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BackgroundPlatelet alpha-granules contain Von Willebrand factor (VWF), which is stored in eccentric alpha-granule nanodomains, and VWF propeptide (VWFpp). Differential release of VWF and VWFpp has been reported from endothelial cells. It is unclear if this also occurs during platelet alpha-granule exocytosis. We have recently developed a 3D super-resolution imaging workflow for quantification of platelet alpha-granule content based on Structured Illumination Microscopy (SIM). With this we can study alpha-granule cargo release following platelet activation in hundreds of platelets simultaneously.AimsTo study release of VWF and VWFpp from alpha-granules using quantitative super-resolution microscopy.MethodsPlatelets were activated with PAR-1 activating peptide (PAR-1 ap) or collagen-related peptide (CRP-XL). Alpha-tubulin, VWF, VWFpp, SPARC and fibrinogen were imaged using 3D-SIM, followed by semi-automated analysis in FIJI. Uptake of anti-VWF nanobody during degranulation was used to identify alpha-granules that partially released content.ResultsVWF+ and VWFpp+ structures overlapped nearly completely (∼90%) in resting platelets, implying they are stored in similar eccentric alpha-granule nanodomains. A subset of VWF+/VWFpp+-structures was released completely at 0.6 µM PAR-1 ap, but at higher concentration (20 µM) significantly more VWFpp (85.3±1.6%) was released than VWF (37.6±1.4%). Release of other cargo was intermediate at 20 µM (SPARC: 62.2±1.4%; fibrinogen: 51.9±2.9%), providing further evidence for differential cargo release. Similar results were obtained using CRP-XL. Anti-VWF nanobody was taken up by VWF+/VWFpp-structures and increased with stimulus strength, demonstrating these were post-exocytotic structures.ConclusionsVWF and VWFpp are differentially released from alpha-granules. This may affect how platelet-derived VWF and VWFpp contribute to formation and stabilization of hemostatic clots.Key pointsVWFpp and VWF are localized in the same, eccentric alpha-granule subdomain in resting platelets and do not overlap with other alpha-granule cargo proteins such as fibrinogenVWFpp and VWF are differentially secreted from individual alpha-granules upon activation with platelet agonists PAR-1 activating peptide and collagen-related peptide

Lowe syndrome (LS) is a rare, X-linked disorder characterised by numerous symptoms affecting the brain, the eyes, and the kidneys. It is caused by mutations in the oculocerebrorenal syndrome of Lowe (OCRL) protein, a 5-phosphatase localised in different cellular compartments that dephosphorylates phosphatidylinositol-4,5-bisphosphate into phosphatidylinositol-4-monophosphate. Some patients with LS also have bleeding disorders, with normal to low platelet (PLT) count and impaired PLT function. However, the mechanism of PLT dysfunction in patients with LS is not completely understood. The main function of PLTs is to activate upon vessel wall injury and stop the bleeding by clot formation. PLT activation is accompanied by a shape change that is a result of massive cytoskeletal rearrangements. Here, we show that OCRL-inhibited human PLTs do not fully spread, form mostly filopodia, and accumulate actin nodules. These nodules co-localise with ARP2/3 subunit p34, vinculin, and sorting nexin 9. Furthermore, OCRL-inhibited PLTs have a retained microtubular coil with high levels of acetylated tubulin. Also, myosin light chain phosphorylation is decreased upon OCRL inhibition, without impaired degranulation or integrin activation. Taken together, these results suggest that OCRL contributes to cytoskeletal rearrangements during PLT activation that could explain mild bleeding problems in patients with LS.

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.

