Second Report of A GPIbα LRR7 Variant (P.Leu194Phe) Supporting the Pathogenicity of Mutations Outside The C-Terminal Domain In Platelet-Type Von Willebrand Disease

Platelet-type von Willebrand disease (PT-VWD) is a rare autosomal dominant platelet bleeding disorder, with fewer than 60 patients reported worldwide so far.[1] While type 2B von Willebrand disease (type 2B VWD) results from a functionally abnormal von Willebrand factor (VWF) molecule arising from mutations in the VWF gene, PT-VWD is caused by hyperresponsive platelets resulting from mutations in the platelet GP1BA gene (GPIα).[2] [3] PT-VWD may be misdiagnosed in approximately 15% of those with type 2B VWD.[4]

GPIbα is the major ligand-binding subunit of the GPIb-IX-V complex that binds VWF.[5] The N-terminal domain of GPIbα consists of eight leucine-rich repeats (LRR) that forms a concave shape.[6] The interaction between VWF A1 domain and GPIbα occurs in the presence of high shear stress.[7]

Affected patients with PT-VWD suffer from mild to moderate mucocutaneous bleeding, low VWF activity compared with antigen, decreased high-molecular-weight (HMW) VWF multimers, variable degree of thrombocytopenia and typically platelet agglutination in response to low concentrations of ristocetin.[8]

To date, only eight variants causing PT-VWD have been described, five in the C-terminal disulfide loop of the VWF-binding domain of GPIbα, two in the LRR domain, and one deletion in the macroglycopeptide.[9] [10] The C-terminal disulfide loop, known as the β-switch, is the region crucial for the binding to the A1 domain of VWF, and it harbors five of the eight gain-of-function (GoF) variants of GPIbα causing PT-VWD described so far.[9]

Recently, Bury et al and Monteiro et al reported the case of two patients with moderate bleeding symptoms, increased ristocetin induced platelet agglutination (RIPA) and GoF GP1BA variants in the LRR domain (c.G380A/p.Arg127Gln and c.580C > T/p. Leu194Phe), outside the C-terminal loop, showing that a single substitution in this LRR structure may also cause a conformational change in the GPIbα binding site, directly affecting the VWF ligation.[9] [10]

Bury et al identified the GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD.[9] Hydrogen-deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure.

The (p.Leu194Phe) variant affecting the LRR7 domain of GPIbα with moderate bleeding phenotype described by Monteiro et al was in a 17-year-old boy adopted patient; thus, there were no other available family members to assess, and segregation studies were not performed.[10] Moreover, study of HMW VWF multimers or functional studies confirming the potential impact of this variant on the structure and/or function of GPIbα were not realized, leaving some uncertainty regarding the pathogenicity of this variant.

Supporting these findings, we herein describe another family with moderate bleeding symptoms, normal platelet counts, and increased RIPA harboring as yet reported p.Leu194Phe GP1BA GoF variant in LRR7.

A 1-year-old boy was referred to us for a possible finding of VWD before a cranioplasty for trigonocephaly. There was no family history of bleeding disorders in his parents, uncles and aunts, first cousin, or grandparents. He was born by cesarean section with vacuum extraction; he did not develop hematomas after intramuscular vaccinations or blood sampling. He has not presented with ecchymosis so far, nor prolonged bleeding from scratches. He has no history of gingival bleeding, no erupted teeth, and no bleeding at umbilical cord detachment. Standard laboratory evaluation showed that platelet counts ranged from 194 × 10⁹/L to 415 × 10⁹/L with normal Mean Platelet Volume (MPV; 8.9 fL). Closure times (platelet function assay–PFA-200) performed using collagen/adenosine diphosphate (153 second) and collagen/epinephrine cartridges (182 second) were moderately increased while FVIII:C (43%), VWF:Ag (immunologic quantification by HemosIL AcuStar VWF Antigen from Werfen; 55%) and VWF:GPIbR (functional activity of VWF measured by ristocetin induced binding to recombinant GPIb fragment; HemosIL VWF Ristocetin Cofactor Activity reagent from Werfen; 36%) were moderately decreased ([Table 1]). His blood group was A⁺. Platelet glycoprotein levels, assessed by flow cytometry, were normal (35,847 sites/plt), whereas RIPA was increased (70% of normal) at a low ristocetin concentration (0.625 mg/mL). VWF multimer pattern was also analyzed by SDS-agarose electrophoresis, showing a normal result ([Fig. 1]).

Table 1 ISTH-BAT bleeding scores and laboratory results of the different reported mild forms of PT-VWD

