Platelet-activating antibodies to the chemokine, platelet factor 4, in cerebral malaria differ from those in heparin-induced thrombocytopenia and vaccine-induced thrombotic thrombocytopenia. Free

Background: Cerebral malaria (CM) is a severe complication of Plasmodium falciparum infection, primarily affecting children, with a mortality rate near 20% and no targeted treatment. Recent studies have identified platelet-activating autoantibodies in patients with CM, detected using commercial heparin-induced thrombocytopenia (HIT) assay (Immunocor), suggesting a potential autoimmune component in CM-associated thromboinflammation (PMID:38652559). We hypothesized, that in CM, platelet factor 4 (PF4) released from activated platelets serves as an immunogenic antigen that triggers the production of anti-PF4 antibodies contributing to thromboinflammation and disease severity.

Aim: To characterize the epitope specificity of anti-PF4 antibodies in CM, and determine whether their properties mirror those associated with HIT or vaccine-induced immune thrombotic thrombocytopenia (VITT).

Methods & Results: We studied 66 plasma samples from Malawi that were previously used in our study (IV, KS & KK; along with five CM samples and five control samples from asymptomatic community children in Uganda. The Malawi samples included 22 CM with positive (OD>0.4) and 22 CM samples negative (OD≤0.4) antibodies to human (h) PF4/polyanion complexes (hPF4/P) and 22 uncomplicated malaria (UM) samples also negative for hPF4/P antibodies, as determined using the Immucor PF4 ELISA. All five Uganda CM samples—and none of the control samples—tested positive for hPF4 antibodies. We found that anti-PF4 antibodies in CM patients react with PF4 alone independent of heparin. To assess the pathogenic potential, we tested whether CM plasma positive for hPF4 antibodies could activate murine platelets derived from PF4-deficient mice, with or without expression of human FcγRIIA. Platelet activation was dependent on the presence of both FcγRIIA and exogeneous hPF4 (p < 0.0001). To determine whether anti-hPF4 antibodies in CM patient samples recognize the previously defined HIT or VITT PF4 epitope, we performed competitive binding assays using increasing amounts of a HIT-like monoclonal antibody KKO (0-5 µg/ml), and a VITT-like monoclonal antibody 1E12 (0-5 µg/ml), respectively. Neither KKO nor 1E12 inhibited the binding of CM-derived anti-hPF4 antibodies. Increasing concentrations of 1E12 actually enhanced CM anti-PF4 IgG binding to hPF4 up to a threefold increase observed at ≥1 µg/ml of 1E12 compared to binding in the absence of 1E12 (p<0.01). Finally, we assessed cross-reactivity to related chemokines stored in platelet α-granules—namely mouse PF4 (mPF4) and both human and mouse neutrophil-activating peptide 2 (NAP2/CXCL7). Using ELISA, we found that CM samples positive for hPF4 antibodies exhibited significantly higher binding to mPF4 (p < 0.05), hNAP2 (p < 0.001), and mNAP2 (p < 0.05), compared to healthy controls.

Conclusions: These findings identify a subset of patients with CM that had high-titer, FcγRIIA-dependent, platelet-activating anti-PF4 antibodies that do not map to the canonical HIT or VITT epitopes. Their cross-reactivity with conserved regions of related chemokines supports the hypothesis that the shared antigenic target lies within the conserved C-terminal heparin-binding domain of these chemokines.and suggests a distinct autoimmune response potentially driven by malaria-induced platelet activation. This highlights a novel mechanism of thromboinflammation in CM and points to PF4-targeted autoimmunity as a contributor to disease severity. Future studies will aim to map this antigenic site and evaluate the thrombogenic potential of CM antibodies in vivo as well as the ability of anti-PF4 blocking antibodies to ameliorate the severity of the CM.