (LPS). Thrombin activation of the platelets significantly enhanced the expression of TLR9, suggesting that at least some TLRs may derive from intracellular compartments. When LPS was administered to LPS-sensitive C3H/HeN and LPS-resistant C3H/HeJ mice, functional TLR4 expression in vivo was shown to be responsible for LPS-induced thrombocytopenia. However, when the C3H/HeN mice were first rendered thrombocytopenic by an antiplatelet antibody and then administered LPS, a significant reduction occurred in their ability to produce TNF-α. The decreased cytokine production in the thrombocytopenic mice was restored with platelet transfusion. These results suggest that platelets express various TLRs and that the functional significance of one of these, TLR4, appears to be a role in the modulation of LPS-induced thrombocytopenia and TNF-α production. This work implicates platelets as important mediators of innate immune responses against invading microorganisms.

Autoimmune hemolytic anemia (AIHA) is an autoimmune disorder in which autoantibodies are directed against an individual's own red blood cells (RBCs), leading to enhanced clearance through Fc receptor (FcR)-mediated phagocytosis. Although there is a large literature relating to clinical aspects of AIHA, relatively little work addresses how IgG autoantibodies are actually produced against RBC autoantigens. This review will first discuss the current understanding of autoimmunity in general and then focus on the knowledge of the immunopathogenic mechanisms responsible for autoantibody production in AIHA. Both human and animal studies will be discussed. Understanding theses mechanism is vital for developing antigen-specific immunotherapies to treat the disease.

BACKGROUND: Immunoglobulin G (IgG) anti-platelet (PLT) immunity has been shown to be initiated by indirect allorecognition where recipient T cells recognize donor PLT antigens presented by class II molecules encoded by the major histocompatibility complex (MHC) on recipient antigen-presenting cells. To understand how the recipient's MHC class II molecules may influence PLT alloimmunity, immune responsiveness against transfused PLTs was tested in different mouse strains. STUDY DESIGN AMD METHODS: Various inbred and mutant mouse strains were transfused with allogeneic PLTs and IgG donor antibodies were measured by flow cytometry. RESULTS: When recipient mice, expressing both MHC class II I-A and MHC class II I-E molecules, were transfused weekly with allogeneic PLTs, high titers of IgG donor antibodies were generated. In comparison, however, recipient mice expressing only MHC class II I-A molecules had significantly (p < 0.001) reduced IgG antibody responsiveness against PLT transfusions. The low IgG responder status against allogeneic PLT transfusions was rescued in transgenic mice expressing I-E molecules and in mice genetically deficient in either β2-microglobulin or CD8+ T cells. CONCLUSION: IgG immune responsiveness against allogeneic PLT transfusions is dependent on recipient expression of I-E MHC class II molecules, whereas I-A expression is linked with CD8-mediated suppression of PLT immunity. The data suggest that strategies to modify recipient MHC class II presentation of donor PLT antigens would be effective in eliminating PLT alloimmunity. (Less)

BACKGROUND: Development and characterization of methods for preventing transfusion-associated GVHD have utilized in vitro studies with human WBCs and in vivo studies in animal models. The limitation of these assays is that the in vivo GVHD response of treated human WBCs has not been tested directly. STUDY DESIGN AND METHODS: PBMNCs isolated from nonleukoreduced RBC units exposed to gamma irradiation, treated with PEN110 or PBS, were tested for their ability to induce xenogeneic GVHD when injected into severe combined immunodeficient (SCID) mice. RESULTS: These studies showed that the SCID mice injected with PBS-treated PBMNCs developed serum levels of human immunoglobulin that were followed by weight loss and display of ruffled fur characteristic of xenogeneic GVHD in these mice. In contrast, SCID mice injected with PEN110-treated or gamma-irradiated PBMNCs did not exhibit any of these responses. CONCLUSIONS: In these studies PEN110 treatment and gamma irradiation were equally effective at preventing in vivo GVHD responses when the treated cells were injected into SCID recipients. These results are consistent with previous results obtained when these two treatment methods were compared with in vitro studies with PBMNCs and in vivo studies in mouse models.