Background: Immune thrombocytopenia (ITP) is an acquired autoimmune disorder against platelets characterized by a low platelet count and increased bleeding risk. ITP is likely to rise from defective immune tolerance in addition to a triggering event, such as vaccination. COVID-19 vaccination is associated with a small increased risk of development of de novo ITP. In patients historically diagnosed with ITP, relapse of thrombocytopenia after COVID-19 vaccination has been described. However, the precise platelet dynamics in previously diagnosed ITP patients after COVID-19 vaccination is unknown Aims: To investigate the effect of the COVID-19 vaccine on platelet count, the occurrence of severe bleeding complications and necessity of rescue medication in patients historically diagnosed with ITP. Methods: Platelet counts of ITP patients and healthy controls were collected immediately before, 1 and 4 weeks after the first and second vaccination. Linear mixed effects modelling was applied to analyse platelet count dynamics over time. Results: We included 218 ITP patients (50.9% women) with a mean (SD) age of 58 (17) years and 200 healthy controls (60.0% women) with a mean (SD) age of 58 (13) years. Healthy controls and ITP patients had similar baseline characteristics (Table 1). 201/218 (92.2%)ITP patients received the mRNA-1273 vaccine, 16/218 (7.3%) the BNT162b vaccine and 1/218 (0.46%) the Vaxzevria vaccine. All healthy controls received the mRNA-1273 vaccine. Fifteen (6.8%) patients needed rescue medication (Table 1). Significantly more ITP patients who needed rescue medication were on ITP treatment prior COVID-19 vaccination compared to patients without exacerbation (56.2% (7/16) vs 27.4% (55/202), p=0.016). We found a significant effect of vaccination on platelet count over time in both ITP patients and healthy controls (Figure 1A). Platelet counts of ITP patients decreased 7.9% between baseline and 4 weeks after second vaccination (p=0.045). Rescue medication and prior treatment significantly increased platelet count over time (p=0.042 and p=0.044). Healthy controls decreased 4.5% in platelet count (p IPT patients with a baseline platelet count of >150x10 9/L had a significant decrease of platelet count 4 weeks after second vaccination compared to baseline (median platelet count (IQR) 205 (94) vs 203 x10 9/L (109) p=0.001). No significant decrease was seen in ITP patients with a baseline platelet count Median (IQR) platelet counts were similar between patients with and without exacerbation, except for 4 weeks after second vaccination (112 (105) vs 45 x 10 9/L (70), p=0.025) (Figure 1B). No significant effect was observed over time in ITP patients with rescue medication (p=0.478) (Figure 1C). In ITP patients without rescue medication, COVID-19 vaccination had a significant effect over time (p=0.001), especially 1 week after second vaccination (Figure1B). Of the 15 patients who needed rescue medication, 8/15 patients (53.3%) received rescue medication within 4 weeks after first vaccination and 4/15 (26.67%) needed rescue medication after the first as well as after the second vaccination. 3/15 (20.0%) patients needed rescue medication after the second vaccination. In the total ITP population, 5/218 (2.2%) experienced a WHO grade 2-4 bleeding complication and 3/218 (1.4%) needed platelet transfusion. 4/5 (80%) bleedings occurred before the second vaccination. One of these patients had fatal varices bleeding, although platelet count was normal. Conclusion: COVID-19 vaccination has a significant effect on platelet count in ITP patients and healthy controls. In 6.8% of ITP patients rescue medication was needed and in 2.2% of ITP patients a WHO grade 2-4 bleeding occurred. The majority of rescue medication was given and the majority bleeding complications occurred in the 4 weeks after the first vaccination. Our results demonstrate that close monitoring of platelet count after COVID-19 vaccination is important in patients historically diagnosed with ITP. Figure 1 Figure 1. Disclosures Westerweel: Pfizer: Consultancy; BMS / Celgene: Consultancy; Incyte: Consultancy; Novartis: Research Funding. Levin: Roche, Janssen, Abbvie: Other: Travel Expenses, Ad-Board. Kruip: Bayer: Honoraria, Research Funding; Daiichi Sankyo: Research Funding. Jansen: Novartis: Consultancy, Other: Travel, Accommodations, Expenses; Advisory Board Novartis: Membership on an entity's Board of Directors or advisory committees; 3SBIO, Novartis: Other: Travel, accomodations, expenses.

Introduction Platelets play a central in hemostasis by facilitating thrombus growth at sites of vascular injury. During activation, platelets secrete a highly diverse cocktail of proteins from their granules, thereby enhancing the hemostatic response. How platelets organize their granules in a resting state as well as during activation and secretion is still poorly understood. We have developed a platform for quantitative analysis of platelet granules employing super-resolution microscopy. Using structured illumination microscopy (SIM), individual alpha- and dense-granule can be separated in platelets. Together with morphological analysis and other quantitative imaging data that can be processed in automatic analysis workflows using ImageJ software, this method yields novel insights in granule organization and has implications for both platelet biology and translational studies on patients with platelet defects. Methods Platelet were isolated from citrated whole blood from consenting healthy donors and select patients. Samples were immediately fixed in 2% paraformaldehyde, washed, and loaded on poly-L-lysine coated high-resolution coverslips. We performed indirect immunostainings for various antigens, including alpha-tubulin, GP1b, Von Willebrand Factor (VWF) and platelet factor 4. Samples were imaged on an Elyra PS1 (Zeiss) with a SIM module using five phases and five rotations. After image acquisition, samples were reconstructed using Zen Software. Reconstructed images were analyzed by ImageJ plugins combined with in house written macro's which allowed for assessment of platelet morphology (volume, surface and shape), granule morphology (counts, volume, surface, shape) and granule metrics (smallest distance between granules and closest distance to membrane). We analyzed platelets from five healthy donors for general variation as well as from selected patients with platelet defects, which allowed us to validate our analytic workflow. Statistics was performed using GraphPad Prism 8. Results We have first assessed the variation in granule content in healthy donors. Segmentation of single resting platelets was done on cells stained for GP1b or alpha-tubulin, while alpha granule content of platelets was assessed by staining for VWF. These analyses revealed that the granule number (defined as unique VWF-positive spots of fluorescence using an optimized threshold) was normally distributed around the average of 16.78 per platelet. While there is considerable variation in alpha granule number (SD = 7.641), the sensitivity of our analysis is retained by analyzing hundreds of platelets per donor (SEM = 0.6923). We then tested whether our analysis could pick up subtle differences in platelet granules. For this purpose, we analyzed platelets from a type 2A Von Willebrand's Disease (VWD) patient with a C1190R mutation, leading to defective multimerization of VWF. Morphological analysis of SIM images revealed that the number of VWF-positive alpha granules was similar when compared to controls (16.7±0.7; p>0.05). Alpha granules in this patient were smaller (granule volume = 42.9±0.9 vs. controls 207.7±9.2; p0.05), nor granule volume (p=0.78). Altogether, this suggests that there is less VWF located in the alpha-granules of the VWD patient which we confirmed by Western blot and confocal microscopy. Our results imply that the packaging of VWF into alpha granules is impaired in VWD patients carrying the C1190R mutation. Discussion We have developed an automated image analysis workflow that allows for evaluation of platelets and their granules in patients with VWD. Our findings show the utility of unbiased, high throughput analyses of platelets and platelet granules for the diagnosis of hemostatic disorders. Disclosures Leebeek: Shire/Takeda: Research Funding; Novo Nordisk: Consultancy; UniQure: Consultancy; CSL Behring: Research Funding; Sobi: Other: Travel grant; Shire/Takeda: Consultancy. Jansen:Novartis: Other: Travel funding; 3SBio: Other: Speaker fee; Celgene: Other: Travel funding.