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.

Public Health Relevance

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.

National Institute of Health (NIH)
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Heart, Lung, and Blood Program Project Review Committee (HLBP)
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Children's Hospital of Philadelphia
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McKenzie, Steven E; Sachais, Bruce S (2014) Advances in the pathophysiology and treatment of heparin-induced thrombocytopenia. Curr Opin Hematol 21:380-7
Cines, Douglas B; Lebedeva, Tatiana; Nagaswami, Chandrasekaran et al. (2014) Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin. Blood 123:1596-603
Yeung, Jennifer; Tourdot, Benjamin E; Fernandez-Perez, Pilar et al. (2014) Platelet 12-LOX is essential for Fc?RIIa-mediated platelet activation. Blood 124:2271-9
Cines, Douglas B; Cuker, Adam; Semple, John W (2014) Pathogenesis of immune thrombocytopenia. Presse Med 43:e49-59
Kowalska, M Anna; Zhao, Guohua; Zhai, Li et al. (2014) Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation. Arterioscler Thromb Vasc Biol 34:120-6
Litvinov, Rustem I; Yarovoi, Serge V; Rauova, Lubica et al. (2013) Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4. J Biol Chem 288:33060-70
Zhi, Huiying; Rauova, Lubica; Hayes, Vincent et al. (2013) Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo. Blood 121:1858-67
Lee, Grace M; Arepally, Gowthami M (2013) Diagnosis and management of heparin-induced thrombocytopenia. Hematol Oncol Clin North Am 27:541-63
Lee, Grace M; Welsby, Ian J; Phillips-Bute, Barbara et al. (2013) High incidence of antibodies to protamine and protamine/heparin complexes in patients undergoing cardiopulmonary bypass. Blood 121:2828-35
Cuker, Adam; Rux, Ann H; Hinds, Jillian L et al. (2013) Novel diagnostic assays for heparin-induced thrombocytopenia. Blood 121:3727-32

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