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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.

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.

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless, many details of the immunopathogenesis of TRALI, particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast, 34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia, severe pulmonary edema, and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8+ T lymphocytes or CD4+ T cells but not CD19+ B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia, lung damage, and mortality, suggesting that T lymphocytes were responsible for the protective effect. Taken together, these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions.

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.

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.

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.

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.

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.

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.

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

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.

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.

Approximately 30% to 40% of patients with acute leukemia receiving repeated pooled random-donor platelet transfusions develop anti-HLA alloantibodies. Over time, however, serum anti-HLA concentrations decrease in approximately 50% of these patients, despite continued exposure to platelet and/or red blood cell transfusions. Using an enzyme-linked immunosorbent assay to measure serum Igs, the present study demonstrates that the sera of 67% of 82 transfused patients exhibiting a decrease in anti-HLA contain antibodies (anti-idiotypes) that react with the variable (V) region of anti-HLA antibodies. Anti- HLA binding to platelet membranes could be inhibited by these serum antibodies in 36% of the patients, indicating they had paratope-related reactivity. Protein G sepharose absorption showed that the anti-HLA V region-reactive antibodies were IgG. Of the 43 patients who had a decrease in anti-HLA levels, that were 16 whose anti-HLA decreased to undetectable levels; 7 (44%) developed anti-idiotypic antibodies that could specifically inhibit their own previously anti-HLA-positive serum. In contrast, antibodies with reactivity to the V region of anti- HLA antibodies (anti-idiotypes) were not demonstrable in patients who developed anti-HLA that did not decrease or disappear. The findings suggest that the development of anti-HLA V region-reactive antibodies (anti-idiotypic antibodies) correlates with a decrease in anti-HLA antibody formation in patients multiply transfused with platelet concentrates. The observations indicate that anti-idiotypic antibodies may downregulate alloimmune responses in patients undergoing repeated allostimulation during platelet transfusion therapy.

Background:Immune thrombocytopenia (ITP) is a common bleeding disorder. Autoantibodies against platelet GPIIbIIIa (integrin αIIbβ3, 70-80%) and GPIb-complex (20-40%) are considered to be the major mechanism leading to autologous platelet destruction. Recent studies demonstrated that in addition to autoantibodies, CD8+ cytotoxic T cells (CTLs) also contribute to thrombocytopenia, either through direct cytotoxicity against platelets or megakaryocytes. However, the roles of CD8+ regulatory T cells (Tregs) in ITP have not been adequately explored. Methods and Results: We developed the first animal models of steroid treatment in ITP, encompassing both the passive and active forms. In the passive model, we injected anti-β3 antibodies to induce transient antibody mediated thrombocytopenia. We found that a single intraperitoneal (IP) injection of steroids post-antibody injection was effective at rescuing platelet counts. We also adapted an active model of ITP whereby wild-type (WT) BALB/c mice were transfused with splenocytes from WT platelet immunized β3-/-mice. This model encompasses both antibody and cell-mediated ITP resulting in sustained thrombocytopenia. In this model, we found steroid treatment (prednisone and dexamethasone) administered daily either orally or through IP-injection were equally efficacious at ameliorating thrombocytopenia. Furthermore, immunophenotyping and cytokine analysis reveal a similar profile as reported of human ITP patients responsive to steroid treatments. Thus, successful steroid treatments in these animal models are representative of the therapeutic effects of steroid treatments seen in human ITP patients. To study the role of CD8+ T cells in the pathogenesis and response to steroid treatments in ITP, we depleted CD8+ T cells from splenocytes prior to its transfusion into WT mice. Unexpectedly,we found CD8+ T cell depleted splenocyte (lacking in CTL cells) engrafted mice had lower, but not higher, platelet counts and were less responsive to dexamethasone (DEX) treatment compared to non-depleted engrafted mice. Furthermore, in the passive ITP model, depletion of CD8+ T cells from mice prior to injection of anti-β3 antibodies resulted in more severe thrombocytopenia, compared with non-depleted mice. Conversely, transfusion of either antigen-primed CD8+ (isolated from immunized β3-/- splenocytes) or WT/β3-/- naïve CD8+ T cells alone was sufficient to rescue platelet counts and improve response to DEX in the passive ITP model. These results indicate for the first time that CD8+ T cells from both antigen-primed and naïve populations play a protective role in attenuating platelet clearance. In further support of these observations, we detected significant increased populations of both CTLs and CD8+ Tregs including, CD8+CD25+Foxp3+, CD8+CD103+, CD8+CD122+ and CD8+CD28- in the blood, and spleen of immunized β3-/- mice. Interestingly, the CD8+ Tregs populations were further increased while CTL population decreased following DEX treatment in the active ITP model. In vitro splenocyte cultures were used to explore putative regulatory mechanisms of CD8+ Tregs. It was found that antigen-primed CD8+ Tregs exerted significantly stronger inhibition CD4+ T- and CD19+ B cell proliferation, platelet apoptosis, and platelet associated IgG production in the presence of platelet antigens, while both antigen-primed and naïve CD8+ Tregs could effectively inhibit macrophage mediated phagocytosis of anti-β3 opsonized platelets. Conclusion: To the best of our knowledge, these are the first reported animal models of effective steroid treatment of ITP. Utilizing these models we uncovered a previously unidentified regulatory role of CD8+ T cells in both ITP and steroid treatment. The increased populations of various CD8+ Tregs following β3-/- immunization exerted a significant inhibitory function against other immune-cell mediated anti-platelet responses. In addition, therapeutic administration of both antigen-primed and naïve CD8+ T cells were able to rescue platelet counts in the passive ITP model. This suggests that CD8+ Treg may play a predominantly protective role in ITP. These data provides significant insights into the understanding of immunopathogenesis of ITP, which may be important in designing effective therapy including the potential usage of CD8+ Tregs as a cellular target in the treatment of ITP. Disclosures No relevant conflicts of interest to declare.

Platelet adhesion and aggregation at sites of vascular injury are key events in thrombosis and hemostasis. Platelet β3 integrins and their ligands are essential in mediating these processes. Therefore the understanding of β3 integrin-ligand interactions is crucial in elucidating mechanisms of thrombosis and hemostasis. In an effort to identify unknown ligands for β3 integrin, we used immobilized human platelet β3 integrin to capture proteins from human plasma. The isolated proteins were further analysed by 2D electrophoresis and mass spectrometry, and apolipoprotein A-IV (apoA-IV) was identified. ApoA-IV is an abundant plasma lipid binding protein secreted by the small intestine during dietary lipid absorption. Several studies in different ethnic populations have suggested that the level of apoA-IV is inversely correlated with cardiovascular diseases. However, the roles of apoA-IV in platelets and thrombosis are completely unknown. A single-molecule technique, biomembrane force probe (BFP), was employed to detect direct interactions between apoA-IV and platelet αIIbβ3 integrin. The BFP adhesion frequency assay demonstrated that apoA-IV bound to αIIbβ3 integrin on ADP treated platelets. ApoA-IV also bound to purified activated αIIbβ3 integrin or the integrin expressed on Chinese hamster ovary (CHO) cells. In comparison, apoA-IV did not significantly bind to αIIbβ3 integrin on resting platelets, GPIb-complex expressed on CHO cells, αMβ2 integrin expressed on K562 cells, nor purified α5β1 and αvβ3 integrins. Importantly, apoA-IV-αIIbβ3 interactions in these experiments could be completely inhibited by a blocking monoclonal antibody (M1) against β3 integrin. These data clearly demonstrated the specificity of apoA-IV for αIIbβ3 integrin. Furthermore, 2D kinetics measurements revealed that the effective 2D affinity of apoA-IV-αIIbβ3 is 43% of that between fibrinogen and αIIbβ3. The BFP competition assay showed that apoA-IV competitively inhibited fibrinogen-αIIbβ3 interactions at its physiological concentration. Platelet functional studies in vitro showed that recombinant apoA-IV significantly inhibited both mouse and human platelet aggregation following stimulation with various agonists. Consistently, platelet aggregation in platelet rich plasma of apoA-IV deficient mice (apoA-IV-/-) was enhanced. Depletion of human plasma apoA-IV also enhanced ADP-induced human platelet aggregation. In ex vivo perfusion chambers, recombinant apoA-IV inhibited human and mouse thrombus growth and dissolved pre-formed thrombi, while absence of apoA-IV in blood enhanced ex vivo thrombus growth under both low and high shear stresses. Using two in vivo intravital microscopy thrombosis models and a carotid artery thrombosis model, we demonstrated that FeCl3- and laser-induced thrombosis were enhanced in apoA-IV-/-mice, while transfusion of recombinant apoA-IV markedly attenuated this process. In addition, we found recombinant apoA-IV significantly decreased platelet P-selectin expression, and consistently more P-selectin expression was observed on ADP treated platelets from apoA-IV-/- mice, suggesting that apoA-IV occupancy may inhibit fibrinogen or other prothrombotic ligands mediated αIIbβ3 outside-in signaling. We further found that the N-terminus of apoA-IV plays a key role in its inhibitory function and the exposure of N-terminus is negatively regulated by its C-terminus. Furthermore, mutation of the two aspartic acid (D) residues at apoA-IV N-terminal 5 and 13 abolished its binding for αIIbβ3 integrin as demonstrated by BFP adhesion frequency assay, resulting in the loss of these inhibitory effects.These findings suggest that D5 and D13 of apoA-IV are the potential binding sites for αIIbβ3 integrin. Thus, apoA-IV is identified as a novel endogenous inhibitor of thrombosis and represents a new link between lipoprotein metabolism and platelet function, both of which play critical roles in cardiovascular diseases. These findings may also contribute to hemostasis, P-selectin mediated postprandial platelet activation and inflammation. Disclosures No relevant conflicts of interest to declare.

