ALBERT

Description: Several adaptor molecules bind to cytoplasmic tails of β-integrins and facilitate bidirectional signaling, which is critical in thrombosis and hemostasis. Interfering with integrin-adaptor interactions spatially or temporally to inhibit thrombosis without affecting hemostasis is an attractive strategy for the development of safe antithrombotic drugs. We show for the first time that the 14-3-3ζ–c-Src–integrin-β3 complex is formed during platelet activation. 14-3-3ζ–c-Src interaction is mediated by the -PIRLGLALNFSVFYYE- fragment (PE16) on the 14-3-3ζ and SH2-domain on c-Src, whereas the 14-3-3ζ–integrin-β3 interaction is mediated by the -ESKVFYLKMKGDYYRYL- fragment (EL17) on the 14-3-3ζ and -KEATSTF- fragment (KF7) on the β3-integrin cytoplasmic tail. The EL17-motif inhibitor, or KF7 peptide, interferes with the formation of the 14-3-3ζ–c-Src–integrin-β3 complex and selectively inhibits β3 outside-in signaling without affecting the integrin-fibrinogen interaction, which suppresses thrombosis without causing significant bleeding. This study characterized a previously unidentified 14-3-3ζ–c-Src–integrin-β3 complex in platelets and provided a novel strategy for the development of safe and effective antithrombotic treatments.

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: 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 (

Description: 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 (

Description: 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.

Description: Background: Platelets are generated from megakaryocytes in the bone marrow and play a key role in hemostasis. GPIba is the major subunit in the GPIb-IX complex expressed on platelets and megakaryocytes (MKs). Deficiency of GPIba results in Bernard Soulier Syndrome (BSS), a severe bleeding phenotype characterized by thrombocytopenia and abnormal platelet morphology. The generation of megakaryocytes and platelets is regulated by the hematopoietic growth factor, thrombopoietin (TPO). In patients with thrombocytopenia, the TPO receptor agonists and recombinant TPO (rTPO) have been used to stimulate thrombopoiesis. TPO production primarily occurs in liver parenchymal cells with minor amounts synthesized in other tissues. The major clearance mechanism of TPO from circulation is via interaction between TPO and its receptor, c-Mpl, following which the c-Mpl-TPO complex undergoes internalization and degradation. However, whether it is the production or clearance of TPO the main regulator of circulating TPO levels has been debated but remains unclear. The prevailing theory was that hepatic TPO is generated constitutively, and the levels of circulating TPO are fine-tuned by its uptake and metabolism in platelets and megakaryocytes. Recently, this concept was modified by studies in Ashwell-Morell receptor (AMR) deficient mice, which highlighted the important role played by platelets in the regulation of hepatic TPO mRNA expression. As the most glycosylated platelet surface protein, GPIba contributes to chilled platelets clearance viahepatocytes, as well as antibody-mediated thrombocytopenia. However, the relationship between GPIba and plasma TPO has not been explored Methods and Results: We observed a significant decrease (approx. 3 fold) of TPO levels in both plasma and sera of GPIba-/- mice compared to wild type (WT) controls. Given the enlarged size of GPIba-/- platelets compared to WT, we investigated whether the low circulating TPO levels could be due to increased TPO clearance. Interestingly, we found similar levels of intracellular TPO in WT and GPIba-/- platelets, and the capacity to adsorb TPO in vitro was not enhanced. Further, we observed GPIba-/- platelets, surprisingly, have less c-Mpl expression compared to WT. Therefore, the decreased circulating TPO levels in GPIba-/- mice is unlikely due to enhanced TPO clearance. We next quantified TPO mRNA from hepatic liver cells as they are known as the major source of TPO. Consistent with the low circulatory TPO levels, we found reduced hepatic TPO mRNA in GPIba-/- mice, suggesting a deficiency in hepatic TPO generation. To determine whether exogenous platelet transfusions could influence steady-state hepatic TPO production, we transfused WT or GPIba-/- mice with WT or GPIba-/- platelets. Post-transfusion of WT, but not GPIba-/- platelets elevated TPO levels to around 150% in GPIba-/- mice. Additionally, our preliminary data showed that co-culture of hepatocytes with WT, but not GPIba-/- platelets in vitro increased hepatic TPO mRNA expression, further supporting a role for GPIba in TPO generation. To test whether TPO can equally induced platelet generation in GPIba-/- and WT mice. We injected rTPO into the two groups of mice, and found that in contrast to WT controls, rTPO did not significantly increase the platelet number of GPIba-/- mice in the circulation. Conclusion: Our data demonstrated that GPIba is important for TPO generation in liver and is necessary for TPO-induced platelet generation. These discoveries may have significant implications in TPO therapy in ITP. Disclosures No relevant conflicts of interest to declare.

