session

63 - Oral Communication
Megakaryocytes, Platelets & VWF
Feb. 25, 2021, 1:45 p.m. - 3:15 p.m., Sydney

Abstract

4
Platelets from patients with MYH9 related disorders are mechanically stiffer
L. Sachs1, J. Baumann2, J. Wesche1, P. Nestler1, C. Zaninetti1, L. Lenkeit1, A. Greinacher1, M. Bender2, O. Otto1, R. Palankar1, Presenter: L. Sachs1 (1Greifswald, 2Würzburg)

Background and 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).
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.
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