Background: Platelets play an essential role in thrombus formation by secreting a variety of prothrombotic proteins from their alpha and dense granules upon activation. The mechanisms that control granule release and organization are still incompletely understood, in part because we lack the ability to obtain quantitative morphometric data from large numbers of platelets imaged at sufficient resolution to discern their individual granules.

Aims: Our aim is to develop an automated method for quantitative analysis of platelet granules using super-resolution microscopy that can be applied to patients with bleeding abnormalities.

Methods: Platelets were isolated from healthy donors and patients with Von Willebrand Disease (VWD). Degranulation was induced by activating platelets with 10 µM PAR-1-AP. Fixed platelets were immunostained for alpha-tubulin (marginal band), VWF and SPARC (both alpha-granules). Samples were imaged employing Structured Illumination Microscopy. After reconstruction, images were analyzed by custom-developed ImageJ macros to quantify platelet (granule) morphometric parameters.

Results: Activation of platelets led to clear morphological and granular changes, such as loss of VWF+ granules (mean granule number 12.6±0.50 vs. PAR-1-AP 7.2±0.35, p< 0.0001) and compression of the marginal band (7.0±0.24 µm² vs. 5.5±0.26 µm², p< 0.0001) (Figure 1). Analysis of platelets from a type 3 VWD patient revealed similar numbers and morphology of SPARC+ granules but no detectable VWF+ granules (Figure 2). Subtle granular changes were observed in a type 2A VWD patient with a C1190R mutation, which leads to defective VWF multimerization. The number of SPARC+ and VWF+ granules was similar to controls, but VWF+ granules were smaller (granule volume = 145.8±3.3 vs. 284.7±4.6). Taken together, this suggests that the C1190R mutation in VWF leads to defective packaging in platelet granules.

Conclusions: We have generated a platform for automated assessment of platelet granules in VWD patients. Our data highlights the potential of such analyses for the diagnosis of hemostatic disorders.

[Figure 1: Representative image (A) and quantification (B,C) of resting vs. PAR-1 AP activated platelets]

[Figure 2: Representative images (A) and quantification (B) of healthy donor and VWD patient VWF+ and SPARC+ granules.]

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/characterizing-platelet-granules-of-von-willebrand-disease-patients-using-super-resolution-structured-illumination-microscopy/