Our laboratory’s research program is focused on understanding the pathogenesis of heparin-induced thrombocytopenia (HIT), a life-threatening immune disorder caused by platelet factor 4 (PF4)/heparin antibodies. To date, we have made the following contributions to the field of HIT research:
Classical Pathway Activation by PF4/Heparin Complexes
Recent studies performed by Dr. Khandelwal indicate that PF4/heparin complexes bind circulating natural IgM and trigger complement activation. Studies reveal an approximately ten-fold variation in natural IgM levels among healthy donors, which may contribute to susceptibility to immune activation by PF4/heparin complexes.
Mechanism of complement activation by PF4/heparin complexes. (A) Heparin displaces PF4 to form ULCs. (B) Polyreactive natural IgM from plasma binds to ULCs, changes conformation and binds C1q to activate the classical pathway of complement activation.
- Khandelwal S, Ravi J, Rauova L, et al. Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway. Blood 2018;132(23):2431-40. PMCID: PMC6284214
- Khandelwal S, Johnson AM, Liu J, et al. Novel Immunoassay for Complement Activation by PF4/Heparin Complexes. Thromb Haemost 2018;118(8):1484-7. PMCID: PMC6288012
Major Role of Complement and CD21 on the PF4/Heparin Immune Response
Our recent work on complement activation in HIT shows that heparinized patients have circulating B-cells coated with PF4/heparin complexes and activated complement fragments (C3/C4). Studies performed by Dr. Khandelwal revealed that PF4/heparin complexes activate complement and bind to B-cells via complement receptor 2/CD21. Because antigen binding to B-cell CD21 enhances immunogenicity by factors of 1,000-10,000. We believe these findings identify an important pathway in the development of HIT antibodies.
Complement activation by PF4/heparin complexes leading to B cell binding via CD21. A. PF4 and heparin interact at over a narrow range of molar ratios to generate ULCs. PF4 alone or PF4 with excess heparin does not make ULCs. B. ULCs activate complement and bind complement activation products (C3/C4). C. Complement-coated ULCs bind to B-cell via CR2/CD21.
- Khandelwal S, Arepally GM. Immune pathogenesis of heparin-induced thrombocytopenia. Thromb Haemost 2016;116(5):792-8. PMCID: PMC6260829
- Khandelwal S, Lee GM, Hester CG, et al. The antigenic complex in HIT binds to B-cells via complement and complement receptor 2 (CD21). Blood 2016;128(14):1789-99.PMCID: PMC5054694
New Class of Heparin-dependent Antibodies in Patients Receiving Heparin (Protamine/heparin Antibodies) 5,6,7
Perhaps the most dramatic example of bi-directional impact of our research was our discovery of a new class of heparin-dependent antibodies to protamine/heparin complexes in patients undergoing cardiac surgery. Based on our murine studies (Chudasama et al., 2010), we predicted that patients receiving protamine during cardiac surgery would develop antibodies to protamine/heparin complexes. Studies performed by Dr. Lee during her time in the laboratory confirmed that 29% of patients undergoing cardiac surgery develop such antibodies(Lee et al., 2013). Dr. Lee received a NIH K08 mentored award to investigate the biological significance of protamine/heparin antibodies.
Incidence of PRT/H antibodies in healthy subjects and patients undergoing CPB. A. PRT/H antibody reactivity was measured by ELISA in healthy subjects (“Controls,” n = 101) and patients after CPB (n = 500) at baseline, day 3-7, and day 30 after CPB. B. The specificity of high titer PRT/H antibodies with PRT/H antibody levels of A450nm > 3 (n=32). Each symbol represents an individual CPB patient.
