ALBERT

Description: 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.

Description: 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

Description: 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.

Description: 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, P

Description: 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.

Description: 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

Description: 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.

Description: 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 (