Background:Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa (70-80%) and/or GPIb-complex (20-40%). Current theory suggests antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc-FcγR interactions. However, evidence from us and others demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP models (Blood 2006) and subsequent IVIG studies in human ITP patients, including our recent large patient cohort study (JTH 2014). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a presently unidentified mechanism different than that of anti-GPIIbIIIa. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa-/- or GPIba-/- mice, which also recognize GPIbα and GPIIbIIIa of different species including human. Flow cytometry, immunofluorescence, and western blotting were used to evaluate whether these mAbs induced platelet activation, neuraminidase-1 translocation and desialylation of the heavily glycosylated GPIbα in the presence of sialidase inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). These experiments were repeated with human platelets and human ITP patient plasma. We further investigated the effects of anti-GPIbα antibodies on platelet activation, desialylation and clearance in vivo; BALB/c mice were injected with anti-GPIbα or anti-GPIIbIIIa mAbs and following, platelet activation and desialylation were measured by flow cytometry. Hepatocytic Ashwell-Morell receptor (AMR) mediated anti-GPIbα platelet clearance in the liver was examined using immunohistochemistry or blocking the AMR with asialofetuin in both wild-type and macrophage depleted mice. Therapeutic administration of DANA in a murine ITP model assessed the significance of Fc-independent anti-GPIbα mediated platelet clearance in ITP. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induced significant P-selectin expression, JON/A binding, neuraminidase-1 translocation and desialylation in murine platelets. Interestingly, certain human platelets were activated (P-selectin expression) and desialylated in the presence of both anti-GPIbα and anti-GPIIbIIIa mAbs or ITP patient plasma. However, we demonstrate that the anti-GPIIbIIIa antibody mediated platelet effects are dependent on the FcγRIIa present exclusively on human platelets as FcγRII blocker IV.3 completely attenuated the response. In contrast, IV.3 had little effect on anti-GPIbα mediated platelet activation or desialylation. Anti-GPIbα Fab fragments and platelet signal pathway inhibitors demonstrate that anti-GPIbα mediated platelet activation and desialylation are consequences of GPIbα cross linking and are reinforced by a positive feedback loop. In vivo, we found significant increases in P-selectin and desialylation in anti-GPIbα injected mice, independent of IgG subclass. A significant role for the hepatic AMR in the clearance of deglycosylated platelets was observed; particularly in macrophage depleted mice whereby, although anti-GPIIbIIIa mediated platelet clearance was completely attenuated, anti-GPIbα mediated platelets clearance still occurred, but was completely rescued with asialofetuin. Immunohistochemistry revealed significant co-localization of anti-GPIbα opsonized platelets with AMR. These suggest the AMR is the dominant Fc-independent anti-GPIbα mediated platelet clearance pathway in the absence of macrophages. Remarkably, sialidase inhibitor DANA ameliorated anti-GPIbα mediated thrombocytopenia in mice. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce platelet activation leading to GPIbα desialyation and platelet clearance via a novel Fc-independent pathway: the hepatic AMR. Our data also suggested that some anti-GPIIbIIIa autoantibodies in human patients may also induce platelet activation and desialylation via the platelet FcR signaling pathway. These findings may lead to novel therapeutic regimens including designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of both anti-GPIIbIIIa and anti-GPIbα mediated and/or refractory ITP. Disclosures No relevant conflicts of interest to declare.

Background Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a severe bleeding disorder that often results in intracranial hemorrhage (ICH), leading to neurological impairments or death. FNAIT has also been linked with miscarriage although the incidence of fetal loss has not been adequately studied. It is the most common cause of severe thrombocytopenia in live born neonates and accounts for up to 35% of all neonates admitted into the intensive care unit. In FNAIT, maternal alloantibodies cross the placenta and target paternally-derived fetal platelet antigens, most commonly platelet GPIIbIIIa (αIIbβ3 integrin). Polymorphisms in human platelet antigen-1a (HPA-1a), located on the integrin β3 subunit are the most common cause of FNAIT and are most widely studied, however the reported incidence of anti-αIIb-mediated FNAIT has recently increased; but the pathogenesis of this disease is unclear. Methods and Results To establish mouse models of anti-αIIb antibody mediated FNAIT, αIIb deficient (αIIb-/-) and human αIIb transgenic mice were employed in this study. We first immunized female αIIb-/- mice by weekly transfusions of wild type (WT) mouse platelets 2 times or 4 times. We demonstrated that the αIIb-/- mice were immunoresponsive to the αIIb antigen and both IgG1 and IgG2 antibodies against αIIb (i.e.TH2-like and TH1-like immune response, respectively) were detected two weeks after platelet transfusion. To test whether αIIb antisera can cause platelet destruction, we injected the antisera into WT mice and found severe thrombocytopenia in the recipient mice (P We found the severity of symptoms was correlated with the level of antibody titers in the maternal circulation. In 2 times and 4 times immunized females, miscarriage was observed (n= 2/9; and n =3/3, respectively). Upon post mortem dissection of miscarrying mothers at embryonic day 15.5, fetuses possessed ICH in 2 times and 4 times immunized females (n=2/14; and 5/12, respectively). Live born neonates from 2 times immunized mothers showed severe thrombocytopenia (P In addition to the knockout mouse model, we also used human αIIb transgenic mice, which do not express mouse αIIb on their platelets, in order to generate an alloimmune model of FNAIT. This model may better mimic the conditions of human FNAIT caused by the polymorphisms in αIIb genes. Following the same protocol as we used in the αIIb-/- mice, we found anti-murine αIIb antibodies were induced although the antibody titer was slightly lower. After breeding with male WT mice, immunized female human αIIb transgenic mice deliver pups with thrombocytopenia and ICH. Miscarriage was also observed, however the severity and symptoms of the disease were less. Furthermore, we also established another model of mouse-anti-human FNAIT by breeding humanized αIIb male mice with immunized female WT mice. We will compare the differences in these iso- and alloimmune models to elucidate the pathogenesis of αIIb mediated FNAIT. Conclusions and Significance We established the first anti-αIIb model of FNAIT and the first alloimmune animal models of FNAIT. Our results indicate that these models recapitulate human disease symptoms such as; lower platelet counts, bleeding diasthesis, and decreased birth weight compared to controls. Both neonates and fetuses possess ICH, and some immunized mothers underwent miscarriage. These models give us the opportunity to compare iso- vs. alloimmune FNAIT, and to compare the pathology between anti- αIIb and our anti-β3 mediated FNAIT. Disclosures: No relevant conflicts of interest to declare.

We have read with interest the paper from Hansen and Balthasar[1][1] regarding the amelioration of thrombocytopenia in a rat model of immune thrombocytopenia (ITP). The authors induce thrombocytopenia with a monoclonal antiplatelet antibody and find that pretreatment with IVIG (intravenous

Abstract 265 Background: Immune thrombocytopenia (ITP) is an autoimmune disease characterized by autoantibodies directed at patient's own platelet antigens, primarily glycoprotein (GP)IIbIIIa-integrin (70–80%) and GPIb-complex (20–40%). Current paradigm suggests that clearance of opsonized platelets through the reticuloendothelial system via Fcγ-receptors results in thrombocytopenia and bleeding disorders. However, evidence from others and our group demonstrated that anti-GPIbα, but not anti-GPIIbIIIa, can induce thrombocytopenia via an Fc-independent pathway, which is resistant to intravenous IgG (IVIG) therapy in murine ITP-models (Blood 2006). These observations are consistent with subsequent IVIG studies in human ITP patients. Interestingly, human anti-GPIb-mediated ITP patients seem also resistant to steroid therapy in our recent retrospective study (American Journal of Hematology 2012). This suggests that binding of anti-GPIbα antibodies may induce platelet clearance through a different mechanism which is currently poorly understood. Methods: We developed unique mouse anti-mouse monoclonal antibodies (mAbs) in GPIIIa or GPIba deficient mice. Some of the mAbs have cross-reactivity to both mouse and human GPIIbIIIa and GPIba. Flow cytometry was used to evaluate whether these mAbs were able to induce platelet activation, apoptosis and desialylation. GPIbα is heavily glycosylated and the role of desialylation and exposure of underlying galactose and β-N-acetyl-D-glucosamine (βGN) residues on GPIbα in platelet clearance was assessed using the sialidase neuraminidase (NA) and it's inhibitor N-acetyl-2,3-dehydro-2-deoxy neuraminic acid (DANA). Desialyation effects on platelet activation and apoptosis was measured by flow cytometry. We also repeated these experiments with human platelets and plasma from human ITP-patients. We also investigated the effects of anti-GPIbα antibodies on platelet activation, apoptosis and clearance in vivo. Briefly, BALB/c mice were injected with anti-GPIbαor anti-GPIIIa mAbs and 24 hrs later, platelet desialylation, activation and apoptosis were measured by flow cytometry. The effect of desialylation on platelet clearance was assessed with DANA. The possible roles of Ashwell-Morell and MAC-1 receptors in GPIbα-mediated platelet clearance in the liver were examined using immunohistochemistry (anti-CD11b) or blocking of the Ashwell-Morell receptor with asialofetuin. Results and Discussion: We found that anti-GPIbα, but not anti-GPIIbIIIa mAbs, induced significant P-selectin expression and phosphatidylserine (PS)-exposure, and increased inner membrane mitochondrial depolarization (ΔYm). Interestingly, platelets were desialylated in the presence of anti-GPIbα but not anti-GPIIbIIIa mAbs. Moreover, we found that desialylation of GPIbα lies directly upstream of platelet activation and apoptosis, as prior treatment with DANA diminished PS-exposure, and P-selectin expression. Most importantly, incubations of human platelets with ITP-patient plasma showed similar effects. In vivo, we found significant increases in PS-exposure and ΔYm induced by anti-GPIbα, but not by anti-GPIIIa mAbs, independent of IgG subclass. Interestingly, prior injection with DANA rescued platelets numbers in anti-GPIbα, but not in anti-GPIIIa injected mice. A significant role for the Ashwell-Morell and MAC-1 receptors in the clearance of deglycosylated platelets was observed; blocking of the Ashwell-Morell receptors by asialofetuin, decreased platelet clearance in anti-GPIbα, but not anti-GPIIbIIIa antibody injected mice, there was also increased staining for MAC-1 on Kupffer cells, exclusively in the presence of an anti-GPIbα mAb tested. Thus, we demonstrate for the first time that anti-GPIbα antibodies induce GPIbα desialyation, leading to platelet activation and apoptosis. Therefore, we identified novel Fc-independent platelet clearance pathways, more specifically, via Ashwell-Morell and MAC-1 receptors on hepatocytes and liver macrophages. These findings may lead to novel therapeutic regimens including the potential use of sialidase inhibitors as a solution for anti-GPIb-mediated ITP patients previously refractory to both steroid and IVIG therapies. Disclosures: No relevant conflicts of interest to declare.

