Cytochalasins modulate donor responsiveness in functional assays for heparin-induced thrombocytopenia

Functional assays are essential for confirming platelet-activating anti-PF4/heparin antibodies in heparin-induced thrombocytopenia (HIT), yet their performance is limited by procedural complexity and donor variability. The actin cytoskeleton regulates platelet activation, but its role in donor responsiveness during functional assays remains unclear.

To investigate donor responsiveness to anti-PF4/heparin antibodies and examine whether cytochalasin B (CB) influences platelet activation responses in HIT functional assays.

Functional assays using light transmission aggregometry and flow cytometry were performed using washed platelets from healthy donors. The effects of cytochalasins on platelet activation were evaluated, and FcγRIIA-, GPVI-, and CLEC-2-mediated activation pathways were compared.

Low concentrations of cytochalasins (1-2 μg/mL) significantly shortened lag time to platelet aggregation without altering maximal aggregation. High concentrations (≥10 μg/mL) prolonged lag time or inhibited aggregation. CB increased platelet responsiveness among low-responder donors and increased the number of donors classified as high responders to weakly positive HIT serum. Among the platelet activation pathways examined, significant lag time shortening following CB treatment was observed in FcγRIIA-mediated activation, whereas GPVI- and CLEC-2-mediated activation did not.

Low-dose cytochalasins shortened lag time during FcγRIIA-mediated platelet activation and increased the number of responsive donors in functional assays. These findings provide insight into donor-dependent variability and may help improve the performance of functional assays for HIT.