BACKGROUND: Immunoadsorption of plasma with Staphylococcal protein A removes immunoglobulins and immune complexes; hence, it should effectively remove inhibitors to FVIII in acquired or congenital hemophilia. The procedure may be cost effective, given the expense of therapies used to treat patients with inhibitors, particularly in an acute setting. STUDY DESIGN AND METHODS: Three patients with inhibitors to FVIII were treated with the Excorim Immunosorba system (two columns used in tandem). Costs for immunoadsorption and for other therapeutic products administered to the patients were calculated. RESULTS: Two patients had acquired hemophilia and severe bleeding associated with low levels of circulating FVIII and high levels of inhibitors to FVIII. They failed to achieve a satisfactory response to management with immunosuppression, pFVIII, recombinant FVIIa or IVIG but responded rapidly, with long-term benefit, to immunoadsorption therapy. The third patient had congenital hemophilia and immunoadsorption was effective in reducing his inhibitor level, allowing him to undergo immune tolerance therapy. Costs of treatment before immunoadsorption were markedly higher than those associated with the immunoadsorption procedures (i.e., >Can$350,000 and >Can$1,000,000 vs.

Previous results have demonstrated that anti-D therapy in children with chronic auto-immune thrombocytopenic purpura (AITP) induced a significant increase in several pro- and anti-inflammatory plasma cytokines within 2 hours of administration. To investigate the biologic basis of these early in vivo responses, we developed a flow cytometric assay to measure Fc-dependent responses of human peripheral leukocytes with fluorescently labeled and anti-D–opsonized red blood cells (RBCs). When anti-D–opsonized RBCs were incubated with peripheral blood leukocytes, the earliest detectible event observed was a significant oxidative burst in both monocytes (P < .05) and granulocytes (P < .0001), characterized by the production of hydrogen peroxide (H2O2), peroxynitrite (ONOO–), superoxide (O –2), and hydroxyl (OH) by 10 minutes which declined by 1 hour. By 2 hours, the opsonized RBCs were phagocytosed, particularly by granulocytes (P < .001), but the phagocytosis subsequently declined by 6 hours of incubation. The decline in phagocytosis was correlated with a significant production of interleukin-1 receptor antagonist (IL1ra) by both monocytes (P = .036) and granulocytes (P = .0002) within 4 hours. None of these events occurred if the RBCs were coated with anti-D F(ab)′2 fragments. When recombinant IL1ra was titrated into the assay, phagocytosis of the opsonized RBCs was significantly inhibited (P = .002). Taken together, these results suggest that at least one mechanism of action of anti-D is via the production of the anti-inflammatory cytokine IL1ra which can negatively regulate the ability of leukocytes to phagocytose opsonized cells.

Apoptosis or programmed cell death was discovered in nucleate cells 30 years ago and has been well documented. In contrast, apoptosis in anucleate platelets has only a five-year research history and as yet but few publications related to it. In this review, we will present the data on platelet apoptosis in several models. These include in vitro models where platelet apoptosis was induced by calcium ionophores, natural platelet agonists, storage in capped tubes at 37 degrees C and storage at room temperature under standard blood banking conditions, and in vivo models where apoptosis was provoked by suppression of thrombopoiesis, malaria infection and injection of tumor necrosis factor or anti-platelet antibodies. Understanding of platelet apoptosis and its role in the platelet storage lesion is an exciting challenge; future research is likely to provide us with further insight into this field.