Abstract 523 Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which antibodies and/or cytotoxic T cells are directed against an individual's own platelets and/or megakaryocytes and lead to enhanced platelet destruction in the periphery and reduced platelet production within the bone marrow. It is becoming increasingly clear that the immune dysregulation leading to ITP is primarily due to T cell disturbances and recently, many reports have demonstrated that active ITP is associated with a peripheral deficiency of tolerance-inducing CD4+CD25+FoxP3+ T regulatory cells (Tregs). Using a murine model of ITP that demonstrates both antibody and T cell-mediated platelet and megakaryocyte destruction, we analyzed the role that Tregs may play in active ITP. BALB/c CD61 knockout (KO) mice were immunized against wildtype (CD61+) platelets and 1.5×104 of their splenocytes were transferred ip into CB.17 severe combined immunodeficient (SCID) mice and their platelet counts, phenotype and percentage of CD4+CD25hi+FoxP3+ Tregs were enumerated in various lymphoid tissues at various times after transfer. Results showed that within 2 weeks post-transfer, 16 of 30 transferred mice became thrombocytopenic ( Disclosures: No relevant conflicts of interest to declare.

Abstract 3351 Transfusion related acute lung injury (TRALI) is a serious complication of transfusion. The pathogenesis of TRALI is not fully understood but previous findings have suggested that platelet depletion can protect mice in a two-hit model of TRALI (Looney et al J Clin Invest 119:3450, 2009). To further understand the role of platelets in preventing antibody-mediated TRALI, two mouse models of immune thrombocytopenia (ITP) were utilized. In the passive ITP model, SCID mice were injected with a monoclonal anti-platelet antibody (MWReg30) intraperitoneally (ip, 18 h before TRALI induction) or intravenously (iv, 2 h before TRALI induction). In the active ITP model, SCID mice were transferred with splenocytes from anti-CD61 immune GPIIIa-knockout mice and thrombocytopenia occurred within 2 weeks post transfer (Chow et al Blood 115;1247, 2010). TRALI induction was performed by injecting the various thrombocytopenic SCID mice with a murine monoclonal MHC class I antibody (mAb, 34-1 -2s) iv and several parameters were observed for up to 2 h post antibody injection. In control, non-thrombocytopenic SCID mice, 34-1 -2s injection caused severe systemic shock as noted by reduced rectal temperatures which was associated with significant lung damage and mortality (45%) within 1 hour of 34-1 -2s infusion as previously shown (Fung et al. Blood DOI 10.1182/blood-2010-05-284570). In contrast, while SCID mice depleted of platelets by the passive ip route had systemic shock, lung damage and a 60% mortality rate, those mice made thrombocytopenic by the iv route were completely protected from mortality. On the other hand, in the active ITP model, where the induced thrombocytopenia is associated with a proinflammatory anti-platelet immune response, no mortality was observed in those mice made thrombocytopenic by antibody-mediated immune mechanisms whereas 80% of mice rendered thrombocytopenic by CD8+ T cell-mediated immunity were dead within 1 hr post 34-1 -2s infusion. These results suggest that thrombocytopenia in itself does not protect against antibody-mediated TRALI severity but the nature of the thrombocytopenia induction (e.g. acute passive iv infusion or active ITP immune transfer) is important. In fact, depending on the inflammatory milieu associated with the thrombocytopenia, platelets may actually increase the severity of TRALI. Disclosures: No relevant conflicts of interest to declare.

Abstract 4671 TTP results from deficiency of ADAMTS13 that leads to accumulation of high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and thrombosis in the microvasculature. Previous studies have shown that HMWM VWF purified from blood group O individuals were cleaved faster by ADAMTS13 compared to HMWM VWF from non-O blood group individuals. We hypothesized that blood group O patients have lower prevalence of TTP and/or less severe form of TTP. We conducted a retrospective chart review of all TTP patients treated at our institution from 1993 to 2010. The patients were identified from the review of plasmapheresis treatment records. Any patients with missing charts were excluded. The diagnosis of TTP was made on the basis of laboratory evidence of microangiopathic hemolytic anemia and thrombocytopenia and neurologic and/or renal abnormalities, following exclusion of alternative diagnoses. ADAMTS13 levels were determined by collagen binding assay in select patients. 94 patients were included in the study. The patient characteristics were as follows: 32 males versus 62 females, mean age 45.4 years. 37/94 (39%) patients had ADAMTS13 analyzed with the following results: low ADAMTS13 (21 patients), normal ADAMTS13 (15 patients), equivocal 1. Presentation laboratory investigations were available for 91 patients and were as follows: median platelet count 16×109/L (min 3, max 274) and median hemoglobin 89 g/L (min 48, max 152). At presentation, 19/91 (20.9%) patients had fever (>38C) and 43/91 (47%) had neurological abnormalities (ranging in severity from headache to seizures and decreased level of consciousness). The blood group distribution in the TTP cohort was blood group O 41 patients, non-O 48 patients, data not available 5 patients. All patients received plasmapheresis with cryosupernatant plasma (CSP) or FFP, with some also receiving steroids and other therapies. The patients received a median of 12 (min 1, max 67) plasmapheresis treatments. Overall mortality rate was 22%. The other outcomes were as follows: death at 6 months follow-up 19%; death at 6 months or relapse 20%, death during treatment or permanent dialysis or permanent neurological disability 21%. The prevalence of blood group O versus non-O in our TTP cohort (n=89) was not significantly different from the prevalence of blood group O versus non-O in the general patient population of our institution over 2000–2010 (n=24,852; p=0.71). As illustrated below, in univariate analysis, TTP patients with blood group O did not have significantly different outcomes compared to TTP patients with non-O group blood. Outcomes Group O patients Non-group O patients p-values Death at 6 months 6/41 11/48 0.32 Death at 6 months or relapse 13/41 20/48 0.16 Death during treatment/permanent disability/permanent dialysis dependence 7/41 10/48 0.38 Conclusion: The prevalence of blood group O vs. non-O was not significantly different between the TTP cohort and our general patient population. Although there appeared to be a trend towards worse outcomes in patients with non-O blood group, there were no statistically significant differences in the outcomes in the patients with blood group O vs. non-O. The outcomes assessed in our study included death at 6 months, death at 6 months or relapse, and death during treatment/permanent neurological disability/permanent dialysis dependence. Disclosures: No relevant conflicts of interest to declare.

Abstract 1433 Introduction: Immune thrombocytopenia (ITP) is a common autoimmune disorder in which autoantibodies are generated against a patient's platelets, leading to decreased platelet counts and bleeding diatheses. Autoantibodies in ITP mainly target platelet receptors GPIIbIIIa (αIIbβ3 integrin) and GPIbα. Current first-line therapy for ITP patients is steroids, while intravenous immunoglobulin (IVIG) is a common second-line therapy. However, not all patients are responsive and we lack useful exclusion criteria for steroid therapy. Appropriate identification of non-responsive patients would facilitate proper treatment and limit side effects. We previously showed anti-GPIbα (versus anti-GPIIbIIIa) mediated ITP is less responsive to IVIG (Blood. 2006;108(3):943-6). Our preliminary data from human patients also suggests that patients with anti-GPIbα antibodies are less responsive to steroids. However, these data need to be confirmed and mechanisms remain to be elucidated. Methods: To examine whether antibody specificity (GPIIbIIIa or GPIbα) correlates with responsiveness to steroids and determine the target immune cells for steroid action, we used two murine ITP models (passive antibody transfer model and active splenocyte transfer model (Blood. 2010;115:1247-53)). In both models, mice are treated with the corticosteroid Dexamethasone (DEX) and platelet counts are monitored. In the passive antibody transfer model: BALB/c or C57 mice were pretreated for 3 days with 0.1mg/kg, 1mg/kg, or 10 mg/kg of DEX prior to injection with our newly developed mouse-anti-mouse monoclonal antibodies (mAbs) targeting GPIIbIIIa or GPIbα. A second group of mice were treated with DEX 1 day following mAb injection (i.e. after becoming thrombocytopenic) and daily thereafter. In both groups, platelet counts were monitored for 4 consecutive days post mAb injection. In the active splenocyte transfer model: β3-/- and GPIbα-/- mice were immunized with wild-type (WT) platelets resulting in an antibody titer of >1:12,800. Purified splenocytes from immunized mice were transferred into syngeneic WT or SCID recipients, such that they developed ITP. Recipient mice were treated with 1mg/kg or 10 mg/kg of DEX starting 1 day after splenocyte transfer, and daily thereafter. Platelet counts were monitored. Results and Discussion: In the passive ITP model, irrespective of DEX dose, route of administration (i.p. or s.c.) or mouse strain, pretreatment with DEX was unable to ameliorate ITP induced by either anti-GPIIbIIIa or anti-GPIbα mAbs. In addition to pretreating with DEX, administration of DEX following ITP induction also appeared to be unable to ameliorate ITP induced by either anti-GPIIbIIIa or anti-GPIbα mAbs. Similar results were obtained in the passive model when using the corticosteroid Prednisolone in a different experimental setting. In the active splenocyte transfer model, we found that varying doses of DEX ameliorated ITP in mice engrafted with anti-GPIIbIIIa reactive splenocytes while untreated mice remained thrombocytopenic. Importantly, DEX failed to show a significant effect on ITP amelioration in mice engrafted with anti-GPIbα reactive splenocytes. Since the passive model involves exogenous administration of mAb, the effect of steroid treatment is largely limited to effects on the reticuloendothelial system (RES) and destruction of opsonized platelets. Conversely, the splenocyte transfer model allows for steroid effects on active immune responses (i.e. it may affect dendritic cells, T cells, B cells, etc.) in addition to the RES. As DEX was efficacious in only the splenocyte transfer model, this suggests steroids primarily affect upstream of the immune responses in ITP. Ongoing experiments involving depletion of CD4+, CD8+, or CD19+ splenocytes will help elucidate specific immune cells as targets of DEX. Conclusions: Our data clearly demonstrated that DEX ameliorated anti-GPIIbIIIa mediated ITP but was not significantly effective for amelioration of anti-GPIbα mediated ITP in the active splenocyte transfer model. We also demonstrated that steroid therapy failed in our passive ITP model. This finding is consistent with our preliminary data in human patients (patients with anti-GPIbα antibodies may be refractory to both IVIG and steroids). This may explain the clinical variability seen in response to steroids and lead to new diagnostic/therapeutic approaches. Disclosures: No relevant conflicts of interest to declare.

