Quantitative 3D microscopy highlights altered von Willebrand factor α‐granule storage in patients with von Willebrand disease with distinct pathogenic mechanisms

Abstract

Background Platelets play a key role in hemostasis through plug formation and secretion of their granule contents at sites of endothelial injury. Defects in von Willebrand factor (VWF), a platelet α‐granule protein, are implicated in von Willebrand disease (VWD), and may lead to defective platelet adhesion and/or aggregation. Studying VWF quantity and subcellular localization may help us better understand the pathophysiology of VWD. Objective Quantitative analysis of the platelet α‐granule compartment and VWF storage in healthy individuals and VWD patients. Patients/Methods Structured illumination microscopy (SIM) was used to study VWF content and organization in platelets of healthy individuals and patients with VWD in combination with established techniques. Results SIM capably quantified clear morphological and granular changes in platelets stimulated with proteinase‐activated receptor 1 (PAR‐1) activating peptide and revealed a large intra‐ and interdonor variability in VWF‐positive object numbers within healthy resting platelets, similar to variation in secreted protein acidic and rich in cysteine (SPARC). We subsequently characterized VWD platelets to identify changes in the α‐granule compartment of patients with different VWF defects, and were able to stratify two patients with type 3 VWD rising from different pathological mechanisms. We further analyzed VWF storage in α‐granules of a patient with homozygous p .C1190R using electron microscopy and found discrepant VWF levels and different degrees of multimerization in platelets of patients with heterozygous p .C1190 in comparison to VWF in plasma. Conclusions Our findings highlight the utility of quantitative imaging approaches in assessing platelet granule content, which may help to better understand VWF storage in α‐granules and to gain new insights in the etiology of VWD.

Quantitative super-resolution imaging of platelet degranulation reveals differential release of VWF and VWF propeptide from alpha-granules

“…VWF is found in platelet alpha-granules (Figure 1A) and in keeping with several ultrastructural studies [18][19][20]29] we observed discrete volumes of VWF in eccentrically located subdomains within the encapsulation of a P-selectin positive alpha-granule membrane (Supplemental Figure 1). As observed previously [20,30,31],…”

“…A number of studies have focused on the role of platelet derived VWF in hemostasis [53][54][55][56][57]. VWD patients with mild and severe circulating VWF deficiencies who still have residual platelet VWF show a milder clinical phenotype [20,58]. Platelet VWF has also been reported to be important for DDAVPrelated amelioration of bleeding times in subgroups of type 1 VWD patients [59].…”

“…Next, we investigated whether the differential release of VWF and VWFpp was dependent on agonist strength by using a semi-automated quantitative workflow on 3D-SIM images [20] of platelets activated with a broad concentration range of PAR-1 and CRP-XL. Using additional agonist concentrations that partially or fully trigger alpha-granule release (Supplemental Figure 3), we found that differential release of VWF and VWFpp was apparent at all stimulus concentrations of (Figure 4A), similar to CRP-XL (Supplemental Figure 5).…”

“…Whole blood is drawn from consenting healthy donors in citrate tubes. Washed platelets were prepared as described previously [20]. In brief, platelet-rich plasma (PRP) is generated by centrifugation at 120 x g for 20 minutes with low acceleration (max.…”

“…In platelets, VWF is zonally packaged within eccentric alpha-granule nanodomains which also contain short VWF tubules [18][19][20]. Platelets also contain VWFpp [21] and are able to secrete it following stimulation with various agonists that induce alphagranule release [22].…”