Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

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Platelets are anucleated blood cells that play an invaluable role in hemostasis. The platelet actin-myosin network generates contractile forces necessary for optimal hemostatic function. Although the significance of cytoskeletal defects in the pathogenesis of inherited platelet disorders affecting platelet function and resulting in increased bleeding risk has been elucidated, deciphering their impact on intrinsic biomechanics of platelets remains challenging. It also represents an unmet need from a diagnostic and prognostic perspective. Recently introduced real-time fluorescence deformability cytometry (RT-FDC) is an enabling biophysical technique for on-chip, high-throughput single-cell mechanophenotyping with molecular specificity. However, it is unclear how pre-analytical variables such as ex vivo anticoagulants used during the collection of peripheral blood impact biomechanical properties of cellular components blood. Here we report on comprehensive high-throughout functional mechanophenotyping of single human platelets by RT-FDC in different ex vivo anticoagulants. We found that the type of ex vivo anticoagulant used to collect peripheral blood is a critical pre-analytical parameter that differentially impacts platelet deformation, size, and functional response to agonists by altering the platelet cytoskeleton. Moreover, we applied our findings to characterize the functional mechanophenotype of platelets using RT-FDC from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.

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