Platelet adhesion and aggregation at the site of vascular injury are two key events in hemostasis and thrombosis. The contribution of several platelet receptors and their ligands has been highlighted in these processes. In platelet adhesion, particularly at high shear stress, GP1b-von Willebrand factor (vWF) interaction may initiate this event, which is followed by firm platelet adhesion mediated by members of the integrin family, such as beta1 (alpha2beta1, alpha5beta1) and beta3 (alphaIIbbeta3) integrins. In platelet aggregation, although GP1b-vWF, P selectin-sulfatides, and other molecules, may play some roles, the process is mainly mediated by beta3 (alphaIIbbeta3) integrin and its ligands, such as fibrinogen and vWF. Recent studies with perfusion chambers and intravital microscopy have revised the dogma established with the static (low shear stress) conditions. It is intriguing that platelet adhesion and aggregation do still occur in mice lacking both vWF and fibrinogen, suggesting that other unexpected molecule(s) may also be important in hemostasis and thrombosis.

The interaction of platelets with von Willebrand factor (VWF) is crucial in the initiation of any hemostatic or thrombotic process. VWF enables the platelet, via its surface glycoprotein receptors, to adhere to exposed subendothelium and to respond to shear stress in the blood. Via VWF that is stored and released from platelet alpha-granules and from Weibel-Palade bodies of endothelial cells, the hemostatic system can respond locally to lesions in the vessel wall and can initiate the activation of other platelets. This review describes the molecular structure of VWF, its functions and its interactions with the platelet membrane glycoprotein receptors GP Ib-IX-V and GP IIb-IIIa. As well, the role of VWF in shear-induced platelet adhesion and aggregation is described, and mechanisms are discussed that control the size of VWF multimers and the responsiveness of platelets to multimeric VWF. Finally, the review discusses the role of locally released VWF from platelet alpha-granules and from Weibel-Palade bodies for platelet activation in neonates and adults.

Autoimmune thrombocytopenic purpura (AITP) is a bleeding disorder in which autoantibodies are directed against an individual's own platelets, leading to enhanced clearance through Fc receptor (R)-mediated phagocytosis by macrophages residing in the reticuloendothelial system (RES), particularly in the spleen. The production of IgG autoantibodies is critically dependent on cellular immune mechanisms particularly relating to T cells. We review the recent literature of the cell-mediated immunology of AITP focusing on platelet phenotype, genetics, T-cell reactivities, and cytokine profiles in patients with AITP. Understanding the interaction between these cell-mediated mechanisms is vital for developing antigen specific immunotherapies to treat this autoimmune disease.

Plasma fibronectin (pFn) has long been suspected to be involved in hemostasis; however, direct evidence has been lacking. Here, we demonstrated that pFn is vital to control bleeding in fibrinogen-deficient mice and in WT mice given anticoagulants. At the site of vessel injury, pFn was rapidly deposited and initiated hemostasis, even before platelet accumulation, which is considered the first wave of hemostasis. This pFn deposition was independent of fibrinogen, von Willebrand factor, β3 integrin, and platelets. Confocal and scanning electron microscopy revealed pFn integration into fibrin, which increased fibrin fiber diameter and enhanced the mechanical strength of clots, as determined by thromboelastography. Interestingly, pFn promoted platelet aggregation when linked with fibrin but inhibited this process when fibrin was absent. Therefore, pFn may gradually switch from supporting hemostasis to inhibiting thrombosis and vessel occlusion following the fibrin gradient that decreases farther from the injured endothelium. Our data indicate that pFn is a supportive factor in hemostasis, which is vital under both genetic and therapeutic conditions of coagulation deficiency. By interacting with fibrin and platelet β3 integrin, pFn plays a self-limiting regulatory role in thrombosis, suggesting pFn transfusion may be a potential therapy for bleeding disorders, particularly in association with anticoagulant therapy.

