Platelets from patients with MYH9 related disorders are mechanically stiffer

L Sachs; J Baumann; J Wesche; P Nestler; C Zaninetti; L Lenkeit; A Greinacher; M Bender; O Otto; R Palankar

doi:10.1055/s-0041-1728123

Hämostaseologie, Table of Contents

Hamostaseologie 2021; 41(S 01): S23
DOI: 10.1055/s-0041-1728123

Oral Communication

Megakaryocytes, Platelets & VWF

Platelets from patients with MYH9 related disorders are mechanically stiffer

Authors

L Sachs

¹Institute for Immunology and Transfusion Medicine, University Hospital Greifswald, Greifswald
J Baumann

²Institute of Experimental Biomedicine – Chair I, University Hospital and Rudolf Virchow Center, Würzburg
J Wesche

¹Institute for Immunology and Transfusion Medicine, University Hospital Greifswald, Greifswald
P Nestler

³ZIK HIKE – Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald
C Zaninetti

¹Institute for Immunology and Transfusion Medicine, University Hospital Greifswald, Greifswald
L Lenkeit

¹Institute for Immunology and Transfusion Medicine, University Hospital Greifswald, Greifswald
A Greinacher

¹Institute for Immunology and Transfusion Medicine, University Hospital Greifswald, Greifswald
M Bender

²Institute of Experimental Biomedicine – Chair I, University Hospital and Rudolf Virchow Center, Würzburg
O Otto

³ZIK HIKE – Center for Innovation Competence, Humoral Immune Reactions in Cardiovascular Diseases, University of Greifswald, Greifswald
R Palankar

¹Institute for Immunology and Transfusion Medicine, University Hospital Greifswald, Greifswald

Abstract

Full Text

Objective The MYH9 gene encodes for the heavy chain of non-muscle myosin IIA (NMMHC-IIA), which is involved in a variety of cellular processes that require biomechanical contractile force generation. Variants of MYH9 cause autosomal dominant MYH9-related disease (MYH9-RD) and are characterized by thrombocytopenia with giant platelets and mild to moderate bleeding tendency. Although the role of the cytoskeleton for platelet function is well known, the consequences of NMMHC-IIA mutations on platelet biomechanics are only poorly understood. To investigate this, high throughput functional mechanophenotyping was performed by real-time fluorescence and deformability cytometry (RT-FDC).

Material and Methods Using RT-FDC we analysed intrinsic biomechanics of single platelets from MYH9-RD patients with point mutations in the MYH9 gene that lead to following amino acid substitutions in NMMHC-IIA at positions p.D1424N and p.E1841K. For functional mechanophenotyping by RT-FDC, platelets were labelled with the platelet marker integrin β3 (CD61-PE) and with the platelet activation markers integrin αIIbβ3 (PAC1-FITC) and P-selectin (CD62P-AlexaFluor 647) while TRAP-6 was used as PAR1 agonist. Platelet F-actin content was determined by phalloidin-Atto 647 binding in flow cytometry.

Results Platelets from MYH9-RD patients showed two fold lower deformation (i. e. stiffer platelets) and were larger (p.D1424N: median deformation 0.0685, median size 9.63 µm2, n = 790 platelets and p.E1841K: 0.0686, 5.77 µm2, n = 955 platelets) than platelets from healthy individual (median deformation 0.1434, size 3.76 µm2, n = 1334 platelets). Upon TRAP-6 activation, platelets from healthy individuals became highly stiffer (Fold change: 2.87 fold, n = 962 single platelets) while platelets from MYH9-RD patients became only marginally stiffer (p.D1424N: 2.27 fold increase in stiffness compared to non-activated and E1841K: 1.9 fold increase in stiffness compared to non-activated). Quantification of basal F-actin content in non-stimulated MYH9-RD platelets was found to be two-fold higher than in the control platelets. With TRAP-6 activation, the F-actin level in control platelets increased more than in MYH9-RD platelets.

Conclusion We demonstrate that platelets from MYH9-RD, individuals with mutations in the MYH9 gene (p.D1424N and p.E1841K), are biomechanically stiffer with higher F-actin content than platelets from healthy individuals.