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Membrane procoagulation and N‑terminomics/TAILS profiling in Montreal platelet syndrome kindred with VWF p.V1316M
Ejaife O Agbani1,2, Daniel Young3, Si An Chen3
1Department of Physiology & Pharmacology, Cumming School of Medicine, University of Calgary, Calgary, AB, Canada. ejaife.agbani@ucalgary.ca.
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.
Insights
Montreal Platelet Syndrome (MPS) patients with the VWF p.V1316M mutation exhibit platelet dysfunction due to chloride channel-1 (CLIC1) loss. This impairs platelet membrane dynamics and contributes to bleeding diathesis.
Area of Science:
- Hematology
- Molecular Biology
- Platelet Physiology
Background:
- Montreal Platelet Syndrome (MPS) with VWF p.V1316M mutation (2B-VWDMPS) is a rare bleeding disorder.
- Patients experience macrothrombocytopenia, platelet clumping, and mucocutaneous bleeding, often requiring VWF concentrate or platelet transfusions.
- Previous research linked platelet procoagulant membrane dynamics (PMD) to Na+ and Cl- influx, followed by water entry.
Purpose of the Study:
- To investigate a potential uncharacterized platelet dysfunction contributing to bleeding diathesis in 2B-VWDMPS patients.
- To analyze platelet procoagulant membrane dynamics (PMD) in MPS patients.
- To compare proteolysis changes in healthy versus 2B-VWDMPS platelets.
Main Methods:
- Quantitative platelet shotgun proteomics and N-terminomics/TAILS were employed.
- Biochemical assays were used to validate findings.
- Systematic analysis of platelet procoagulant membrane dynamics (PMD) was performed.
Main Results:
- 2B-VWDMPS platelets showed a loss of transmembrane chloride channel-1 (CLIC1) and reduced chloride ion influx post-collagen stimulation.
- Diminished membrane ballooning, phosphatidylserine externalization, and thrombin formation were observed.
- Platelets exhibited basal activation, partial degranulation, and loss of regulatory, cytoskeletal, and contractile proteins.
Conclusions:
- Loss of CLIC1 and impaired chloride influx contribute to platelet dysfunction in 2B-VWDMPS.
- These molecular defects may explain the structural disorganization, giant platelet formation, and weakened hemostatic response.
- Further understanding of platelet membrane dynamics is crucial for managing bleeding disorders.
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