Abstract 484 Background: Plasma fibronectin (pFn) is an abundant protein in the blood. It has long been suspected that pFn plays a role in thrombosis/hemostasis, but this has remained controversial. Our previous study using pFn deficient mice demonstrated that pFn supports thrombosis (PNAS. 2003; 100: 2415-9). Unexpectedly, depletion of pFn in fibrinogen (Fg) and von Willebrand factor (VWF) double deficient (Fg/VWF−/−) mice enhanced, rather than abolished, platelet aggregation and thrombus formation, revealing a functional switch of pFn in the presence and absence of Fg and VWF (Blood. 2009;113:1809-17). However, the mechanism that controls this switch is not known. Furthermore, the hemostatic function of pFn in VW disease (VWD) or afibrinogenemia is unclear. Methods: To address these questions, we bred pFn conditional knockout mice with VWF−/− or Fg−/− mice, establishing 2 new strains of mice: Fg/pFn−/− and VWF/pFn−/−. We also extended our studies of pFn in the triple knockout (TKO, Fg/VWF/pFn−/−) mice. PolyI-polyC was injected into Cre+ and Cre- mice, which resulted in the depletion of plasma pFn (>98%) and platelet pFn (>80%) in Cre+ mice but not in Cre- littermate controls. Aggregometry, a perfusion chamber system, thromboelastography (TEG), tail vein bleeding assay and intravital microscopy were used to study these mice. Results: We first observed a significantly higher mortality in TKO (25%, P We further found that the mortality rate in Fg/pFn−/− mice was also higher than their Cre- Fg−/− littermates (29%, P We also found that pFn supports hemostasis in VWF−/− mice, although no significant mortality difference was observed (P>0.05). The tail vein bleeding time was longer in VWF/pFn−/− mice than in Cre- VWF−/− littermates (P pFn was also found to support hemostasis in a fibrin-dependent manner. We first demonstrated with TEG that fibrin clot strength was significantly stronger in Cre- littermates than in pFn−/− mice (P Very interestingly, in keeping with our earlier observation in TKO mice, pFn also inhibited platelet aggregation when fibrin was absent. In Fg−/− mice, we found that pFn depletion enhanced gel-filtered platelet aggregation induced by both thrombin and thrombin receptor activating peptide (TRAP, AYPGKF; P Conclusion: Our data demonstrated that pFn is a critical factor for the survival of Fg−/− mice and supports hemostasis in VWF−/− mice via both fibrin-independent and dependent pathways. We clearly showed that fibrin, likely in the form of covalently-linked fibrin-pFn complexes, is required for pFn to support platelet aggregation. Through inhibition of platelet aggregation, non-fibrin-linked soluble pFn may play an important role in the prevention of excessive thrombus formation at the site of vessel injury and thus maintains blood circulation. pFn is therefore likely a crucial supportive factor in hemostasis (for afibrinogenemic and VWD patients), and an important regulator in thrombosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 3686 In the International Consensus Report (Rodeghiero et al. Blood 2009;113:2386-2393), platelet transfusions are only recommended in patients with auto-immune thrombocytopenia (ITP) as an adjunctive therapy for life-threatening bleeding. However, the clinical evidence of post-transfusion increments in ITP suggests that in some cases of ITP, platelet transfusions may be a useful prophylactic therapy. To understand the importance of platelet transfusion in this setting, we utilized our murine model of ITP that demonstrates both antibody- and CTL-mediated thrombocytopenia. BALB/c CD61 knockout (KO) mice were immunized by transfusions of three different platelet populations that were either single antigen positive (e.g. CD61+ or MHC class I+) or double antigen positive (CD61+/MHC class I+). Splenocytes from the immune KO mice were either not depleted (ND) or depleted of CD19+ B cells and then transferred into SCID mice and the development of thrombocytopenia and bleeding diathesis was monitored. When ND splenocytes from MHC class I+ immune KO mice were transferred, no thrombocytopenia or bleeding was observed. In contrast, transfer of ND splenocytes from CD61+ immune mice induced a significant thrombocytopenia with a bleeding mortality of 40% within 3 weeks post-transfer; depletion of B cells was used to analyze CD8+ T cell- mediated thrombocytopenia and these depleted splenocytes also induced thrombocytopenia and bleeding. In addition, compared with healthy mice, the bone marrow of the transferred mice showed elevated numbers of megakaryocytes with some of them appearing to be apoptotic. On the other hand, when splenocytes from the double immunized (CD61+/MHC class I+) mice were transferred, the CD8+ T cell- mediated thrombocytopenia and bleeding mortality was not observed. In addition to preventing the thrombocytopenia and bleeding, histological analysis of the bone marrow showed normal numbers of megakaryocytes. These results suggest that both antibody- and CD8+ T cell-mediated immune thrombocytopenia exist and that when anti-CD61 specific CD8+ T cells interacted with allogeneic MHC antigens on platelets they were prevented from inducing CD8+ T cell-mediated thrombocytopenia. Thus, allogeneic MHC platelet transfusions may be responsible for inhibiting CD8+ T cell-mediated thrombocytopenia and may be a therapeutic option for those patients who suffer from cell-mediated ITP. Disclosures: No relevant conflicts of interest to declare.

Abstract 156 Platelet adhesion and aggregation at sites of vascular injury are key events required for haemostasis and thrombosis. It has been documented that von Willebrand factor (VWF) and fibrinogen (Fg) are required for platelet adhesion and aggregation. However, we previously showed that occlusive thrombi still form in mice deficient for both Fg and VWF (Fg/VWF−/−) via a β3 integrin-dependent pathway. Here, we have investigated novel, non-classical ligands of β3 integrin that may regulate platelet adhesion and aggregation. To identify potential ligand(s) of β3 integrin, latex beads were coated with purified human platelet β3 integrin and incubated with human plasma. Protein(s) specifically associated with β3 integrin were electrophoresed and apolipoprotein AIV (ApoA-IV) was identified by mass spectrometry. We found that ApoA-IV binds to the surface of stimulated platelets, but not to quiescent platelets or β3−/− platelets, and ApoA-IV/platelet association was blocked by the addition of a specific anti-β3 integrin monoclonal antibody. It appears that ApoA-IV binds to, but is not internalized by platelet β3 integrins. ApoA-IV-deficient (ApoA-IV−/−) mice exhibited enhanced platelet aggregation induced by ADP, Collagen, and TRAP in plasma (but not PIPES buffer) compared to wild type (WT) littermates. This enhancement was diminished when ApoA-IV−/− plasma was replaced by WT plasma, indicating that the reduction was due to plasma ApoA-IV and not an unrelated platelet effect. When platelets were incubated with FITC-Fg, ApoA-IV was able to reduce platelet/Fg association, indicating that ApoA-IV may act to displace pro-thrombotic β3 integrin ligand(s). In support of this, ApoA-IV reduced the number of adherent platelets on immobilized Fg in perfusion chamber assays and enhanced thrombus formation was observed when ApoA-IV−/− mouse blood was perfused over collagen. We found that addition of recombinant ApoA-IV inhibited platelet aggregation and thrombus formation in vitro, while the control apolipoprotein ApoA-I did not. Using intravital microscopy, we further demonstrated that early platelet deposition was increased, and the time for thrombus formation and vessel occlusion were shorter in ApoA-IV−/− mice, which can be corrected by recombinant ApoA-IV transfusion. Furthermore, recombinant ApoA-IV inhibited WT platelet aggregation, thrombus formation and enhanced thrombus dissolution both in vitro and in vivo. Our data demonstrate for the first time that ApoA-IV is a novel ligand of platelet β3 integrin that negatively regulates thrombosis. These new data are consistent with the reported association between ApoA-IV and reduced cardiovascular diseases, and establish the first link between ApoA-IV and thrombosis. Disclosures: No relevant conflicts of interest to declare.

