Study: Functional study

Model: HEK293 cell lines

Variant: VWF NM_000552.5: c.7664C>G, p.(Pro2555Arg), described as GOF mutation

rare missense variant, MAF = 0.00007

RefSNP_ID:rs915754316

Not reported in ClinVar

Cells lines were transfected and expressed one of the following: wtVWF, p.Pro2555Arg, both variants, or a negative control variant p.Asp2509Glu

Main result:

p.Pro2555Arg is the first VWF GOF variant that increases platelet aggregate size (AS), which is shear-dependent and independent of fibrinogen (fb). May provide a novel risk factor for thromboembolic disease.

Additional results:

Variant does not affect the binding affinity of the C4 domain for BPIIb/IIIa

VWF stem region mutations can enhance VWF's prothrombotic properties

C4 domain of VWF may be a novel antithrombotic drug target.

functional assays

Chemiluminescence assays (measuring binding capacity)

VWF-collagen binding assay (CBA)

Electrophoresis (Western blot)

Bidirectional direct sequencing of PCR products

Paternity test

PCR and restriction assays to detect SNVs

in vitro expression of recombinant WT and p.P1127S VWF variants in HEK293 cells

Platelet aggregation studies

DDAVP test

Binding assays

Proteolysis assays

in silico modeling

Patient phenotypes listed: mild bleeding history normal platelet number increased mean platelet volume increased RIPA (les than typical PT-VWD) enhanced binding of VWF to platelets (assessed by flow cytometry)

Has functional and HXMS data to support classification to pathogenic

Disease: platelet-type von Willebrand disease (PT-VWD)

Patient: 14 yo, Male

Variant: GP1BA NM_000173.7:c.389G>A p.(Arg127Gln), Heterozygous, Gain-of-Function (GOF)

Located in LRR5 domain of GP1BA

Family: Mother did not refer any bleeding symptoms (variant absent in mother) Father not available for collection of clinical history or platelet function testing

Pavord, S., Hunt, B. J., Horner, D., Bewley, S., & Karpusheff, J. (2021). Vaccine induced immune thrombocytopenia and thrombosis: Summary of NICE guidance. BMJ, 375, n2195. https://doi.org/10.1136/bmj.n2195

Greinacher, A., Thiele, T., Warkentin, T. E., Weisser, K., Kyrle, P. A., & Eichinger, S. (2021). Thrombotic Thrombocytopenia after ChAdOx1 nCov-19 Vaccination. New England Journal of Medicine, 384(22), 2092–2101. https://doi.org/10.1056/NEJMoa2104840

the Guardian. ‘How UK Doctor Linked Rare Blood-Clotting to AstraZeneca Covid Jab’, 13 April 2021. http://www.theguardian.com/society/2021/apr/13/how-uk-doctor-marie-scully-blood-clotting-link-astrazeneca-covid-jab-university-college-london-hospital.