Description: Background: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a life threatening disease often leading to severe bleeding diatheses, and/or miscarriage; although the incidence of miscarriage has not been adequately studied. FNAIT is caused by a maternal immune response against fetal platelet antigens, in which 〉80% reported cases have antibodies against β3 integrin. Maternal antibodies and Natural Killer (NK) cells may target antigen positive trophoblast cells and cause miscarriage, but this hypothesis has never been explored.In this study, we investigated whether platelet antibodies and NK cells impaired trophoblast invasion, and placental angiogenesis and function. Methods: β3 integrin deficient female mice were immunized with wild-type (WT) platelets and bred with WT males. Placental vascularisation and function were investigated by echography and micro-computered tomography (CT). Angiogenic and inflammatory cytokines, placenta growth factor (PlGF) and fms-like tyrosine receptor levels (Flt-1) were detected by ELISA. Placental function such as nutrient transport was detected by maternal intravenous administration of biotin and its perfusion to the fetus. NK cell phenotype was assessed by FACS. Placenta pathology was investigated by hematoxylin & eosin staining and immuno-histochemistry for cytokeratin-7, NK cell perforin and smooth muscle actin. Apoptosis and decidual remodeling were investigated by TUNEL assays. In vitro, NK cells were co-cultured with trophoblast cell lines and cytotoxicity was detected by flow cytometry. Results and discussion: Growth restriction and fetal loss/miscarriage only occurred in immunized mothers around embryo day E14.5. Placenta of affected fetuses had significantly reduced vascularization and materno-placental perfusion as demonstrated by ultrasound. CT scan also confirmed shallow development of placental sponge capillaries. These observations are validated by significantly reduced biotin perfusion to the fetuses which had a high hypoxia level. Immunized mice exhibited enlarged spleens as well as an increased Th1 and Th17 immune responses. These pro-inflammatory responses may contribute to trophoblast Flt-1 over-expression and a decreased plasma PlGF/sFlt-1 ratio. E.14.5, the end of organogenesis, is concomitant with trophoblast invasion into spiral arteries. The remodeling of spiral arteries lowers maternal vascular resistance and increases utero-placental blood flow which is critical for healthy pregnancy. Expression of trophoblast marker, Cytokeratin 7, was decreased in the placentas of immunized mice, suggesting scanty invasion invasion. Histology of affected placenta confirmed ischemic tissues, as revealed by significantly reduced numbers of maternal red blood cells in the labyrinth. Unexpectedly, decidual NK cell number remained elevated by day E14.5 in the placenta of immunized mice. Importantly activated NK cells released significant amount of perforin in the placenta, which may destroy target cells expressing β3 integrin. These observations support an abnormal remodeling process mediated by maternal antibodies and NK cells, since increased apoptosis was found in the decidua of immunized pregnant mice compared to naive control. Interestingly, maternal intravenous immunoglobulin therapy ameliorated survival of FNAIT fetuses.. To investigate the molecular and cellular mechanism involved, human choriocarcinoma cells (BeWo) were incubated with both murine anti-β3 antibodies and human HPA-1a IgG. Our data demonstrated, for the first time, that anti-platelet antibodies induced over-expression of the anti-angiogenic molecule, Flt-1 by BeWo cells. Conclusion: Our data suggest that maternal β3 integrin antibodies and NK cells impaired placental pro-angiogenic signalling and function. Reduced trophoblast invasion may cause poor materno-placental perfusion and fetus loss/miscarriage in FNAIT. Disclosures No relevant conflicts of interest to declare.