Summary. Although the mechanism of action of intravenous immunoglobulin (IVIg) in treating antibody-dependent thrombocytopenia remains unclear, most studies have suggested that IVIg blocks the function of Fc receptors in the reticuloendothelial system (RES) and/or the protective effect may be due to the presence of variable region-reactive (anti-idiotype) antibodies within IVIg. We evaluated the effect of IVIg on platelet counts in a murine model of passively induced immune thrombocytopenia (PIT). Although IVIg was unable to neutralize the binding of two platelet-specific monoclonal antibodies to their target antigens either in vivo or in vitro, it was able to prevent PIT as well as ameliorate pre-established PIT mediated by these antibodies. IVIg adsorbed against the antibody used to induce thrombocytopenia or endogenous murine immunoglobulin also protected against PIT, indicating that antibodies with anti-idiotype activity present in IVIg are not necessary for its effective treatment of PIT. IVIg significantly blocked the ability of the RES to clear antibody-sensitized red blood cells. F(ab 0 )2 fragments of IVIg, which are unable to block the RES but retain the idiotypic regions, were ineffective at protecting mice from PIT. Our data suggest that IVIg exerts its rapid effect by inhibiting RES function and that anti‐idiotype interactions are not required.

Presensitization of donor platelets with allo-specific immunoglobulin (Ig)G results in a diminished immune response against subsequent transfusions of platelets. To understand better the mechanism of how alloantibody presensitization results in a decreased alloimmune response, we have used murine monoclonal antibodies directed to polymorphic and non-polymorphic regions of human leucocyte antigen (HLA) as well as platelet-specific molecules. Here, we demonstrated that presensitization with anti-human HLA class I antibodies, as well as beta2-microglobulin-specific antibody, protected against alloantibody production to five subsequent untreated platelet challenges. Use of complement fixing, non-fixing or F(ab')2 fragments of HLA-specific antibody also resulted in complete inhibition of alloantibody production. This protection was not seen when the platelets were presensitized with monoclonal antibodies to CD42a (GPIX), CD32 (low-affinity IgG/Fcgamma receptor) or murine IgG and was thus independent of B-cell FcgammaRII-mediated immune suppression. The inhibition observed was independent of HLA alloantigenic specificity as antibodies directed at the beta2-microglobulin portion of HLA class I were as effective as antibodies against any of the HLA-alpha regions (either polymorphic or non-polymorphic) of class I. This work demonstrates that monoclonal antibody-mediated suppression of the human HLA alloimmune response to platelet transfusion is antigen specific and is independent of FcgammaRII-mediated immune regulation, complement fixing or HLA alloantigenic specificity.

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen-activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine–threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal-related kinases (ERKs), c-Jun amino-terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF-induced human platelet activation. It is not known whether VWF-dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF-dependent platelet activation, we functionally inhibited ERK-dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK-1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF- or thrombin-induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF- or thrombin-dependent platelet activation.

Recipient IgG immunity against leukoreduced donor platelets is dependent on indirect T-cell allorecognition and is suppressed in vivo by inhibitors (aminoguanidine, AMG) of inducible nitric oxide synthase (iNOS). To examine recipient processing pathways of donor platelet antigens, enriched macrophages (antigen-presenting cells [APC]) from BALB/c (H-2d) mice were pulsed with allogeneic C57BL/6 (H-2b) platelets and transfused weekly into naive BALB/c mice. Platelet-pulsed APC stimulated IgG antidonor antibody production in 45% of recipients by the second transfusion and in 100% by the sixth transfusion; this response was enhanced by pulsing in the presence of interferon-γ. By the sixth transfusion, high-titer IgG1 (mean titer 4990) and IgG2a (1933) isotypes specific for donor major histocompatibility complex (MHC) class I antigens were detected. Platelet pulsing in the presence of AMG or colchicine significantly inhibited the ability of APC to stimulate IgG alloantibodies; only 50% (P

We have investigated the role of p38 mitogen-activated protein kinase (MAPK) in von Willebrand factor (VWF)-dependent platelet activation. The interaction of platelets with subendothelial VWF, especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. As a model of VWF-dependent platelet activation, porcine VWF was employed. Porcine VWF induced p38 MAPK activation by 1 min post-addition; assessed by phosphorylation of a recombinant p38 MAPK fusion protein substrate termed glutathione S-transferase-MAPK activated protein kinase-2. To determine if p38 MAPK was necessary for porcine VWF-induced platelet activation, we functionally inhibited p38 MAPK activity with SB203580 before exposure of the platelets to porcine VWF. Inhibition of p38 MAPK had no effect on VWF-induced platelet alpha or lysozomal granule release, expression of activated GPIIb IIIa, modulation of membrane glycoprotein CD41, expression of phosphatidylserine as assessed by annexin V binding, microparticle formation, or platelet agglutination. It was concluded that SB203580-inhibitable p38 MAPK activity induced by porcine VWF is not necessary for platelet activation.

