Abstract Number: PB1809
Meeting: ISTH 2020 Congress
Background: Gram-positive Staphylococcus aureus is a leading cause of blood stream infection. Platelet activation and aggregation by S. aureus proteins plays an important role in the pathogenesis of infective endocarditis.
Aims: To quantify S. aureus protein induced changes in cytoskeleton dependent biomechanical properties and activation status of platelets.
Methods: High-throughput (>1000 single platelets/second) mechano-phenotyping of single platelets was performed in microfluidic real-time 1D-imaging fluorescence and deformability cytometry (RT-FDC). 50 µL of washed human platelets (300,000/µL) were incubated for 10 minutes with S. aureus proteins: phospholipase C (Plc, 4 µM), major autolysin (AtlA-1, 2 µM), extracellular adherence protein (EapD3D4 domain, 4 µM), chemotaxis inhibitory protein of S. aureus (CHIPS, 4 µM), and formyl peptide receptor-like 1 inhibitory protein (FLIPr, 4 µM). Thrombin receptor activator peptide 6 (TRAP6) was used as PAR1 agonist. Platelet deformation and activation (CD62P expression) were detected simultaneously in live-platelets by RT-FDC and F-actin by flow cytometry and confocal fluorescence microscopy.
Results: Mechano-phenotyping by RT-FDC revealed, except for FLIPr (median deformation = 0.145), that all tested S.aureus proteins lowered platelet deformability (i.e. increased stiffness) compared to the internal negative control Plc (median deformation 0.154) (Figure 1, A). While, EapD3D4 and CHIPS decreased deformation (median deformation 0.058 and 0.101) and increased CD62P expression (median fold increase 17.7 and 13.25) and F-actin content (median fold increase 1.43 and 2.54), intriguingly, AtlA-1 induced lower deformation (median fold increase 0.107) albeit without significant increase in CD62P expression but with increased F-actin content (median fold increase 1.65)
(Figure 1, A & B).
Conclusions: Changes in cytoskeleton dependent viscoelastic properties of platelets in the presence of S. aureus proteins reveal heterogeneity in platelet activation response in individual donors. Platelet deformation can be used as sensitive label-free activation marker for platelet activation even in the absence of CD62P expression.
To cite this abstract in AMA style:Lenkeit L, Sachs L, Kohler T, Wesche J, Greinacher A, Hammerschmidt S, Otto O, Palankar R. High-throughput Mechano-Phenotyping of Single Platelet Activation Induced by Bacterial Proteins [abstract]. Res Pract Thromb Haemost. 2020; 4 (Suppl 1). https://abstracts.isth.org/abstract/high-throughput-mechano-phenotyping-of-single-platelet-activation-induced-by-bacterial-proteins/. Accessed November 30, 2021.
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