- Chudasama SL, Espinasse B, Hwang F, et al. Heparin modifies the immunogenicity of positively-charged proteins. Blood 2010;116 (26):6046-53. PMCID:PMC3031390
- Lee GM, Welsby IJ, Phillips-Bute B, Ortel TL, Arepally GM. High incidence of antibodies to protamine and protamine/heparin complexes in patients undergoing cardiopulmonary bypass. Blood 2013;121(15):2828-35. PMCID:PMC3624931
- Lee GM, Joglekar M, Kuchibhatla M, et al. Serologic characterization of anti-protamine/heparin and anti-PF4/heparin antibodies. Blood Advances 2017;1(11):644-51. PMCID: PMC5727817
Modeling the HIT Immune Response in Vivo
Our laboratory has pioneered the first murine immunization model to study the anti-PF4/heparin immune response (Suvarna et.al. 2005 and 2009). With this animal model, we demonstrated:
- Requirements for T cells (Suvarna et.al. 2005)
- The effects of heparin-dependence on antibody formation (Suvarna et.al. 2007)
- Immunogenicity of other heparin-like molecules (Joglekar et al., Thromb Haemost 2012) and protein/heparin complexes (Chudasama et al., 2010)
In vivo immune response to PF4/UFH is heparin dependent. A. Mice were immunized with mPF4 and UFH daily for five days and antibodies to mPF4/heparin (mP+H) were assayed by ELISA. B. Murine cohorts (n=7/cohort for PF4 alone, and UFH 50 U/mL, with remaining cohorts at n=10/cohort) were injected with mPF4 at 100 mg/mL alone or in combination with heparin at various PHRs as shown (PHRs from left to right after PF4 alone, 20:1, 10:1, 1:1 and 1:5). Significant differences between cohorts are indicated at the top of the figures.
We also generated critical pre-clinical data for the approval of the first generic enoxaparin compounds by the Food and Drug Administration (FDA). Our pivotal biophysical studies described in Suvarna et al (Blood, 2007) serve as the industry standard for testing of biosimilar low molecular weight heparins as recommended by the FDA.
- Suvarna S, Rauova L, McCracken EK, et al. PF4/heparin complexes are T cell-dependent antigens. Blood 2005;106(3):929-31. PMCID: PMC1895158
- Suvarna S, Espinasse B, Qi R, et al. Determinants of PF4/heparin immunogenicity Blood 2007;110:4253-60. PMCID: PMC2234783
- Joglekar MV, Quintana Diez PM, Marcus S, et al. Disruption of PF4/H multimolecular complex formation with a minimally anticoagulant heparin (ODSH). Thromb Haemost. 2012;107(4):717–725. PMCID: PMC4441624
- Suvarna S, Qi R, Arepally GM. Optimization of a murine immunization model for study of PF4/heparin antibodies. J Thromb Haemost 2009;7(5):857-64. PMCID: PMC3711941
- Chudasama SL, Espinasse B, Hwang F, et al. Heparin modifies the immunogenicity of positively-charged proteins. Blood 2010;116 (26):6046-53 PMCID: PMC3031390
First Monoclonal HIT Antibody
Our laboratory was the first to develop a monoclonal antibody to PF4/heparin complexes, KKO.12 Using KKO, our group was the first to demonstrate that PF4/heparin antibodies recapitulate disease manifestations of thrombocytopenia and thrombosis in a mouse model.17 KKO has served as a critical reagent for advancing insights into cellular mechanisms of thrombosis,13,(Kasthuri et al., Blood 2012) platelet activation(Rauova et al, Blood 2006) and diagnostic assays.14 KKO is currently licensed to Biokit Barcelona as diagnostic testing for HIT.
Binding characteristics of KKO, an anti-PF4/heparin monoclonal antibody. A. Binding of KKO to microtiter wells coated with PF4 (red circles) or PF4 and heparin (blue squares) expressed as absorbance at 405 nm. B. 14C-labeled platelet-rich plasma was incubated with 80 µg/ml KKO (or isotype control, IC) in the presence of PF4 (10 µg/ mL) and the indicated concentrations of heparin.
- Arepally GM, Kamei S, Park KS, et al. Characterization of a murine monoclonal antibody that mimics heparin-induced thrombocytopenia antibodies. Blood 2000;95(5):1533-40. PMID: 10688805
- Reilly MP, Taylor SM, Hartman NK, et al. Heparin-induced thrombocytopenia/thrombosis in a transgenic mouse model requires human platelet factor 4 and platelet activation through FcgammaRIIA. Blood 2001;98(8):2442-7. PMID: 11588041
- Cuker A, Rux AH, Hinds JL, et al. Novel diagnostic assays for heparin-induced thrombocytopenia. Blood 2013;121(18):3727-32. PMCID:PMC3643769
- Cai Z, Yarovoi SV, Zhu Z, et al. Atomic description of the immune complex involved in heparin-induced thrombocytopenia. Nat Commun 2015;6:8277. PMCID:PMC4580983
- Gollomp K, Kim M, Johnston I, et al. Neutrophil accumulation and NET release contribute to thrombosis in HIT. JCI Insight. 2018;3(18):e99445. PMCID:PMC6237233