Severe combined immune deficient (SCID) mice were engrafted with human (Hu) peripheral blood lymphocytes (PBL) from a previously alloimmunized donor and transfused with HLA-mismatched platelets. We have previously shown this to be a useful model for platelet transfusion. These engrafted mice (Hu-PBL-SCID mice) produced high levels of alloantibody in response to standard platelet preparations. However, when the first platelet challenge was presensitized with anti-HLA antibody and then transfused there was a marked reduction in the amount of alloantibody produced to five subsequent untreated platelet transfusions. Platelets pretreated with platelet-specific anti-HPA-1a (PLA1) sera did not induce a decrease in the anti-HLA alloantibody response. This demonstrated that platelet-induced HLA alloimmunization can be blocked by anti-HLA antibody-sensitized cells.

In a murine model of platelet alloimmunization, we examined the definitive role that mononuclear cells (MC) have in modulating platelet immunity by using platelets from severe combined immunodeficient (SCID) mice. CB.17 (H-2d) SCID or BALB/c (H-2d) mouse platelets were transfused weekly into fully allogeneic CBA (H-2k) mice and antidonor antibodies measured by flow cytometry. MC levels in BALB/c platelets were 1.1 ± 0.6/μL and SCID mouse platelets could be prepared to have significantly lower (

Platelets express Toll-like receptor 4 (TLR4), and this has been shown to be responsible for the thrombocytopenia induced by lipopolysaccharide (LPS) administration in vivo. We studied the role of LPS in mediating platelet phagocytosis by THP-1 cells in vitro by flow cytometry. Opsonization of platelets with an IgG monoclonal (W6/32) antibody or with IgG autoantibody-positive sera from patients with autoimmune thrombocytopenia (AITP) significantly enhanced platelet phagocytosis (P < .001). In contrast, platelet phagocytosis did not occur if platelets were bound with only LPS. If, however, the LPS-bound platelets were also opsonized with either W6/32 or autoantibody-positive sera with titers greater than 4, there was a significant and synergistic increase in Fc-dependent platelet phagocytosis (P < .001, P = .003, P = .048, and P = .047). These results suggest that, in the presence of antiplatelet antibodies, bacterial products can significantly alter platelet phagocytosis, and this may have relevance to how Gram-negative infections enhance platelet destruction in some patients with AITP.

Sera from HIV-1-infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate, HIV-p24 and HIV-gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid-sensitive and alkaline-sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV-lysate columns; 96% (25/26) of the acid-eluted antibodies were HIV-specific but 48% (12/25) of the alkaline-eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline-eluted HIV-p24 antibodies, 40% (8/20) reacted specifically with HIV-p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline-eluted HIV-gpl20 antibodies (n=17), 88% (15/17) were HIV gpl20-specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti-p24 antibodies recognized three bands with approximate molecular weights of 72 000 to 95 000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti-HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline-sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV-infected haemophiliacs.

Chronic autoimmune thrombocytopenic purpura (AITP) is an organ specific autoimmune bleeding disease in which autoantibodies are directed against the individual's own platelets, resulting in increased Fc-mediated platelet destruction by macrophages in the reticuloendothelial system. Although AITP is primarily mediated by IgG auto-antibodies, their production is regulated by the influence of T lymphocytes and antigen presenting cells (APC). This review argues that enhanced T helper cell/antigen presenting cell interactions in patients with AITP may be responsible for IgG anti-platelet auto-antibody production. Understanding these cellular immune responses in AITP may lead to the development of more immune specific therapies for the management of this disease.

