Determinants of pathogenic anti-PF4 antibody-mediated platelet activationathophysiology, diagnosis and treatment

The National Heart, Lung, and Blood Institute awarded Mayo Clinic Arizona $249,000 on August 1, 2026, under the Cardiovascular Diseases Research program (CFDA 93.837) to investigate the pathogenic mechanisms of platelet factor 4 (PF4)–reactive antibodies in antibody-mediated thrombotic disorders including heparin-induced thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia, and monoclonal gammopathy of thrombotic significance.

The project, titled "Determinants of Pathogenic Anti-PF4 Antibody-Mediated Platelet Activation: Pathophysiology, Diagnosis and Treatment," aims to define how platelet-surface PF4 antigen presentation and anti-PF4 antibody Fc N-glycan structure jointly determine FcγRIIA-dependent platelet activation. Current standard anti-PF4 ELISA assays measure antibody binding rather than platelet-activating potential, missing some pathogenic antibodies and delaying risk stratification during clinical decision-making. Aim 1 will define platelet-surface determinants required for PF4 antigenic complex assembly using protein-directed perturbation, glycosaminoglycan class dissection, and platelet activation readouts. Aim 2 will determine how Fc N-glycan structure controls FcγRIIA-dependent platelet activation using glycoengineered patient-sequenced monoclonal antibodies, PF4/heparin immunopurification, and LC-MS endogenous glycoform profiling.

The award obligates $249,000 with a period of performance from August 1, 2026, through June 30, 2029. Work is performed at Mayo Clinic Arizona's Scottsdale, Arizona facility.

Abstract

(see instructions): antibody-mediated thrombotic disorders targeting platelet factor 4 (PF4), including heparin-induced thrombocytopenia (HIT), vaccine-induced immune thrombotic thrombocytopenia (VITT), and monoclonal gammopathy of thrombotic significance (MGTS), cause clinically important thrombosis but remain difficult to classify with current frontline tests. Standard anti-PF4 elisas measure antibody binding rather than platelet-activating potential, and some pathogenic anti-PF4 antibodies in HIT and MGTS are missed by standard assays. This diagnostic blind spot can delay appropriate risk stratification and leads to unnecessary interruption of heparin and use of alternative anticoagulants while specialized platelet activation testing is pending. The objective of this R00 project is to define how platelet-defined PF4 antigen presentation and anti-PF4 FE N-glycan structure jointly determine fcyriia-dependent platelet activation, the central hypothesis is that anti-PF4 antibodies become pathogenic only when PF4 is displayed in a platelet-defined antigenic complex and the antibody FE domain supports productive fcyriia engagement. This hypothesis is supported by K99 data showing that patient-derived anti-PF4 monoclonal antibodies can be expressed and glycoengineered, PF4 forms multivalent platelet-surface complexes, and FE N-glycan remodeling changes platelet activation despite preserved PF4/polyanion elisa binding. Aim 1 will define platelet-surface determinants required for PF4 antigenic complex assembly using protein-directed perturbation, glycosaminoglycanclass dissection, and platelet activation readouts. Aim 2 will determine how FE N-glycan structure controls fcyriiadependent platelet activation using glycoengineered patient-sequenced monoclonal antibodies, PF4/heparin immunopurification, LC-MS endogenous glycoform profiling, and selected microfluidic thrombosis validation. Aim 3 will develop a prototype paired-well diagnostic framework that separates total anti-PF4 binding from fcyriia-engaging activity and reports a pathogenicity index benchmarked against PF4 elisa and platelet activation testing. Successful completion will establish an independent mechanistic and translational program focused on anti-PF4 antibody pathogenicity, FE glycoform biology, and receptor-engagement diagnostics. The expected outcome is a framework for distinguishing detectable from pathogenic anti-PF4 antibodies, supporting improved classification, risk stratification, and diagnostic development in HIT, VITT, MGTS, and related immune thrombotic disorders.