Compromised PAR1 Activation—A Cause for Bleeding in XMEN?

XMEN (X-linked immunodeficiency with magnesium defect, Epstein–Barr virus infection, and N-linked glycosylation [NLG] defect) is a rare monogenetic disease due to MAGT1 pathogenic variants, which is potentially curable by allogeneic blood or bone marrow transplantation (BMT).[1] Affected patients may experience increased bleeding tendency and fatal hemorrhage after BMT has been described.[2] [3] However, bleeding appears disproportionate to the degree of thrombocytopenia, suggesting a qualitative platelet defect.[2] [4] [5] This is the first case report to describe platelet dysfunction in XMEN (three related males with the same novel hemizygous loss-of-function variant in MAGT1 and one unrelated male with a different hemizygous loss-of-function variant in MAGT1) identifying the same qualitative platelet defect which may explain the increased bleeding tendency in these patients.

The index patient's, P1, age was 29 years. His bleeding history, with platelets in the 300,000/μL range, included: frequent nosebleeds as a child and a severe perioperative hemorrhage at age 19 after the removal of a nasopalatine ductal cyst, which required four blood transfusions. Three years later, posttraumatic reconstructive surgery was complicated by postoperative bleeding. No family history of bleeding disorders was reported. After removal of a large dermal naevus in the shoulder region, the patient experienced delayed bleeding and was treated with tranexamic acid. The patient had normal coagulation (see the [Supplementary Material] [available in the online version]) and hematology parameters and was negative for von Willebrand disease.

Trio-based whole-exome sequencing identified the patient with hemizygosity for a novel disease-causing frameshift variant in MAGT1 introducing a premature stop-codon (NM_032121.5: c.334del p.(Ala112Leufs*52), hg38/GRCh38 position: NC_000023.11:g.77875462del). The variant has not been observed among the over 125,000 individuals in the gnomAD database (v2.1.1, accessed: November 10, 2022).

P1 had two male relatives (brothers and maternal cousins to P1), identified as “P2,” age 28, and “P3,” age 21 ([Fig. 1A]). Both P2 and P3 had normal hematological parameters (data not shown) and no previous surgical history or severe hemorrhagic history. They were subsequently tested for the MAGT1 c.334del variant by Sanger sequencing identifying hemizygosity for the variant in both ([Fig. 1B]).

Fig. 1 Family pedigree (A), hemizygosity for NM_032121.5 (MAGT1): c.334del, identified in trio-based WES analysis (P1) and subsequently by Sanger sequencing (5′ to 3′ direction) in P2 and P3. Nucleotide range shown, NC_000023.11: g.77875457_77875467 (B), hemizygosity for NM_032121.5:c.712C > T, (NP_115497.4:p.Arg238Ter, hg38/GRCh38 position: NC_000023.11:g. 77112286G > A), identified in trio-based WES analysis (P4) (C), Platelet activation-dependent expression of the active conformation of GPIIb/IIa (D), antigen CD62p (E), and CD63 (F) induced by PAR1 agonist, PAR4 agonist, CRP, ADP, and ristocetin. Platelet aggregation of P1's (closed circles) isolated platelets measured by flow cytometry as compared with that of healthy individuals (open circles) (G) and in whole blood by Multiplate (H). ADP, adenosine diphosphate; CRP, collagen-related peptide.

Patient 4 is an unrelated male, age 13. Trio-based whole-genome sequencing identified a hemizygous, nonsense variant in MAGT1 introducing a premature stop-codon (NM_032121.5:c.712C > T, NP_115497.4:p.Arg238Ter, hg38/GRCh38 position: NC_000023.11:g. 77856789G > A). The variant was found to be de novo in the patient ([Fig. 1C]) and has previously been described in two families in male patients with XMEN disease.[2] [6] Besides thrombocytopenia, P4 had normal hematological parameters (data not shown) and no previous surgical history or severe hemorrhagic history besides intermittent minor self-limiting nosebleeds.

Platelet function was initially evaluated in P1, and certain parameters were selected for subsequent evaluation in P2–P4. A full description of methods is available in the [Supplementary Material] (available in the online version). In brief, we evaluated hematological parameters; platelet surface glycoprotein expression (CD41, CD42b, CD49b, CD61, and CD42a) and activation (percentage of platelets positive for CD62P as a measure of α granule release, CD63 reflecting dense granule release, and procaspase activating compound-1 [PAC-1] binding [detecting the active conformation of GPIIb/IIa]) was determined in whole blood using flow cytometry. Further, platelet aggregation was determined in whole blood (Multiplate, Roche Diagnostics, Mannheim, Germany) and with isolated platelet by flow cytometry, previously explained in detail.[7] The platelet surface glycoprotein expression was within the normal range for all patients (P1–P4; data not shown). Failure of the activation of platelets from P1 by protease-activated receptor (PAR)1 agonist peptide prompted the measurement of PAR1 receptor expression as cytosolic free calcium [Ca²⁺] _i function is an important second-messengers in platelets, flow cytometric measurement of platelet [Ca²⁺] _i was used to further evaluate PAR1 and PAR4 platelet activation and reactivity in the index patient P1. As MAGT1 deficiency abolishes steady-state expression of the immune response protein NKG2D (natural killer group 2 D),[8] NKG2D expression on NK cells was measured by flow cytometry.