Expression of the T-cell receptor (TCR) zeta chain in normal individuals was studied by two-color flow cytometric analysis using digitonin-permeabilized human peripheral blood lymphocytes. Optimal detection of the TCR zeta chain involved fixation of cells in 0.25% paraformaldehyde for 2 min and permeabilization with 500 microg/ml of digitonin at 4 degrees C. Permeabilized lymphocytes and monocytes displayed decreased forward light scatter properties. Neutrophils did not survive this permeabilization/fixation/washing procedure. Permeabilization did not affect the ability of antibodies to CD3, CD4, CD8, CD16, and CD19 to detect CD antigens on lymphocytes, and the percentage of lymphocytes reacting with these antibodies was comparable between untreated and permeabilized cells. Staining of the TCR zeta chain was accomplished by incubating permeabilized cells with unconjugated antibody to the zeta chain, followed by an FITC-conjugated F(ab')2 goat anti-IgG. Following TCR zeta chain staining, cells were blocked with 25 microg/ml of mouse IgG for 20 min to saturate the goat anti-mouse antibody and then incubated with a phycoerythrin-conjugated mouse antibody to a variety of lymphocyte surface antigens. The TCR zeta chain was observed in lymphocytes displaying CD3, CD4, CD8, CD16, or TCRgammadelta markers. This permeabilization/fixation/washing procedure was also validated for detection of another intracellular T-cell protein known as the cytolytic granule-associated protein, recognized by the TIA-1 antibody. Thus the technique can be applied to detection of at least two different intracellular T-cell markers.

The pathophysiology of platelet dysfunction in the Wiskott-Aldrich immune deficiency syndrome (WAS) remains unclear. Using flow cytometry, we have characterized the functional properties of platelets from 10 children with WAS. Patients with WAS had thrombocytopenia, small platelets, increased platelet-associated IgG and reduced platelet-dense granule content. Levels of reticulated 'young' platelets were normal in the WAS patients. Although the mean numbers of platelet glycoprotein (GP) Ib, GPIIbIIIa and GPIV molecules per platelet appeared lower in WAS patients than in healthy controls, analysis of similar-sized platelets revealed the mean number of GPIb molecules per platelet to be comparable in patients and normal controls. Surface GPIIbIIIa and GPIV expression was, however, significantly lower on the WAS platelets than on normal platelets. Compared with normal platelets, WAS platelets showed a reduced ability to modulate GPIIbIIIa expression following thrombin stimulation. In addition, thrombin- and ADP- induced expression of CD62P and CD63 was defective in WAS platelets. Phallacidin staining of the WAS platelets revealed less F-actin content than in normal platelets. Together, these data suggest that the reduced platelet number and function in WAS reflects, at least in part, a defect in bone marrow production as well as an intrinsic platelet abnormality. (Less)

The roles of the G-protein-linked thrombin receptor and platelet glycoprotein Ib (GPIb) as alpha-thrombin-binding sites on platelets remain controversial. alpha-Thrombin has been proposed to bind to both GPIb and the hirudin-like domain of the G-protein-linked receptor (from which it cleaves the NH2-terminal extracellular domain to release a 41-mer peptide (TR-(1-41), where TR is alpha-thrombin receptor)) to initiate platelet activation. Using affinity-purified rabbit anti-human TR-(1-41) IgG and immunoblotting, we demonstrated TR-(1-41) release from platelets suspended in Tyrode's buffer containing 2 mM CaCl2 and incubated with >/=0.5 nM alpha-thrombin for 10-60 s at 37 degrees C. As quantified by enzyme-linked immunosorbent assay, 0.32-0.59 nM TR-(1-41) was released from washed platelets (5 x 10(11) platelets/liter) after their incubation with 10 nM alpha-thrombin for 10 s. Parallel binding of alpha-thrombin to and activation of the platelets were confirmed by flow cytometry. A monoclonal antibody against the hirudin-like domain of the G-protein-linked receptor abrogated alpha-thrombin binding to platelets, cleavage of TR-(1-41), and platelet activation by