Abstract 2106 Poster Board II-83 Background Phenotyping blood donors for human platelet antigens (HPAs) can be limited by the availability of antibodies and methodologies. Robotic high throughput multiplex polymerase chain reaction (PCR) single-nucleotide polymorphism (SNP) technology provides the opportunity to perform mass genotyping for HPAs to screen apheresis donors who are negative for antigens implicated in alloimmune thrombocytopenia. This technology needs to be validated by standard polymerase chain reaction-SSP to establish a repository of platelet donors to allow for timely and adequate platelet support for patients requiring HPA-matched platelets. The objective of this study is to validate the use of high throughput SNP technology. Methods This is a prospective cohort study of 750 regular apheresis donors. Platelet apheresis donors were identified from Canadian Blood Services' Toronto Centre and genotyped for HPA 1-5 and 15 using high throughput SNP (SNPStream®) technology. HPAs were genotyped using both the sense and antisense DNA strands of each polymorphism to ensure maximum specificity. The genotypes identified by SNP were confirmed by standard methods thus all donors found to be negative for antigens implicated in alloimmune thrombocytopenia were retested using manual (sequence specific SSP-PCR at Canadian Blood Services' Platelet Immunology Laboratory and at the Toronto Centre. Results Of the 750 donors that were screened, 130 donors were found to be negative for antigens implicated in alloimmune thrombocytopenia based on SNP technology. The table illustrates genotyping results using SNP technology and SSP-PCR confirmation to date. In some instances, donors were homozygous for two low frequency HPA genotypes: 2 donors were homozygous for both HPA-1b/b and -2b/b, 6 donors for HPA-1b/b and -3b/b, 1 donor for HPA-2b/b and -3b/b, 1 donor for HPA-1b/b and -5b/b, 10 donors for HPA-1b/b and -15b/b, 4 donors for HPA-5b/b and -15b/b and 1 donor for HPA-2b/b and 15-b/b. HPA System Number of Genotypes Identified by SNP Technology Discrepant results Identified by SSP HPA-1 b/b 15 2 (13%) HPA-2 b/b 8 3 (27%) HPA-3 b/b 25 1 (4%) HPA-5 b/b 1 0 HPA-15 b/b 9 1 (11%) Conclusions: Robotic high throughput multiplex PCR SNP is potentially useful for mass genotyping of apheresis platelet donors and can identify both low frequency antigen-negative donors and combinations of these genotypes. This technology needs to be further developed. Disclosures: No relevant conflicts of interest to declare.

Abstract 2998 Poster Board II-976 Introduction: The platelet surface receptor glycoprotein (GP) IIbIIIa (integrin αaIIbβ3) mediates platelet aggregation and plays a key role in hemostasis and thrombosis. Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation. Two of these antagonists, eptifibatide (Integrilin) and tirofiban (Aggrastat) have been approved by the U.S. Food and Drug Administration (FDA) and widely used for preventing and treating thrombotic complications in patients undergoing percutaneous coronary intervention and in patients with acute coronary syndromes. It has been reported, however, that some GPIIbIIIa antagonists, such as orbofiban and xemilofiban, promote apoptosis in cardiomyocytes by activation of the apoptosis executioner caspase-3, raising the possibility that platelets also may be susceptible to pro-apoptotic effects of eptifibatide and tirofiban. Over the past decade it has been well-documented that apoptosis occurs not only in nucleated cells but also in anucleated platelets stimulated with thrombin, calcium ionophores, very high shear stresses and platelet storage (Leytin et al, J Thromb Haemost 4: 2656, 2006; Mason et al, Cell 128: 1173, 2007). It has been further reported that platelet activation and apoptosis may be induced by different mechanisms and/or require different levels of triggering stumuli (Leytin et al, Br J Haematol 136: 762, 2007; Br J Haematol 142: 494, 2008). Recently, we have shown that injection of anti-GPIIb antibody induced caspase-3 activation in mouse platelets in vivo (Leytin et al, Br J Haematol 133: 78, 2006), suggesting that direct GPIIbIIIa-mediated pro-apoptotic signaling is able to trigger caspase-3 activation within platelets. Study Design and Methods: The current study aimed to examine, for the first time, the effect of eptifibatide and tirofiban on caspase-3 activation in human platelets. We studied the effects of eptifibatide and tirofiban on caspase-3 activation in resting platelets, which express GPIIbIIIa receptors in their non-active (“closed”) conformation, and in platelets stimulated with thrombin or calcium ionophore A23187, which induce transition of GPIIbIIIa receptors into active (“open”) conformation. Resting platelets were treated with control buffer, 0.48 μM eptifibatide or 0.48 μM tirofiban, and stimulated platelets were treated with 1 U/mL thrombin or 10 μM A23187, or preincubated with eptifibatide or tirofiban before treatment with thrombin or A23187. Caspase-3 activation was determined by flow cytometry using the cell-penetrating FAM-DEVD-FMK probe, which covalently binds to active caspase-3. Results and Discussion: We found that treatment of resting platelets with eptifibatide and tirofiban did not affect caspase-3 activation (P>0.05, n=7). In contrast, a 2.3-2.7-fold increase of caspase-3 activation was observed in platelets after thrombin or A23187 stimulation (P Conclusions: We have demonstrated a novel platelet-directed activity of two clinically used GPIIbIIIa antagonist drugs, eptifibatide (Integrilin) and tirofiban (Aggrastat), with ability to inhibit apoptosis executioner caspase-3 induced by potent platelet agonists, thrombin and A23187, and the absence of adverse pro-apoptotic effects on resting platelets. Taken together with earlier reported data (Leytin et al, Br J Haematol 133: 78, 2006), the current study indicates that, aside from their well-known participation in platelet activation and aggregation, GPIIbIIIa receptors are involved in the modulation of platelet apoptosis. This GPIIbIIIa-mediated mechanism of apoptosis modulation may be very efficient given the extremely large number of GPIIbIIIa copies (≈80,000) on the platelet surface. Disclosures: No relevant conflicts of interest to declare.

Although anucleate, platelets are recognized to undergo apoptotic-like events, with some characteristics of activated platelets resembling those of apoptotic nucleated cells, e.g. membrane blebbing (microparticle (MP) formation), exposure of phosphatidylserine (PS). There are reports in the older literature that platelets from BSS patients expose more PS and have enhanced senescence. In the present study, using flow cytometry, we investigated apoptotic-like events, specifically PS exposure, MP formation, cell shrinkage and loss of mitochondrial inner membrane potential (ΔΨm) in platelets from 2 BSS patients (BSS-Ps), compared with platelets from a patient with another inherited macrothrombocytopenia - an MYH9-related disorder (MYH9-RD-P) - and from normal controls. Investigations were done using whole blood to avoid platelet manipulation, especially of the large platelets. Citrated blood was mixed with GPRP and Ca2+, and the agonists collagen (C; 20 μg/mL), thrombin (T; 1 U/mL), SFLLRN (PAR1 activating peptide (AP); 50 μM), AYPGKF (PAR4 AP; 250 μM), or combinations of C with T or the PAR APs. Platelets and MPs were identified as CD41-positive events; platelet size was expressed as median forward scatter (mFSC); MPs were defined as events % PS Exposure Agonist(s) BSS-P1 BSS-P2 MYH9-RD-P controls none (resting platelets) 3.0 3.5 3.0 1.3±0.4 C 27.0 29.9 37.9 7.0±1.5 C+T 11.3 35.3 64.5 13.5±1.7 C+PAR1 AP 20.1 43.6 49.5 16.4±3.1 C+PAR4 AP 31.6 47.8 49.7 18.4±3.7 C+PAR1 AP+PAR4 AP 33.4 49.5 54.8 20.6±3.4 Interestingly, the platelets in the PS-exposing subpopulation were small (mFSC of BSS-Ps: 27, 30; MYH9-RD: 32; controls 18±1) and the annexin A5 MFI of this subpopulation of BSS and MYH9-RD platelets was .5-fold higher than that of controls irrespective of the platelet agonist(s). Unstimulated blood samples from patients and controls had 1–2% MPs; adding combinations of agonists led to the highest MP production, with control platelets producing 5–6% MPs, and BSS and MYH9-RD platelets, a 1.5–2.5-fold greater amount. Another apoptosis hallmark, cell shrinkage, was observed upon platelet activation in the non-PS-exposing population; in response to the various agonists, the size of this platelet population in patients and controls correlated inversely with the amount of PSexposing platelets formed (P

Fetal and neonatal immune thrombocytopenia (FNIT) is a life-threatening bleeding disorder, resulting from fetal platelet opsonization and destruction by maternal antibodies developed during pregnancy. The frequency of FNIT has been estimated at 0.5–1.5/1,000 liveborn neonates. However, the incidence of fetal mortality is currently unknown, as the rate of miscarriage in affected pregnant women has not been well studied. Integrin αIIbβ3 and Glycoprotein (GP) Ibα are major glycoproteins expressed on the platelet surface and are the two major antigens targeted by anti-platelet antibodies in autoimmune thrombocytopenia (ITP). However, it is unclear why the incidence of FNIT caused by anti-GPIbα antibodies is far lower than that of FNIT mediated by anti-β3 integrin antibodies. This difference cannot be well explained by the frequency of genetic polymorphisms of the two antigens. We hypothesized that: 1) GPIbα is less immunogenic, leading to less maternal antibody production during pregnancy, or 2) anti-GPIbα antibodies cause a less severe pathology, and thus have a lower chance of being reported, or 3) anti-GPIbα antibodies cause higher incidence of miscarriage, resulting in reduced reported cases. To test these hypotheses, the maternal immune response against fetal platelet GPIbα versus β3 integrin were compared in FNIT models, using syngeneic background BALB/c GPIbα−/− and β3−/− mice. The FNIT models were established by transfusing female GPIbβ −/− or α3−/− mice with 108 gel-filtered platelets from wild-type (WT) BALB/c mice weekly. After two platelet immunizations, flow cytometry assays were used to detect the titers of anti-GPIbα and anti-β3 antibodies, and the immunized females were bred with WT BALB/c male mice. We found that there was no significant difference in mean antibody titer between the two groups (P>0.05). However, miscarriage occurred more frequently in anti-GPIbα-mediated FNIT (14/16 versus 8/16, P0.05). Our data suggest that fewer reported FNIT cases mediated by anti-GPIbα antibodies cannot simply be explained by less immunogenicity of GPIbα, or less severe pathology caused by anti-GPIbα antibodies. Higher incidence of miscarriage caused by maternal immune response to fetal GPIbα likely masks the reported frequency and severity of this life-threatening disease. The mechanisms leading to miscarriage in FNIT, and the potential therapeutic effect of intravenous immunoglobulin (IVIG) in this disorder are currently being investigated. (Li C and Piran S contributed equally to this work).