Mass spectrometry-based proteomic analysis of isolated platelets showed a total lack of MAGT1 protein in P1, P2, and P3 (P4 not investigated) ([Fig. 2F]).

Fig. 2 Flow cytometry analysis of surface expression (MFI) of NKG2D in CD3⁺CD16⁺ NK cells from five healthy controls and the four patients (A). Flow cytometry analysis of surface expression (MFI) of PAR1 in platelets from five healthy controls, P1, and P4 (B). Intracellular Ca2⁺ mobilization in platelets presented as mean of three repetitive measurements (black lines: healthy control; green lines: P1) induced by PAR1 (C) and PAR4 (D). Serum levels of carbohydrate-deficient transferrin (CDT) (%) in P1–P3 (gray area indicates normal values <2%) (E). Reporter ion signals for the peptide GFSAEQIAR measured at m/z 604.33493 (+2) from MAGT1 in the three healthy controls (quan channels 126, 127N, and 128N) and P1–P3 (quan channels 129N (P3), 130N (P1), and 131N (P2)) (F). Signals in latter quan channels are most probably caused by the interference of reporter ion signals from peptides not derived from MAGT1. MFI, mean fluorescence intensity.

Moreover, all patients showed normal collagen-related peptide-, adenosine diphosphate-, and ristocetin-induced platelet activation ([Fig. 1D–F]). Human platelets express two PARs: PAR1 and PAR4. Remarkably, while cleavage-independent PAR activation and aggregation by PAR4 agonist peptide was normal in all three patients, each of them showed a total lack of platelet activation by PAR1 agonist peptide ([Fig. 1D–F]), despite near normal PAR1 expression (only investigated for the index patients P1 and P4; [Fig. 2B]). This discrepancy was further shown by a total lack of [Ca²⁺] _i release in P1's platelets upon cleavage-independent PAR1 activation and normal PAR4-induced platelet [Ca²⁺] _i release ([Fig. 2C, D]). The platelet aggregation response in P1, 2, and 3 was normal in whole blood and in isolated platelets for all agonist, except for decreased platelet aggregation in response to PAR1 agonist peptide in both platelet aggregation tests ([Fig. 1G, H]). While the platelet aggregation response to a PAR1 agonist in P4 was highly decreased, the aggregation response to other agonists was decreased to a lesser degree. However, the platelet count of 80 × 10⁹/L is below the recommended platelet concentration of 150 × 10⁹/L using Multiplate.[9] PARs belong to the seven transmembrane (TM) domain G protein-coupled receptor family and are unique in their lack of soluble ligands. In contrast to classical receptors, PARs are activated by N-terminal proteolytic cleavage. Upon removal of specific N-terminal peptides, the resulting N-termini serve as tethered activation ligands that interact with a conserved region on the extracellular loop 2 (ECL2) domain. This interaction initiates conformational changes and alters affinity for intracellular G proteins initiating receptor signaling.[10] [11] [12] The used PAR-1 and 4 agonists are synthetic peptides that mimic the first six amino acids of tethered N-terminal ligands and act as agonist peptides that activate PAR-1 and 4, respectively, in the absence of cleavage events.

Interestingly, ECL2 of PAR1, and not PAR4, contains two conserved N-glycosylation sites and it has been shown that NLG of PAR1 at the surface of ECL2 is important for ligand docking interactions that enhance the stabilization of the activated receptor complex and activation of G protein signaling.[13]

MAGT1 is a noncatalytic subunit of the oligosaccharyltransferase (OST) complex and facilitates asparagine (N)-linked glycosylation of specific substrates, making XMEN a congenital disorder of glycosylation (CDG).[14]

MAGT1 forms, in combination with the catalytic subunit STT3B, the OST complex responsible for endoplasmic reticulum-mediated N-glycosylation of sites close to the C-terminus or in short membrane loops. Recent glycoproteomic analysis of T lymphocytes from 23 XMEN patients showed that affected N-glycosylation sites were near TM domains and included the short loops between two TM regions.[15]

Although no glycoproteomic analysis has been conducted in the present study, the analysis of serum transferrin glycosylation status (carbohydrate-deficient transferrin [CDT]) has historically been used as a screening and classification tool for CDGs that affect NLG. All three investigated patients (P1–P3) had markedly elevated serum levels of CDT ([Fig. 2E]). In conjunction with the total lack of complete MAGT1 protein and NKG2D expression ([Fig. 2A]), which is related to hypoglycosylation in MAGT1-deficient patients,[8] this strongly indicates affected NLG in all four patients.

As NLG appears to play a key role in PAR1 receptor function, and abnormalities in NLG can be found in XMEN patients, this is one possible mechanism, which could explain our findings that these patients have defective platelet activation, and that it is specifically their response to PAR1 signaling which is dysfunctional. This is the primary thrombin receptor on human platelets, and therefore we hypothesize that the observed abnormality in PAR1 signaling could be sufficient to cause the subsequent increased bleeding tendency observed in affected individuals.

This is the first case report to investigate platelet dysfunction in XMEN patients and to show a qualitative platelet defect, which may explain the increased risk of hemorrhage in these patients. The finding in unrelated XMEN patients may suggest that defective PAR-induced platelet activation is a characteristic of MAGT1 deficiency.

Publication History

Received: 21 November 2022

Accepted: 25 January 2023

Accepted Manuscript online:
31 January 2023

Article published online:
09 March 2023

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