Platelet transfusions are life-saving treatments for many patients with thrombocytopenia; however, their use is generally discouraged in the autoimmune disorder known as immune thrombocytopenia (ITP). We examined whether allogeneic platelet major histocompatibility complex (MHC) class I transfusions affected antiplatelet CD61-induced ITP. BALB/c CD61 knockout mice (CD61(-)/H-2(d)) were immunized against platelets from wild-type syngeneic BALB/c (CD61(+)/H-2(d)), allogeneic C57BL/6 (CD61(+)/H-2(b)), or C57BL/6 CD61 KO (CD61(-)/H-2(b)) mice, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP. When nondepleted splenocytes were transferred to induce antibody-mediated ITP, both CD61(+) platelet immunizations generated immunity that caused thrombocytopenia independently of allogeneic MHC molecules. In contrast, when B-cell-depleted splenocytes were transferred to induce T-cell-mediated ITP, transfer of allogeneic MHC-immunized splenocytes completely prevented CD61-induced ITP development. In addition, allogeneic platelet transfusions into SCID mice with established CD61-induced ITP rescued the thrombocytopenia. Compared with thrombocytopenic mice, bone marrow histology in the rescued mice showed normalized megakaryocyte morphology, and in vitro CD61-specific T-cell cytotoxicity was significantly suppressed. These results indicate that antibody-mediated ITP is resistant to allogeneic platelet transfusions, while the T-cell-mediated form of the disease is susceptible, suggesting that transfusion therapy may be beneficial in antibody-negative ITP.

SummaryFetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life-threatening bleeding disorder caused by maternal antibodies against fetal/neonatal platelets. FNAIT is also linked with miscarriages, although the incidence and mechanisms of fetal death have not been well studied. Integrin αIIbβ3 (GPIIbIIIa) and the GPIbα complex are major glycoproteins expressed on platelets and are also major antigens targeted in autoimmune thrombocytopenia (ITP), but reported cases of anti-GPIb-mediated FNAIT are rare. Bacterial and viral infections have been causally linked with the pathogenesis of immune-mediated thrombocytopenia (ITP); however, it is unknown whether these infections contribute to the severity of FNAIT. Here, immune responses against platelet antigens were examined by transfusing wild-type (WT) mouse platelets into β3-/- or GPIbα-/- mice. To mimic bacterial or viral infections, lipopolysaccharide (LPS) or polyinosinic:polycytidylic acid (Poly I:C) were injected intraperitoneally following platelet transfusions. The FNAIT model was established by breeding the immunised female mice with WT male mice. We demonstrated for the first time that the platelet GPIbα has lower immunogenicity compared to β3 integrin. Interestingly, co-stimulation with LPS or Poly I:C markedly enhanced the immune response against platelet GPIbα and caused severe pathology of FNAIT (i.e. miscarriages). LPS or Poly I:C also enhanced the immune response against platelet β3 integrin. Our data suggest that bacterial and viral infections facilitate the anti-platelet GPIbα response, which may lead to a severe non-classical FNAIT (i.e. miscarriage but not neonatal bleeding) that has not been adequately reported in humans.

