Heparin-induced thrombocytopenia (HIT) is a life-threatening immune-mediated thrombotic disorder caused by antibodies to PF4/heparin complexes. The immune response is common and yet little is understood about its occurrence. In the last funding period, we made fundamental observations related to early events surrounding the host?s encounter with the PF4/heparin antigenic complex. Specifically, we showed: 1) preferential binding of PF4/heparin to circulating B-cells, compared with other leukocytes or platelets, 2) heparin-dependent binding of PF4/heparin to B-cells in patients receiving heparin, 3) complement fixation by PF4/heparin antigen, 4) complement-mediated PF4/heparin binding to circulating B-cells in patients receiving heparin, and 5) binding of complement-coated antigen to B-cells via complement receptor 2 (CR2/ CD21). In the following aims, we will test the hypothesis that complement activation by PF4/heparin complexes and deposition of antigen on B-cells via CD21 is essential for development of HIT autoantibodies.
Specific Aim 1 : Mechanism of complement activation by PF4/heparin complexes. In preliminary data, we show that mannose-binding lectin and ficolin-2 bind PF4/heparin complexes. Based on these findings, we will test the hypothesis that PF4/heparin complexes activate complement via the lectin pathway. We will define the structural basis of PF4/heparin-lectin interactions, study functional interactions of lectins with PF4/heparin complexes, and examine the role of complement inhibition in HIT.
Specific Aim 2 : Cellular consequences of CD21 engagement by PF4/heparin and complement. Binding of complement-coated antigen to CD21 augments humoral immunity by 103 to 104 fold. We hypothesize that binding of complement-coated multivalent PF4/heparin to the CD21 complex facilitates recruitment and signaling of antigen-specific B-cells. We will examine downstream signaling, activation and proliferation of cognate and non-cognate B-cells, perform in vivo studies in mice with a fixed B-cell receptor and characterize the contribution of CD21-expressing follicular dendritic cells to Ab formation.
Specific Aim 3 : Examine host predictors of PF4/heparin seroconversion. Complement-coated antigen can be detected on B-cells in some, but not all, patients receiving heparin therapy. Using a novel C3 capture immunoassay, we also show significant donor to donor variation in healthy donor plasma incubated with a fixed dose of PF4/heparin. Based on these observations, we will examine host susceptibility to anti- PF4/heparin seroconversions by characterizing the complement proteome and using antigen-positive B-cells as a marker for seroconversions in heparinized patients. By defining the cellular pathways that initiate formation of PF4/heparin Abs, we hope to uncover mechanisms relevant to the immunogenicity of other auto- or exogenous antigens.
Heparin is a commonly used blood thinner in hospitalized patients. Patients develop a life- threatening allergy to this drug. This grant seeks to understand the mechanisms by which patients develop allergic reactions to this drug.
| Khandelwal, Sanjay; Johnson, Alexandra M; Liu, Jian et al. (2018) Novel Immunoassay for Complement Activation by PF4/Heparin Complexes. Thromb Haemost 118:1484-1487 |
| Rauova, Lubica; Arepally, Gowthami; Poncz, Mortimer et al. (2018) Molecular and cellular pathogenesis of heparin-induced thrombocytopenia (HIT). Autoimmun Rev 17:1046-1052 |
| Khandelwal, Sanjay; Ravi, Joann; Rauova, Lubica et al. (2018) Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway. Blood 132:2431-2440 |
| Gunaratne, Ruwan; Kumar, Shekhar; Frederiksen, James W et al. (2018) Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass. Nat Biotechnol 36:606-613 |
| Gollomp, Kandace; Kim, Minna; Johnston, Ian et al. (2018) Neutrophil accumulation and NET release contribute to thrombosis in HIT. JCI Insight 3: |
| Dai, Jing; Madeeva, Daria; Hayes, Vincent et al. (2018) Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia. Blood 132:727-734 |