Background Utilization of intravenous immune globulin (IVIG) is increasing in Canada and worldwide despite few and no new labeled indications. In 2007, Canadian Blood Services in collaboration with the National Advisory Committee on Blood and Blood Products convened a panel of solid organ transplantation (SOT) experts (kidney, heart, lung, and liver) and methodologists to develop an evidence-based practice guideline for the use of IVIG in patients undergoing SOT. The objectives of this guideline are to examine the evidence for the use of IVIG in patients who are candidates for SOT and are sensitized to HLA or ABO antigens, to provide guidance for Canadian practitioners involved in the care of these patients and transfusion medicine specialists on the use of IVIG. Methods: The panel identified clinical areas of SOT that would benefit from treatment with IVIG and generated key clinical questions. A systematic, expert and bibliography literature search up to July 2008 was conducted to ensure all relevant publications were included. The panel generated recommendations based on the evidence. The levels of evidence and grading of recommendations were adapted from the Canadian Task Force on Preventative Health Care. To validate conclusions and recommendations, the practice guideline will be sent to physicians involved in solid organ transplantation in Canada and a patient representative. Recommendations from practitioner feedback will be incorporated, and the guideline will be disseminated to all physicians involved in the care of patients receiving solid organ transplantation in Canada to aid implementation of the guideline. The National Advisory Committee of Blood and Blood Products in Canada will subsequently assess the performance of the guideline and will renew the guideline at timely intervals. Results and Conclusions: The research questions developed by the panel were: Is there evidence that the use of IVIG reduces morbidity and mortality for patients undergoing SOT who are sensitized (HLA or ABO) in the perioperative setting and are sensitized experiencing acute graft rejection or experiencing chronic graft rejection? 791 citations were retrieved, and panel members identified 3 additional citations. 51 reports and a systematic review were used for this guideline. These reports were limited by inconsistent definitions of sensitization, inconsistent reporting of the type and titre of the antibody, the assays used to detect HLA antibodies, the response criteria and dosing schedules for IVIG. Thus, a consensus process was used to account for the poor evidence. The use of IVIG was associated with decreased sensitization and acceptable morbidity and mortality in living donor kidney transplantation. IVIG has been used with several other modalities for ABO-incompatible kidney transplantation and it was difficult from the existing literature to separate outcomes based on a single modality. There was also limited data on the perioperative use of IVIG in renal transplantation. IVIG was shown to be effective in combination with plasmapheresis for acute antibody mediated rejection of the kidney; however the role of IVIG was not clear for other forms of rejection. There were also several methodological limitations in the literature assessing IVIG for cardiac transplantation and only limited data were available to assess the use of IVIG for lung or liver transplantation. Future studies are needed to define the role of IVIG in solid organ transplantation and should capture the following elements: impact on antibody (specificity and titres), transplant rates, time to transplantation, graft function, graft survival, and rejection (cellular and antibody mediated).

Platelet storage lesion is a serious problem limiting clinical use of platelet concentrates (PC) after extended and long-term storage. Platelet activation is a well-known manifestation of platelet storage lesion. However, over the last decade, platelet apoptosis has been also recognized in stored PCs and in platelets following exposure to thrombin, calcium ionophores, anti-platelet antibodies and very high shear stresses. The aim of this study was to elucidate the contribution of platelet activation and apoptosis to the platelet storage lesion during conventional (Days 2–5), extended (Days 6–8) and long-term (Days 11–16) PC storage. We prepared seven prestorage-leukoreduced PC by the platelet-rich plasma (PRP) method, stored PC for 2–16 days at 22°C, and used flow cytometry for determining platelet activation as P-selectin (CD62) exposure and platelet apoptosis as depolarization of mitochondrial inner membrane potential (ΔΨm), activation of executioner caspase-3, exposure of phosphatidylserine (PS) and release of apoptotic platelet fragments microparticles (MP). Platelet activation and apoptotic responses were also determined in fresh (Day 0) PRP using thrombin titration. We found a significant increase of platelet activation under conventional PC storage for 2–5 days (38.6 ± 3.1% CD62 positive cells, P < 0.0001). With extended (Days 6–8) storage, platelet activation was increased to 66.5 ± 5.3% and reached the maximal level of 92.0 ± 1.1% after 11–12 storage days (P < 0.0001). In contrast, ΔΨm depolarization and caspase-3 activation did not increase in comparison with Day 0 platelets, even after PC storage for 12 days (P > 0.05) and storage for 13–16 days was required for significant triggering these apoptotic events (P < 0.05-0.0001). Similarly, although we observed a slight increase of PS exposure (5–10%) and MP release (9–11%) during PC storage for 2–12 days, incubation for 13–16 days was required for a stronger (30–60%) stimulation of these apoptotic manifestations (P < 0.001-0.0001). Paired comparison between the effects of PC storage on CD62 exposure and apoptotic events clearly demonstrated for all storage times a significantly higher level of platelet activation than levels of ΔΨm depolarization, caspase-3 activation, PS exposure and MP release (P < 0.01-0.0001). Furthermore, we found that when fresh (Day 0) PRP was treated with different thrombin doses, ranging from 0.05 to 10 U/ml, a much higher maximal level of platelet activation (~90%) was reached, in comparison to the level of apoptosis (30–40%), and 100- to 200-fold lower dose of thrombin were required for maximal induction of activation (0.05–0.1 U/ml) than for stimulation of apoptosis (10 U/ml). Taken together, these data indicate that (i) during PC storage, platelet activation is triggered much earlier than platelet apoptosis, (ii) platelet activation rather than apoptosis contributes most to the platelet storage lesion during conventional (Days 2–5) and extended (Days 6–8) PC storage whereas during long-term (Days 13–16) storage both responses are involved in platelet deterioration, and (iii) platelet activation and apoptosis are different phenomena; they may be induced by different mechanisms and/or require quite different levels of triggering stimuli.

Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by IgG autoantibody-opsonized platelets prematurely being destroyed in the spleen although recent evidence suggests that thrombocytopenia in perhaps as many as 40% of patients with ITP can be mediated by CD8+ T cells. Although several animal models of immune thrombocytopenia have been developed, few are induced by platelet-specific autoimmune mechanisms nor have any demonstrated cell-mediated platelet destruction. We developed a murine model of ITP by first immunizaing GPIIIa (CD61) knockout (KO) mice against wildtype (WT) CD61+ platelet transfusions and subsequently transferring their splenocytes (5×104 cells/transfer) intraperitonealy into irradiated SCID mouse recipients. The SCID mice developed significant bleeding mortality and thrombocytopenia within 2 weeks post-transfer. Lower doses (

Platelet P-selectin plays important roles in inflammation and contributes to thrombosis and hemostasis. While 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, but not VWF, deficiency. The impaired P-selectin expression on fibrinogen-deficient platelets was further confirmed in a human patient with severe hypofibrinogenemia and his heterozygous parents. 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. This effect seems to be specific for P-selectin, as other alpha-granule proteins (e.g. thrombospondin-1, vitronectin, platelet factor 4) were not decreased in fibrinogen-deficient mouse or human platelets. We found that fibrinogen transfusion recovered the P-selectin expression in fibrinogen-deficient mice to >80% of the normal level within 48 hours, and four days post-transfusion, the P-selectin levels were nearly completely recovered. Furthermore, engagement of the C-terminus of the fibrinogen γ chain with β3 integrin was required for this process since a similar impairment of P-selectin expression was also observed in fibrinogen γΔ5 mice and β3 integrin-deficient mice. To determine whether fibrinogen affects the biogenesis of platelet alpha-granules, platelets were examined via electron microscopy. No statistically significant difference in the number of platelet alpha-granules was observed in fibrinogen-deficient or β3 integrin-deficient mice compared to controls (P>0.05). These data suggest fibrinogen may play important roles in inflammation, including immune-mediated inflammation, thrombosis and hemostasis via enhancement of platelet P-selectin expression. Furthermore, there are 2–3 fold variations in human plasma fibrinogen levels in healthy populations, and there are patients with hypo- and afibrinogenemia, hyperfibrinogenemia, and Glanzmann’s Thrombasthenia (β3 integrin deficiency). Our data suggest that platelet P-selectin expression can be affected by all these conditions. Therefore, P-selectin as a platelet activation marker should be used with caution.

