Our Research

1) Thrombosis and hemostasis

We established intravital microscopy thrombosis models and demonstrated that thrombosis occurs in mice lacking two key molecules in thrombosis: fibrinogen (Fg) and von Willebrand factor (VWF). This work significantly modified the theory of thrombosis/hemostasis (JCI 2000). I found that GPV in the GPIb-V-IX complex plays an inhibitory role in thrombin-mediated platelet activation, but a supportive role in thrombus stabilization (Blood 2001); both Fg and fibrin play roles in thrombus stabilization and plasma Fg inhibits platelet fibronectin (Fn) internalization (Blood 2003); plasma Fn promotes thrombus growth in injured arterioles (PNAS 2003). My lab further showed that Fg/VWF-independent platelet aggregation occurs in vitro under more physiological conditions (i.e. non-anticoagulated blood), and both platelet and plasma proteins contribute to this novel platelet aggregation pathway (J Thromb Haemost 2006). Interestingly, we found that soluble plasma Fn is a potent inhibitor of platelet aggregation (Blood 2009). By switching between a soluble and insoluble form, Fn plays a self-limiting regulatory role in thrombosis/hemostasis (JCI 2014). Most recently, we found that “Apolipoprotein A-IV  is a novel ligand of platelet β3 integrins and an endogenous inhibitor of thrombosis” (invited revision by Nature).

Fig. 1. Platelets are key players in hemostasis and thrombosis.
At the site of vascular injury, the “protein wave of hemostasis”, a process in which plasma fibronectin (pFn) and likely other proteins deposit onto the vessel wall, occurs earlier than platelet accumulation (the classical first wave of hemostasis). Platelet translocation is initiated by GPIb-IX-V complex and VWF interactions. Following GPVI-collagen binding and other platelet activation events, stable adhesion is mediated by several platelet integrins and their ligands, such as integrin αIIbβ3-VWF/fibronectin/fibrinogen/fibrin, α2β1-collagen, α5β1-fibronectin, α6β1-laminin. Platelet adhesion, activation and aggregation constitute the classical “first wave of hemostasis”. In addition, platelets actively contribute to the “second wave of hemostasis” (blood coagulation) promoting cell-based thrombin generation.
(Xiaohong Ruby Xu, Dan Zhang, Brigitta Elaine Oswald, Naadiya Carrim, Xiaozhong Wang, Yan Hou, Qing Zhang, Christopher Lavalle, Thomas McKeown, Alexandra H. Marshall & Heyu Ni. Platelets are versatile cells: New discoveries in hemostasis, thrombosis, immune responses and beyond. Critical Reviews in Clinical Laboratory Sciences. 2016, 9: 1-69. )

2) Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT)

We established the first animal model of Fetal and Neonatal Alloimmune Thrombocytopenia (FNAIT); clarified the correlation between maternal antibody titers and pathological severity in FNAIT; and found that IVIG is a useful therapy for this life-threatening disease (Blood 2006). We further demonstrated that the neonatal Fc receptor (FcRn) plays a key role in FNAIT pathogenesis and can be targeted for therapy (Blood 2010); maternal immune response against GPIbα causes frequent miscarriage due to thrombus formation in placenta (JCI 2011, with editorial commentary and media coverage); antibody-mediated immune suppression can prevent FNAIT (Transfusion 2012, with editorial commentary; initiation of a large clinical trial in 4 European countries); impairment of angiogenesis but not thrombocytopenia is the major cause of intracranial hemorrhage in FNAIT and can be prevented with IVIG (JCI 2015). Our preliminary data suggest that maternal immune response against fetal β3 integrins (αIIbβ3 or αvβ3) on vessels, trophoblasts, and placental stem cells may play key roles in intrauterine growth restriction and miscarriage.

Fig. 2. Diagram of some major human platelet antigens (HPAs) on various glycoprotein complexes that are responsible for alloimmunization of pregnant women leading to fetal and neonatal alloimmune thrombocytopenia (FNAIT).
(Darko Zdravic, Issaka Yougbare,  Brian Vadasz,  Conglei Li,  Alexandra H. Marshall,  Pingguo Chen,  Jens Kjeldsen-Kragh,  Heyu Ni. Fetal and neonatal alloimmune thrombocytopenia. Seminars in Fetal and Neonatal Medicine, 2016, 21(1):19–27.)

3) Immune thrombocytopenia (ITP)

We established models of immune thrombocytopenia (ITP) using our unique mouse anti-mouse αIIbβ3 and GPIbα antibodies. We found ITP induced by anti-GPIbα may fundamentally differ from that of anti-β3 integrin antibodies. The former can deliver signals to platelets and cause platelet activation/desialylation, leading to Fc-independent phagocytosis, which can be resistant to IVIG therapy. Some of these data were published (Blood 2006) and confirmed in humans by others (Haematologica. 2007) and ourselves (J Thromb Haemost 2014). Most recently, we found this Fc-independent platelet clearance occurred in liver via hepatocyte Ashwell-Morell receptors (Nature Communications 2015). This research could save Canada $10 million/year ($100 million in US and Europe/year) by identifying patients that are refractory to IVIG therapy. Two patents have been approved stemming from this project, and new sialidase inhibitor therapies have been initiated in clinical trials in China and Canada. We also identified several CD8 T + regulatory cells from this project (Blood 2015).

Fig 3. (A) The schematic diagram of GPIb-IX-V complex and GPIIbIIIa integrin. Binding of GPIbα may induce downstream signalling involving recruitment of the 14-3-3-ζ complex. This may lead to inside-out signalling that activates the GPIIbIIIa integrin. (♦) N-linked glycosylation. (●) O-linked glycosylation. (B) Anti-GPIIbIIIa opsonized platelets are likely cleared via Fc receptors in the RES. (C) Anti-GPIbα antibodies may cause platelet activation, PS exposure, and aggregation leading to clearance via Fc independent mechanisms in the liver and spleen.
(June Li, Dianne E. van der Wal, Lingyan Zhu, Brian Vadasz, Elisa K. Simpson, Conglei Li, Michelle Lee Webster, Guangheng Zhu, Sean Lang, Pingguo Chen, Qingshu Zeng, Heyu Ni. Fc-independent phagocytosis: Implications for IVIG and other Therapies in immune-mediated thrombocytopenia. Cardiovascular & Haematological Disorders-Drug Targets, 2013, 13: 50-58.)

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