Alterations of the platelet proteome in type I Glanzmann thrombasthenia caused by different homozygous delG frameshift mutations in ITGA2B

Abstract

Glanzmann thrombasthenia (GT) is one of the best characterised inherited platelet function disorders but global platelet proteome has not been determined in these patients. We investigated the proteome and function of platelets from two patients with type I GT, caused by different homozygous ITGA2b mutations, from family members and unrelated controls. The global proteome of highly purified washed platelets was quantified by liquid chromatography-mass spectrometry (LC-MS) and targeted MS-methods. Platelet function was analysed by flow cytometry, light transmission aggregometry and flow-based assays. Platelets from GT patients showed less than 5 % relative levels of the integrin subunit α and 5-9 % fibrinogen compared to controls. These patients demonstrated loss of αβ-dependent platelet function, but normal platelet granule secretion induced by physiological agonists. Platelets from heterozygous family members of a patient expressed 50-60 % of control α levels which were sufficient for normal αβ-dependent platelet function. Studying type I GT as model disease we established quantitative LC-MS to detect and clearly distinguish normal platelets, platelets from GT heterozygotes and platelets from GT patients. Diminished levels of factor XIIIB chain, plasminogen and carboxypeptidase 2B were identified in thrombasthenic platelets. Additionally, GT platelets showed up to 2.5-fold increased levels of FcγRIIA and laminin-α4 chain. Elevated levels of platelet FcγRIIA was associated with increased CD63-surface expression after FcγRIIA-crosslinking in one GT-patient which might present a compensatory mechanism of platelet activation in GT. We demonstrate that quantitative LC-MS based proteomics is suitable to validate known but also to identify previously unknown protein level changes of dysfunctional platelets.

Mass Cytometry Reveals Distinct Platelet Subtypes in Healthy Subjects and Novel Alterations in Surface Glycoproteins in Glanzmann Thrombasthenia

“…In agreement with previous findings 7 , 8 , 50 , both MC and FFC demonstrated CD41, CD61 and activated αIIbβ3 expression to be significantly reduced on GT platelets compared to control platelets under both non-stimulating and stimulating conditions. MC enabled us to survey an array of additional surface antigens and, in agreement with previous reports 51 – 55 , we found CD29, CD36, CD62P, and CD107a membrane expression to be similar on GT and control platelets following agonist stimulation.…”

“…Similar to CD9, CD63 expression may be limited by membrane protein crowding, and in the absence of αIIbβ3 there would be less crowding. Interestingly, studies have shown that some GT patient platelets show increased surface expression of CD63, but not CD107a or CD62P following FcγRIIA crosslinking 55 . The investigators of these studies hypothesized that increased dense granule exocytosis was responsible for the increased surface expression of CD63 55 .…”

cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets

“…Many signaling platelet pathways are affected by integrin out-side-in signaling, especially by the integrin α IIb β 3 . To address the possible role of the integrin α IIb β 3 for GPIbα-induced Syk activation we studied washed platelets from a patient with Glanzmann thrombasthenia (GT), which showed severe reduction of the major fibrinogen receptor α IIb ß 3 and which has been studied previously [51]. As expected, EB did not induce any aggregation response of platelets from a patient with GT, whereas platelets from a healthy control showed clear aggregation in response to EB, which was completely inhibited by the cAMP-elevating agent iloprost (Fig.…”

Detecting post-translational modification signatures as potential biomarkers in clinical mass spectrometry

“…Sometimes sample size is inherently limited, for instance in case of rare disorders with low prevalence, rendering the estimation of the variance uncertain. Nevertheless, such rare disorders may still be analyzed by proteomics [223]. For small sample sizes, the t-test may consider that a potential marker showing strong regulation but (concurrently) high intra-group variance is not significant [224] (Figure 6).…”