Leukoreduced allogeneic platelet transfusions have been previously shown to initially stimulate an in vitro cellular cytotoxicity and subsequently Induce the formation of immunoglobulin G (IgG) antidonor alloantibodies. To further characterize these responses and determine if they are related, recipient BALB/c H-2d mice were treated with aminoguanidine (AMG), a selective inhibitor of inducible nitric oxide synthase (iNOS), and transfused weekly with 2 x 10(8) C57BL/6 H2b platelets. In control, non-AMG-treated mice, transfusion significantly (P < .01) increased serum levels of interferon-gamma (IFN-gamma) by day 1 posttransfusion (PT). IFN-gamma returned to pretransfusion levels by day 3 PT, and its production was not affected by AMG treatment. Serum interleukin-4 (IL-4), on the other hand, was undetectable before and during the transfusion protocol. By day 3 PT, recipient spleen cells could mediate in vitro anti-P815 (auto), anti-EL4 (allo), and anti-R1.1 (third-party MHC) cytotoxicity, and these responses were maximal by day 7 PT. Concurrently, a significant reduction in the vitro ability of recipient splenocytes to respond to Concanavalin A (ConA) was observed; this was not seen with lipopolysaccharide (LPS) stimulation. Elevated levels of NO2- were found in the ConA culture supernatants from transfused mice at day 3 PT. Serum antidonor alloantibodies were detected by the fifth platelet transfusion. AMG treatment of recipient mice significantly inhibited the transfusion. Induced cytotoxicity and ConA-stimulated NO2- production, and restored ConA-induced proliferation to normal levels. AMG appeared to selectively inhibit platelet-induced alloantibody production in that it did not affect antibody production induced by transfusions with 10(5) allogeneic leukocytes or by immunization with a foreign protein antigen, human gamma globulin, in adjuvant therapy. These results indicate that an in vivo AMG-sensitive mechanism is essential for recipients to initiate a humoral IgG immune response against allogeneic platelets.

Thrombin accelerates its own production by activating platelets (and perhaps other cells) to provide coagulant surfaces on which prothrombinase, the enzyme complex that activates prothrombin in plasma and consists of equimolar factor Xa (serine protease) and factor Va (cofactor), assembles in a Ca(2+)-dependent reaction. Thrombin also activates factor V to provide the cofactor for factor Xa in prothrombinase, and factor VIII to provide the cofactor for factor X activation by factor IXa. Even when factor X activation is initiated by tissue factor, efficient propagation of factor X activation is critically dependent on intrinsic tenase (the factor IXa and factor VIIIa enzyme complex). These procoagulant actions of thrombin explain why good antithrombins can inhibit both intrinsic and extrinsic prothrombin activation. Because of the low level of thrombin (1 nM) required to initiate platelet activation and the activation of platelet-bound factor V, only highly efficient antithrombins, such as hirudin, can abrogate platelet-dependent prothrombinase assembly.

Flow cytometric analysis of blood leukocytes is currently used for both routine clinical measurements as well as for cutting edge research applications. This technology has enabled rapid and accurate determination of leukocyte antigens and quantitative analysis of leukocyte subsets, tests of leukocyte function, determination of the presence of antineutrophil and antilymphocyte antibodies in plasma and on cells, measurement of CD34+ hematopoietic stem cells in peripheral blood and bone marrow samples, measurement of apoptosis, and detection of virusinfected leukocytes. This review will focus on the use of the flow cytometer for investigations of blood leukocytes in transfusion medicine.

Platelet apoptosis and activation have been studied in human platelets treated with BH3-only mimetic ABT-737 and calcium ionophore A23187, agents triggering apoptosis through the intrinsic mitochondrial pathway. Platelet apoptosis was determined as activation of crucial apoptosis-associated caspases, initiator caspase-9 of intrinsic apoptosis pathway, executioner caspase-3 and initiator caspase-8 of extrinsic death receptor pathway, and platelet activation was detected by P-selectin (CD62) exposure on the platelet surface. We found that ABT-737 predominantly induced activation of caspases-9, -3 and -8 rather than CD62 exposure, whereas A23187 induces both caspases activation and CD62 exposure. Caspase-8 activation was stimulated independently of the extrinsic apoptosis pathway via mitochondrial membrane permeabilization and depolarization. These data suggest that (i) caspase-8 activation is triggered in ABT-737- and A23187-treated anucleate platelets through the mitochondria-initiated caspase activation cascade bypassing the death receptors, and (ii) ABT-737-treated platelets are a useful experimental tool for discerning the role of platelet apoptosis in platelet function and survival.