Apoptosis, or programmed cell death, is the physiologic mechanism that serves for controlled deletion of unwanted cells. Apoptosis was initially attributed exclusively to nucleated cells but over the past decade it has been recognized that apoptosis also occurs in anucleated cytoplasts and platelets. In this study, using flow cytometry we analyzed in human platelets three critical manifestations of mitochondrial, cytoplasmic and plasma membrane apoptosis, mitochondrial inner transmembrane potential (Δψm) depolarization, caspase-3 activation and phosphatidylserine (PS) externalization, respectively. We found that these hallmarks of apoptosis can be induced in human platelet suspension by diverse stimuli, including human α-thrombin (1, 10, 100 nM), calcium ionophore A23187 (3, 5, 10 μM), high shear stresses generated by cone-and-plate viscometer (120, 200, 390 dyn/cm2) and prolonged storage of platelet concentrates in blood banking conditions at 22°C for 6 and 13 days. We also demonstrated that these apoptotic markers can be induced in mouse platelets in vivo in a murine model of immune thrombocytopenia caused by injection of anti-glycoprotein (GP) IIb (rat anti-mouse GPIIb, MWReg30) antibody. Other manifestations of apoptosis were detected in human platelets, including expression of proapoptotic members of Bcl-2 family proteins (Bax and Bak) induced by thrombin, and platelet shrinkage and shedding of microparticles induced by high shear stresses. In addition to apoptosis in fluid-phase platelets, apoptosis was also revealed by confocal fluorescent microscopy in adherent human platelets and thrombi-like platelet aggregates deposited on thrombogenic immobilized human vascular collagen types I and III, as detected by PS exposure and shedding of PS-exposed microparticles. Taken together, these data suggest that platelet apoptosis is a phenomenon that can be triggered by a wide diversity of chemical and physical stimuli using different mechanisms mediated by thrombin-, collagen- and integrin GPIIbIIIa-receptors, mechanoreceptors and Ca2+-overloading. These stimuli trigger platelet apoptosis by impacting on several intracellular apoptotic targets, including shifting the balance between Bcl-2 regulatory proteins in a proapoptotic direction, depolarizing the inner mitochondrial membrane, activating the executioner caspase-3, stimulating aberrant PS exposure on the platelet surface and, eventually, resulting in ‘terminal’ stages of platelet apoptosis, such as platelet shrinkage and shedding of PS-exposed microparticles resembling apoptotic bodies. Platelet apoptosis can be induced both in fluid-phase and adherent platelets and thrombi-like platelet aggregates. These data also indicate that natural PL agonists thrombin and subendothelial vascular collagens and hemodynamic shear forces, can be involved not only in the processes of hemostasis, thrombosis and blood coagulation but also can trigger platelet death via apoptosis. Platelet apoptosis may contribute to the pathophysiology of thrombocytopenia in diseases associated with enhanced thrombin generation, such as sepsis and disseminated intravascular coagulation, as well as in autoimmune and alloimmune thrombocytopenias.

We have previously demonstrated that IVIg-primed leukocytes can, upon transfer to naïve mice, completley recapitulate the therapeutic effects of IVIg in treating immune thrombocytopenia in a murine model. Dendritic cells (DC) appear to be the primary cellular target of IVIg in this model. The exact pathway by which IVIg-primed DC inhibit platelet clearance remains, however, unknown. DC are professional antigen presenting cells of haematopoietic origin that are specialized for the capture, processing and presentation of antigens to T cells via their MHC Class II. This primary function of DC is absolutely dependent on MHC Class II expression. Herein, we have evaluated the efficacy of action of IVIg-primed splenocytes from MHC mismatched mice in the amelioration of murine ITP. Mice expressing the Class II haplotype I-Ab (C57BL/6) were injected intravenously with IVIg-primed splenocytes from syngeneic mice, or Balb/C mice (Class II haplotype I-Ad /I-Ed) or CD1 mice (outbred, mixed Class II haplotype I-A/E) prior to the induction of thrombocytopenia by an anti-platelet antibody. We found that IVIg-primed splenocytes from all strains of mice tested were able to successfully ameliorate thrombocytopenia in C57BL/6 mice, regardless of Class II haplotype. None of the splenocytes primed with a control protein, BSA, ameliorated thrombocytopenia. To confirm our findings that IVIg-primed splenocytes function independent of MHC haplotype, we next employed IVIg-primed splenocytes from MHC Class II deficient mice and found these splenocytes were also able to successfully ameliorate murine thrombocytopenia. These data demonstrate a non-MHC-restricted function for DC in IVIg function, and suggest that IVIg-primed DC function occurs independent of MHC Class II expression in the amelioration of murine ITP. In addition, since this potential new cell-based therapy for ITP is not MHC-restricted, we speculate that IVIg-primed DC from any donor (including autologous) could theoretically be used to treat individuals with ITP or other autoimmune diseases without the requirement for donor matching.

Immune thrombocytopenic purpura (ITP) is an autoimmune-mediated bleeding disorder in which platelets are opsonized by autoantibodies and prematurely destroyed by Fc receptor (R)-mediated phagocytosis in the reticuloendothelial system (RES). Although the pathogenesis of the disorder has been considered to be primarily antibody mediated, recent reports have indicated that cell-mediated (e.g. CD8+ T cell) mechanisms can also lead to platelet destruction at least in those patients with ITP who have no detectible antibodies. To study these mechanisms, we developed a murine model of immune thrombocytopenia by transferring spleen cells (SC) from GPIIIa-knockout (KO) mice immunized against wild type (WT; GPIIIa-positive) platelets into syngeneic severe combined immunodeficient (SCID) mice. Results show that compared with naive SC, transfer of as few as 5x104 immune GPIIIa-KO SC caused significant thrombocytopenia and a bleeding diathesis and death in 60 percent of recipient SCID mice within 10 days post-transfer. Bleeding occurred primarily in the gut, lungs, subcutaneous tissues and brain. When the immune GPIIIa-KO SC were first depleted of CD4+T cells and transferred, thrombocytopenia or bleeding mortality occurred. In contrast, CD8+ T cell depletion of the GPIIIa-KO SC before transfer did not affect their ability to cause thombocytopenia nor bleeding. Interestingly, depletion of CD19+ B cells before transfer did not affect the ability of the SC to induce thrombocytopenia but prevented all bleeding. These results suggest that both antibody-mediated and CD8+ T cell-mediated platelet destruction occur in this animal model of immune thrombocytopenia, however, it appears that only antibody-mediated thrombocytopenia is associated with bleeding mortality. Because the immune thrombocytopenia is against a platelet-specific antigen e.g. GPIIIa (like in ITP), it will not only allow for the study of disease pathogenesis but may also be important for testing new immunospecific therapeutics to increase platelet counts in patients with ITP.

Background: Platelets are critical for maintaining hemostasis, but inappropriate platelet activation can lead to pathogenic thrombosis. It has been demonstrated that the platelet integrin αIIbβ3 is essential for platelet aggregation and is also a major target antigen in immune thrombocytopenias (e.g. ITP). Current monoclonal antibodies (mAbs) against this protein complex have been generated using traditional methods involving cross-species immunization (e.g. mouse proteins into rat hosts). These approaches may generate a limited repertoire of anti-β3 mAbs since the antigenicity of the protein and the variety of epitopes targeted are based on amino acid sequence differences between the two species and integrin family members are highly conserved. Additionally, studies in murine models of ITP are hampered by the use of xenogeneic antibodies rather than syngeneic antibodies. Methods: We developed a method to generate mouse anti-mouse β3 integrin mAbs utilising β3 gene deficient mice (β3−/−) immunized with wild-type platelets. To generate antibodies specific to the PSI domain (HPA-1 region) of β3 integrin, β3−/− mice were immunized with the recombinant murine PSI domain of β3 integrin. Platelet binding and specificity were determined by flow cytometry and western blot. In vitro effects on platelet function were measured using aggregometry. Different doses of mAbs (5, 10, and 15 μg/mouse) were injected intravenously to induce thrombocytopenia in vivo. Results: A total of twelve mAbs were generated against native β3 integrin (JAN A1, B1, C1, D1 and DEC A1 and B1, 9D2, M1) or recombinant PSI domain (PSI A1, B1, C1, E1). The mAbs were specific for β3 integrin; no binding was observed using β3−/− platelets. Isotyping showed that DEC A1 and DEC B1 are IgG3, PSI E1 is IgG2b, and all other mAbs are IgG1. The anti-PSI domain mAbs recognized linear epitopes and the anti-native β3 mAbs recognized conformational epitopes. All mAbs, with the exception of JAN A1 and B1, cross-reacted with human platelets. JAN C1, JAN D1, DEC A1, 9D2, M1, and all anti-PSI antibodies inhibited mouse platelet aggregation. These antibodies, except DEC A1, 9D2 and M1, also inhibited human platelet aggregation. One anti-PSI domain antibody (PSI B1), however, directly induced human platelet aggregation in the absence of agonist in platelet rich plasma but not in PIPES buffer. This suggests that PSI B1 may initiate conformational changes in β3 integrin and promote fibrinogen binding. Six anti-β3 mAbs (JAN A1, B1, C1 and D1, 9D2 and M1) induced severe dose-dependent thrombocytopenia in mice, while the anti-PSI domain mAbs induced only a mild decrease in platelet count. Interestingly, the two IgG3 mAbs (DEC A1 and B1) did not induce thrombocytopenia. Conclusion: This approach to generating mouse anti-mouse β3 integrin mAbs using β3−/− mice was successful. Different anti-β3 mAbs had different effects on platelet aggregation, and on the induction of thrombocytopenia. These mAbs may be useful reagents for research in thrombosis and immune thrombocytopenia and as novel anti-thrombotic therapeutics.

