Complement Activation As a Biomarker for Platelet Activating Antibodies in Heparin-Induced Thrombocytopenia (HIT)
Authors: Myoung Sooho S , Francis Samuel , Chen Jonah , Lee Grace M , Rauova Lubica , Poncz Mortimer , Cines Douglas B. , Kuchibhatla Maragatha , Khandelwal Sanjay , Arepally Gow 
Published: December 2024

Background: IgG antibodies (Abs) to platelet factor 4 and heparin (PF4/H) commonly occur after heparin exposure, but cause life-threatening complications of HIT in only a subset of patients. To date, only platelet activation assays can reliably distinguish anti-PF4/heparin Abs that cause disease (HIT Abs) from those more likely to be asymptomatic (AAbs). In recent studies, we showed that complement activation by HIT Abs is essential for downstream FcgRIIA-mediated cellular activation.

Study Objectives: As pathogenic HIT Abs also bind and cross-link platelet FcgRIIA, we asked if there was a correlation between complement and platelet activating properties of anti-PF4/heparin Abs. To address this question, we studied a clinically annotated cohort of patients with (HIT; n=8) or without HIT (AAb+, n=14) to compare clinical and laboratory features with serologic properties of Ab titers, platelet and complement activation.

Methods: Patients testing positive for polyclonal anti-PF4/heparin Abs in the Duke Coagulation Laboratory were consented and included in the study based on sample availability. Plasma or whole blood from consented healthy donors was used as controls or as a source of complement in complement activation assays. Demographics, platelet counts, 4Ts and HEP score, % decline in platelet count, serologic data (anti-PF4/heparin polyclonal and IgG assay, and serotonin release assay or SRA results) were recorded. Endpoint titers were calculated using an in-house ELISA. Complement activation was measured, as previously described (Khandelwal, Blood 2021), using patient plasma (10% v/v) added to undiluted healthy donor plasma with buffer or PF4 (25 µg/ml) and heparin to generate in situ immune complexes (ICs). Complement-fixed ICs were measured using an immunocapture assay, using KKO, a monoclonal anti-PF4/heparin Ab, as the capture Ab and an anti-C3c (Quidel, San Diego) Ab for detection of captured immune complexes. MMP9 and IL-8 were measured after incubation of patient plasma (10% v/v) added to undiluted whole blood for 30 minutes (MMP9) or 6 hours (IL-8), with respective proteins detected using commercial immunoassays (R&D Systems, Minneapolis, MN).

Results: As compared to AAb+ patients, HIT patients had significantly lower mean + standard deviation (SD) platelet counts (AAb+ vs. HIT: 67 +31 v39 +16; p&lt;0.02), greater % drop in platelet counts (AAb+ vs. HIT: 65% + 14% v 79% +12%; p&lt;0.03), higher 4Ts (AAb+ vs. HIT: 3.4 + 0.8 v 6.5 +1.1; p&lt;0.0001), HEP scores (AAb+ vs. HIT: 4.4 +1.7 v 11.1 +2.5; p&lt;0.0002), anti-PF4 polyclonal (AAb+ vs. HIT: 3.9 + 4.0 v 8.5 + 5.8; p&lt;0.03) and IgG Ab levels (AAb+ vs. HIT: 0.7 +0.7 v 2.2 +0.9; p&lt;0.008). Serologic properties also significantly differed between the two cohorts, with HIT patients showing median higher Ab titers (AAb+ vs. HIT: 900 v 7400; p&lt;0.009), higher mean + SD% serotonin release or SRA+ (AAb+ vs. HIT: 5+7 v 91 +8; p&lt;0.0001) and greater mean + SD complement activation, as determined by a C3c immunoassay (AAb+ vs. HIT: 0.8 +0.6 v 3.4 +0.6; p&lt;0.0002). All HIT patients (8/8) showed strong complement activation, while 2/14 AAb+ patients showed increased complement activation over background levels of healthy donors. The extent of complement activation closely correlated with % serotonin release by anti-PF4/heparin Abs (r=0.754; p&lt;0.001 by Spearman), as well as other clinical and laboratory parameters including, clinical 4Ts score (r = 0.64, p = 0.001), % drop in platelet count (r = 0.256, p = .023) and anti-PF4/heparin IgG levels (r = 0.444, p = .038). Complement activation also showed strong correlation with properties of cellular activation as gauged by MMP9 secretion (r = 0.680, p = 0.001) and release of IL-8 (r = 0.799, p = 0.0001) in whole blood.

Conclusions: Our findings support that complement activation strongly correlates with other cellular activation endpoints, including platelet and monocyte/neutrophil activation and importantly distinguish anti-PF4/heparin Abs with and without disease potential. If these findings are confirmed in a larger cohort, complement activation can likely serve as “functional” biomarker for detecting pathogenic HIT Abs, perhaps obviating the need for a functional platelet activation assay.