The development of platelet factor 4 (PF4)/heparin (H) antibodies initiates the morbidity of heparin-induced thrombocytopenia (HIT). This application seeks to elucidate the cellular basis of the PF4/H immune resposne using an optimized murine immunization model developed in our laboratory. Studies with this model have shown that PF4/H ultra large complexes (ULCs) potently induce PF4/H antibody (Ab) formation, directly activate DCs and induce proliferation of antigen-naive T-cells. Based on these observations, we hypothesize that PF4/H ULCs are not processed and presented as conventional antigens, but activate the immune system as SAGS. To test this hypothesis, we propose the following specific aims:
Specific Aim 1. Mechanisms of APC activation by PF4/H ULCs. Our studies show Ab responses to mPF4/H depend on the structural features of the antigen, that PF4/H complexes interact with variety of cell-surfaces through charge-dependent interactions, and that DCs are directly activated by PF4/H ULCs. Based on these observations, we hypothesize that biophysical attributes of PF4/H ULCs (charge and size) are critical for its SAG-like properties, enabling intact antigen to directly activate: DCs and T-cells without need for MHC-restricted antigen processing and presentation. To test this hypothesis, we will examine: effects of antigen composition (size and charge) on DC activation and DC binding, requirements for cell-surface MHC class II molecules on DCs and role of ULCs on promoting heterocellular interactions.
Specific Aim 2. Effects of PF4/H ULCs on T-cell activation, T/B cell interactions and T-cell memory. Our preliminary data show that PF4/H ULCs, like SAGs, elicit APC-dependent T-cell activation and proliferation, and that proliferation is CD28 dependent. In this aim, we hypothesize that PF4/H ULCs, as SAGs, elicit polyclonal activation of Vp restricted T-cell subsets and potentiate T-cell helper activity, but fail to produce memory T-cells. To test this hypothesis, we will examine proliferation of V(3-restricted T-cell subsets, T-helper activity leading to in vivo cytokine production, and T-cell memory.
Specific Aim 3. Germinal center formation and B- and T-cell fate in HIT. In this aim, we show that despite robust GC formation and production of isotype-switched Abs, immune recall is impaired in animals receiving mPF4/H ULCs. Based on these findings, we hypothesize that impaired immune recall in our murine model and human HIT is consistent with SAG induced T-cell anergy and failure to form memory B-cells. To test this hypothesis, we will investigate mechanisms of T-cell anergy and examine the fate of GC activated B-cells in the HIT immune response. With the availability of an optimized murine model, we are poised to address fundamental questions regarding the HIT immune response, specifically, how PF4, a self-antigen, becomes a potent immunogen in the presence of heparin, why antibody responses occur commonly in certain settings and why the immune response appears to be self-limited. We hope that insights from these studies will lead to novel interventions that will maintain anticoagulation but mitigate PF4/H Ab formation.
These proposed studies address why this allergic response occurs in response to a commonly used blood-thinner (heparin). In the context of this Program Project, these studies may lead to new preventative or diagnostic test and better therapies.
|Ostertag, Eric M; Bdeir, Khalil; Kacir, Stephen et al. (2016) ADAMTS13 autoantibodies cloned from patients with acquired thrombotic thrombocytopenic purpura: 2. Pathogenicity in an animal model. Transfusion 56:1775-85|
|Khandelwal, Sanjay; Arepally, Gowthami M (2016) Immune pathogenesis of heparin-induced thrombocytopenia. Thromb Haemost 116:792-798|
|Cai, Zheng; Zhu, Zhiqiang; Greene, Mark I et al. (2016) Atomic features of an autoantigen in heparin-induced thrombocytopenia (HIT). Autoimmun Rev 15:752-5|
|Khandelwal, Sanjay; Lee, Grace M; Hester, C Garren et al. (2016) The antigenic complex in HIT binds to B-cells via complement and complement receptor 2 (CD21). Blood :|
|McKenzie, Steven E (2016) Syk Inhibition in Ischemic Stroke. Arterioscler Thromb Vasc Biol 36:1054-5|
|Tutwiler, Valerie; Madeeva, Daria; Ahn, Hyun Sook et al. (2016) Platelet transactivation by monocytes promotes thrombosis in heparin-induced thrombocytopenia. Blood 127:464-72|
|ReppschlÃ¤ger, Kevin; Gosselin, Jeanne; Dangelmaier, Carol A et al. (2016) TULA-2 Protein Phosphatase Suppresses Activation of Syk through the GPVI Platelet Receptor for Collagen by Dephosphorylating Tyr(P)346, a Regulatory Site of Syk. J Biol Chem 291:22427-22441|
|Piatt, Raymond; Paul, David S; Lee, Robert H et al. (2016) Mice Expressing Low Levels of CalDAG-GEFI Exhibit Markedly Impaired Platelet Activation With Minor Impact on Hemostasis. Arterioscler Thromb Vasc Biol 36:1838-46|
|Belay, Eyayu; Miller, Chris P; Kortum, Amanda N et al. (2015) A hyperactive Mpl-based cell growth switch drives macrophage-associated erythropoiesis through an erythroid-megakaryocytic precursor. Blood 125:1025-33|
|Lambert, M P; Meng, R; Xiao, L et al. (2015) Intramedullary megakaryocytes internalize released platelet factor 4 and store it in alpha granules. J Thromb Haemost 13:1888-99|
Showing the most recent 10 out of 51 publications