Although primarily known as a coagulation factor and as an inducer of platelet activation and aggregation, thrombin can modulate apoptosis in nucleated cells. Over the last decade, it has been recognized that apoptosis occurs not only in nucleated cells but also in anucleated cytoplasts and platelets. The current study investigated whether thrombin can impact apoptosis in anucleated human platelets. Using flow cytometry, we studied platelet apoptosis at the single cell level, analyzing markers of mitochondrial and cytoplasmic apoptosis (Leytin et al, Biochem Biophys Res Commun320:303, 2004; Leytin et al, Br J Haematol133:78, 2006). Western blotting was also employed, in addition to flow cytometry, for determining the expression of proapoptotic Bax and Bak proteins. We found that, in comparison to untreated platelets, human alpha-thrombin (1 U/mL) significantly induced four key manifestations of platelet apoptosis: (i) mitochondrial inner transmembrane potential depolarization (P

Fibrinogen (Fg) has been considered essential for platelet aggregation. We demonstrated, however, that thrombi do form in Fg-deficient mice and in mice doubly deficient for both fibrinogen and von Willebrand factor (Fg/VWF−/−). We further reported that β3 integrin and thrombin are critical for this Fg/VWF-independent platelet aggregation. In Fg−/− or Fg/VWF−/− mice, platelet fibronectin (Fn) content is increased 3–5 fold. Furthermore, thrombus growth and stability are impaired in plasma Fn conditional deficient (M×-Cre, Fnflox/flox) mice. These data are consistent with the most recent studies of Fn assembly and suggest that Fn may support platelet thrombus formation. To examine whether Fn is the alternative key molecule which mediates platelet aggregation and thrombus formation in Fg/VWF−/− mice, we developed a novel strain of triple knockout (TKO) mice by breeding Fg/VWF−/− mice with M×-Cre+/− Fnflox/flox conditional knockout mice. Cre- littermates delivered from the same parents were used as a control. Fn depletion was induced by i.p. injections of polyinonic-polycytidylic acid. We found that TKO mice are viable with dramatically decreased levels of Fn in both the plasma (

Fetal and neonatal alloimmune thrombocytopenia (FNAITP) is a life-threatening bleeding disorder which results from maternal anti-platelet antibodies that cross the placenta and destroy fetal platelets. In most cases FNAITP is mediated by anti-β3 integrin antibodies, whereas reported cases of anti-GPIbα mediated FNAITP are rare. This difference can not be solely explained by the frequency of their respective polymorphisms. It is unclear whether this is due to:the GPIbα antigen being less immunogenic, resulting in less maternal antibody generation during pregnancy, oranti-GPIbα antibodies mediating a less severe pathology, resulting in a reduced number of reported cases. To study the immunogenecity and antibody response to the GPIbα antigen, GPIbα deficient mice (GPIbα−/−, Black Swiss background) were transfused with wild-type (WT) platelets. No detectable anti-GPIbα IgG antibody was induced even after 8 transfusions (108 platelets/transfusion). This clearly differs from the immune response generated in β3 integrin deficient (β3−/−, BALB/c background) mice, in which anti-β3 integrin antibody can be easily detected after 2–4 WT platelet transfusions. This suggested that the MHC complex in Black Swiss mice might not be able to present the GPIbα antigen. We thus introduced the BALB/c background to the GPIbα−/− mice via backcrossing these mice with BALB/c mice for 9 generations (F9). To further minimize genetic background differences, the F9 BALB/c GPIbα−/− mice were bred with F9 BALB/c β3−/− mice to generate heterozygous BALB/c GPIbα+/− and β3+/− mice. These mice were subsequently used to generate littermate GPIbα−/− or β3−/− mice for the following studies. We found that BALB/c GPIbα−/− mice are immunoresponsive to the GPIbα antigen, but antibody titers after 2 and 4 platelet transfusions were significantly lower than those seen with the anti-β3 integrin model (1:50 & 1:200 vs 1:400 & 1:3200 respectively, P

Using a murine model of ITP, we have previously demonstrated that both IVIg and a monoclonal antibody which reacts with red cells (TER-119), ameliorates thrombocytopenia. We have also recently demonstrated that passive transfer of IVIg-primed splenic leukocytes can completely recapitulate the effects of standard IVIg therapy in the amelioration of murine ITP, and further showed that CD11c+ dendritic cells were the primary cell mediating IVIg functions (Nat Med2006;12:688–692). However, the duration of action of IVIg, TER-119 and IVIg-primed leukocytes in the inhibition of murine ITP has not been evaluated. Herein, we have evaluated the efficacy and duration of action of IVIg-primed leukocytes compared with standard therapeutic delivery of IVIg and TER-119. On day 1, mice were pre-treated with 50 ug TER-119 or 50 mg IVIg, or with only 106 leukocytes that had been primed for 30 minutes with IVIg, followed by extensive washing to remove all extraneous IVIg. Alternatively, leukocytes were also primed as above with BSA as a negative control. On days 2, 5, 8 and 11, mice from each group were injected with an antibody directed to integrin αIIβ to induce immune thrombocytopenia and bled the following day for platelet enumeration. On days 3 and 6, platelet counts revealed that mice receiving IVIg-primed leukocytes, IVIg or TER-119 were all protected from thrombocytopenia to the same extent. Mice that were pre-treated with leukocytes primed with BSA were not protected, as expected. However, at day 9, mice that received IVIg-primed leukocytes were still completely protected from thrombocytopenia, while mice receiving IVIg or TER-119 were not protected. By day 12, none of the treatment regimens conferred protection from thrombocytopenia. We are currently repeating these experiments with IVIg-primed bone marrow-derived dendritic cells and these results will also be presented. These data confirm that in the treatment of murine immune thrombocytopenia, pre-treatment of mice with IVIg-primed leukocytes offers up to 50% longer duration of protection from thrombocytopenia than standard therapies with IVIg or TER-119.

Recent studies of sickle cell disease (SCD) suggest that hemostatic activation is a key aspect of pathophysiology, leading to clinical consequences such as vaso-occlusive crises and stroke. In many of these studies a SCD-associated hypercoagulable state has been inferred from markers suggestive of increased hemostatic activity, such as elevated levels of plasma thrombin-antithrombin complexes and tissue factor-containing microparticles. As part of a study using a broad-spectrum approach to explore the relationships among various aspects of normal and abnormal hemostasis in SCD we used a whole-blood coagulation assay (thromboelastography, TEG) to directly assess global hemostatic activation in children with SCD defined by genotype (SS and SC), together with a group of unaffected children. We also assessed platelet activation and procoagulant surface expression in platelets and red blood cells (RBCs) using flow cytometry. Eligible SCD subjects included:patients with painful crisis assessed at two time points: hospital admission (crisis group) and clinic follow-up appointment (follow-up group);patients not in crisis attending a regular clinic appointment (steady-state group). Patients were ineligible if they had received a recent blood transfusion, hydroxyurea, anticoagulants or aspirin. The results of TEG assays with citrated whole blood showed that compared to SC patients (n = 16) and normal children (n = 16), SS patients (n = 45) had significantly (p

The use of polyclonal anti-D is the established treatment for prophylaxis against rhesus immunization in Rh(D) negative pregnant women and is effective in raising the platelet counts in patients with autoimmune thrombocytopenic purpura (AITP). Since the supply of plasma derived anti-D antibodies is limited and plasma products carry the risk of infectious complications, replacement with monoclonal anti-D antibodies is a promising alternative. This study analyzed the efficacy of polyclonal anti-D (WinRho®SDF, Cangene) and six human monoclonal anti-D antibodies with differeing isotypes and specificities (Table 1) for their ability to opsonized erythrocytes and modulate monocyte phagocytosis of opsonized platelets in a flow cytometric assay. Human platelets were labeled with CellTracker CMGreen, opsonized with various HLA or HPA1a specific antibodies, incubated with the monocytic leukemic cell line THP-1 for 2 h and intracellular fluorescence was compared at 4°C and 37°C. Various concentrations of anti-D opsonized erythrocytes were added to the assay and the changes in intracellular fluorescence was compared. The results demonstrate that the THP cells significantly phagocytosed opsonized platelets in an Fc-dependent manner e.g. the fold change in median channel fluorescence between 37°C and 4°C for intact IgG W6/32 opsonized platelets was 51.5 ± 5.2 (mean+SD, n=74) and only 2.1+2.1 if the platelets were labeled with F(ab’)2 fragments. When erythrocytes were opsonized with the monoclonal anti-D antibodies that shared specificity but differed in IgG isotype (RhG1/RhG3 and BRAD3/BRAD5) and added to the assay, the IgG3 isotypes had a significantly greater ability to inhibit opsonized platelet phagocytosis compared with IgG1 isotypes and this inhibition was similar to that observed with polyclonal anti-D opsonized erythrocytes. However, in the one case where the monoclonal anti-D antibodies shared isotypes but differed in specificity (113/178), rRh9B8-113 monoclonal antibody significantly inhibited platelet phagocytosis whereas the rRh429-178 antibody did not. These results suggest that some human monoclonal antibodies can be as efficient as polyclonal anti-D in inhibiting opsonized platelet phagocytosis and the degree inhibition depends on the IgG isotype and specificity of the monoclonal antibodies. Thus, a defined cocktail of monoclonal anti-D antibodies could be prepared to mimic the effects and replace plasma-derived polyclonal anti-D. Table 1: Characteristic of the monoclonal anti-D antibodies. Antibody Isotype Specificity rRh9B8-113 IgG1 Loop 3 rRh429-178 IgG1 Loop 4 RhG1 IgG1 Loop 6 RhG3 IgG3 Loop 6 BRAD3 IgG3 Loop 6 BRAD5 IgG1 Loop 6

Toll-like receptors (TLR) comprise a family of transmembrane proteins characterized by multiple copies of leucine-rich repeats in the extracellular domain and an IL-1 receptor motif in their cytoplasmic domain. The TLR family is a phylogenetically conserved mediator of innate immunity that is essential for microbial recognition and the stimulation of adaptive immune responses. Recently, our laboratory demonstrated that TLR4 expression on platelets was responsible for lipopolysaccharide (LPS)-mediated thrombocytopenia and tumour necrosis factor-α production in vivo (Aslam et al. Blood107:637, 2006). To understand the mechanism of how platelet TLR4 may mediate RES activation, platelets from wild type (WT) and TLR4 knockout mice were incubated with various concentrations of LPS in vitro, washed extensively and transfused into WT mice. Results suggest that only the WT platelets could significantly stimulate TNF-α production in vivo. In vitro flow cytometric analysis of phagocytosis by the monocytic cell line THP-1 demonstrated that platelet TLR4 could efficiently present LPS to THP-1 cells and stimulated them to engulf the LPS-coated platelets. The phagocytosis of the platelets was correlated to elevated levels of intracellular TNF-α production in the THP-1 cells. These results suggest that platelets, via TLR4 expression, can act as initial sentinels of the innate immune system by presenting bacterial products such as LPS to phagocytes of the RES.