Super-resolution imaging of platelet-activation process and its quantitative analysis

Understanding the platelet activation molecular pathways by characterizing specific protein clusters within platelets is essential to identify the platelet activation state and improve the existing therapies for hemostatic disorders. Here, we employed various state-of-the-art super-resolution imaging and quantification methods to characterize the platelet spatiotemporal ultrastructural change during the activation process due to phorbol 12-myristate 13-acetate (PMA) stimuli by observing the cytoskeletal elements and various organelles at nanoscale, which cannot be done using conventional microscopy. Platelets could be spread out with the guidance of actin and microtubules, and most organelles were centralized probably due to the limited space of the peripheral thin regions or the close association with the open canalicular system (OCS). Among the centralized organelles, we provided evidence that granules are fused with the OCS to release their cargo through enlarged OCS. These findings highlight the concerted ultrastructural reorganization and relative arrangements of various organelles upon activation and call for a reassessment of previously unresolved complex and multi-factorial activation processes.


Supplementary Note 2
Aggregation between the spread platelets was also observed at early time points, when they were nearly located ( Supplementary Fig. S3). However, we tried to image the separated platelets at each activation time point to observe the resolved ultrastructure of individual activated platelets. Figure S3.
STORM images of actin filaments in the aggregated platelets at different activation time points. Scale bar: 5 μm.

Supplementary Note 3
We also performed STORM imaging of acetylated microtubules, and found that these were mainly present in the small microtubular ring and not in the depolymerized microtubules outside the small ring (Fig. 2i).

Supplementary Note 4
We performed 3D STORM imaging to elucidate the ultrastructural changes of the other cytoskeletons during the activation process, including spectrin and vimentin, since their localization has not been well explored in superresolution. To observe the spectrin, we used the II-spectrin antibody since II-spectrin is known to be abundantly expressed in platelets as the major spectrin isoform of nonerythroid cells 1 . From the STORM images of 2IIspectrin in a resting platelet, we observed their punctate and speckled pattern and uniform distribution, which were also previously observed from the STORM images of erythrocytes, thus suggesting that they were real structures and not random dots ( Supplementary Fig. S6a). 2 Upon activation, they were concentrated in the center of the activated platelets at a relatively high position, probably localized in the 'yolk' spot of 'fried egg' morphology of activated platelets. Since the spectrin-based membrane skeleton is known to line the inner plasma membrane, the hollow shape in the swollen central region of the activated platelet was observed from the x-z cross-section of the 3D STORM images. Their increased density in the center was slightly decreased at the later stages of activation (~20 min), which could be due to the released microparticles encapsulated by spectrin.
We also performed 3D STORM imaging for vimentin intermediate filaments. We observed that they exhibit a punctate localization in platelets, as previously observed ( Supplementary Fig. S6c) 3 . It is known that the vimentin network is exposed on the surface of activated platelets, and we could also observe that it is bound to the surface of the activated platelets from the x-z cross-section of 3D STORM images 3 . Moreover, the higher level of vimentin at 5 min compared to levels in the resting platelet could be possibly due to the protein synthesis induced by various needs within the platelet as previously suggested 4 . We also found that they are centralized in the 'yolk' region of 'fried-egg' morphology with increased density during the activation process, in a similar fashion to II-spectrin.
( Supplementary Fig. S6d) We also observed a high density of vimentin on the released platelet microparticles from the activated platelets, probably wrapping the proteins released from the α-granules or organelles such as mitochondria, as previously suspected.

Supplementary Note 5
To quantify the α-granule clusters, we set 200 nm diameter of the cluster as the minimum size criterion to distinguish α-granules from other background signals. The average area of α-granules was 0.04 -0.20 μm 2 , which is consistent with previous results (200 -400 nm in diameter). To quantify the dense granule clusters, we used 150 nm, the average diameter of a cluster, as the minimum size criteria to distinguish the dense granule from other background signals. Under these criteria, on average, three dense granules were counted per resting platelet, which is consistent with previously reported values (Fig. 6g).

Supplementary Note 6
For membrane staining, the sample was stained with 100 nM Nile Red solution in DPBS for 20 -30 min at RT, and immediately imaged using STORM. We tested both permeabilized and non-permeabilized platelets, and found that the permeabilized sample exhibits both of the OCS and the membrane organelles, as reported before 5 . (Fig.   S13) Since the permeabilized platelets showed stained membrane organelles more clearly than the OCS, we used the non-permeabilized sample to quantify OCS, which exhibits the OCS more clearly. Scale bar: 1 μm. Figure S13.

Supplementary Note 7
Since the three-dimensional optical diffraction tomography is based on the measurement of three-dimensional refractive index distributions of samples, it clearly reveals these balloons in platelets. (Supplementary Video 2) Although previous attempts to assess ballooning in platelets have been limited by the methods of investigation due to the fragility of the balloon structure, platelets have long been reported to transform to form balloons upon activation.

Supplementary Video 2.
The 3D optical diffraction tomography video showing a platelet undergoing ballooning on adhesion to a glass surface as the beginning step of platelet activation. Scale bar: 1 μm.
The 3D reconstructed HV-EM images of the activated platelet using tomographic slices.