Membrane procoagulation and N‑terminomics/TAILS profiling in Montreal platelet syndrome kindred with VWF p.V1316M mutation

BACKGROUND: The Montreal platelet syndrome kindred (MPS) with VWF p.V1316M mutation (2B-VWDMPS) is an extremely rare disorder. It has been associated with macrothrombocytopenia, spontaneous platelet clumping, mucocutaneous, and other bleeding, which can be largely prevented by von Willebrand factor (VWF) concentrate infusion. However, supplemental platelet transfusion has been required on occasion, particularly for severe gastrointestinal bleeds. This raised the question of whether a previously uncharacterized platelet dysfunction contributes to bleeding diathesis in 2B-VWDMPS patients. We have previously shown that membrane ballooning, a principal part of the platelet procoagulant membrane dynamics (PMD) after collagen stimulation, is driven by the influx of Na(+) and Cl(-), followed by the entry of water. METHODS: We study two members (mother and daughter) of the MPS kindred with severe bleeding phenotype and address this question by coupling quantitative platelet shotgun proteomics and validating biochemical assays, with the systematic analysis of platelet procoagulant membrane dynamics (PMD). Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compare changes in proteolysis between healthy and 2B-VWDMPS platelets. RESULTS: Here, we report in 2B-VWDMPS platelets, the loss of the transmembrane chloride channel-1 (CLIC1), and reduced chloride ion influx after collagen stimulation. This was associated with diminished membrane ballooning, phosphatidylserine externalization, and membrane thrombin formation, as well as a distinct phenotypic composition of platelets over fibrillar collagen. We also identify processing differences of VWF, fibronectin (FN1), and Crk-like protein (CRKL). 2B-VWDMPS platelets are shown to be basally activated, partially degranulated, and have marked loss of regulatory, cytoskeletal, and contractile proteins. CONCLUSIONS: This may account for structural disorganization, giant platelet formation, and a weakened hemostatic response.