Bleeding scores and laboratory parameters	Normal values	Our patient	Mother	Patient from Monteiro et al[10]	Patient from Bury et al[9]
GP1BA variant	–	c.580C > T/p. Leu194Phe	c.580C > T/p. Leu194Phe	c.580C > T/p. Leu194Phe	c.G380A/p.Arg127Gln
GPIbα domain	–	LRR7	LRR7	LRR7	LRR5
Sex, age (y)	–	Male, 1 y	Female, 39 y	Male, 17 y	Male, 14 y
Bleeding score (International Society of Thrombosis and Haemostasis-Bleeding Assessment Tool)	<3 in children; <4 in adult males; <6 in adult females	0 Normal	0 Normal	3 Sub-normal	4 Sub-normal
Platelet count (x109/L)	150–400	194–415 Normal	281 Normal	127–161 Sub-normal	208 Normal
MPV (fL)	<10	8.9 Normal	8.9 Normal	14.2 Sub-normal	12 Sub-normal
VWF:Ag (%)	50–150	55 Normal	200 Sub-normal	105 Normal	Value not reported, but normal
VWF:GP1bR (%)	50–150	36 Sub-normal	155 Sub-normal	66 Normal	Value not reported, but normal
VWF:GP1bR/VWF:Ag ratio	≥0.7	0.65 Sub-normal	0.77 Normal	0.62 Sub-normal	Not reported
PFA-CEPI (s)	<150	182 Sub-normal	128 Normal	131 Normal	Value not reported, but normal
PFA-CADP (s)	<100	153 Sub-normal	Not performed	96 Normal	Value not reported, but normal
Ristocetin-induced platelet agglutination	Normal	Abnormal	Abnormal	Abnormal	Abnormal
VWF multimers	Normal	Sub-normal	Not performed	Not reported	Not reported
Platelet GPIb levels (sites/platelet)	22,450–42,490	35,847 Normal	39,407 Normal	58[a] Sub-normal	Value not reported, but normal

^a Normal Reference Range: 70-130 (% Normal).

Fig. 1 Western blot analysis of von Willebrand factor showing a normal profile of high-molecular-weight multimers in the patient (P) relative to a control subject (C).

For genetic analysis, genomic deoxyribonucleic acid was extracted from the peripheral blood of the patient following informed consent. The entire coding and flanking intronic regions of VWF exon 28 were amplified by PCR. Results obtained by Sanger sequencing revealed no pathogenic variant, except the previously described D1472H polymorphism interfering with VWF:RCo measurement, but having less effect on VWF:GPIbR testing,[11] as it is the case here with almost normal VWF:GPIbR/VWF:Ag ratio (ratio = 0.65).

Next Generation Sequencing of different human genes implicated in inherited platelet disorders revealed, in the GP1BA gene, a single missense substitution at nucleotide 580, corresponding to the change of a Leucine to Phenylalanine at residue 194 (c.580C > T; p.Leu194Phe), which was previously reported by Monteiro et al.[10] No other relevant variants were detected. Segregation study was performed on both parents, showing that this variant was only inherited from his mother.

Mother's standard laboratory evaluation showed that platelet count (281 × 10⁹/L) and MPV (8.9 fL) were normal. The PFA closure time performed using collagen/epinephrine cartridges was also normal (128 seconds, as FVIII:C (145%), VWF:Ag (200%), and VWF:GPIbR (155%). Her blood group was A⁺. Platelet glycoprotein levels were normal (39,407 sites/plt), whereas RIPA ([Fig. 2]) was increased (85% of normal) at a lower ristocetin concentration (0.5 mg/mL) than her son. This might be due to the presence of the interfering D1472H variant; however, this could not be confirmed as VWF genotyping was not performed in the mother.

Fig. 2 Ristocetin-induced platelet agglutination (RIPA) assays. RIPA traces from the patient's platelets and his parents, with different concentrations of ristocetin. All tests were performed using ristocetin from Stago and an APACT-4004 aggregometer (Labitec), as previously described in detail.[12] Platelet agglutination is expressed as a percentage.

As previously reported by Monteiro et al, according to sequence alignment analysis, although the Leu194 residue might not be highly conserved, the amino acid substitution can cause structural adjustments affecting the ligand domain function. Leu194 forms stabilizing hydrogen bonds within LRR7, and substitution with phenylalanine may disrupt local structure and protein dynamics, potentially leading to a more open conformation with increased binding affinity.[10] This LRR variant, together with the one reported by Bury et al, expands the typical PT-VWD phenotype by exhibiting a mild clinical presentation with a common feature, the increased RIPA at a low ristocetin concentration.[9]

In addition to the increased RIPA, patients showed normal or sub-normal bleeding score, platelet count, MPV, GPIbα expression, as well as normal or sub-normal VWF levels ([Table 1]). Phenotypes and variants' localization sustain the hypothesis that GoF variants in this region can be associated with a mild form of PT-VWD, and this second report also confirms what Monteiro et al had previously observed.[10] As these variants in LRR are immediately adjacent to the C-terminal region, this proximity may translate into an intermediate phenotype compared with the most severe ones previously associated with C-terminal variants.

These new family cases harboring the c.580C > T/p. Leu194Phe expands the spectrum of GoF GP1BA variants outside the C-terminal loop and adds to the documented number of PT-VWD reports. Although further evidence is needed to establish robust genotype–phenotype correlations, distinguishing between PT-VWD and type 2B VWD remains critical for clinical management, as therapeutic strategies differ: patients with type 2B VWD may benefit from VWF replacement, whereas those with PT-VWD require platelet transfusion.[2]

Data Availability Statement

Data are available upon request.

Contributors' Statement

All authors were responsible for data collection and creating the article.

Ethical Approval

Blood samples from patients were collected and obtained in accordance with the Declaration of Helsinki.

Informed Consent

All participants provided written informed consent for blood sampling.

Publication History

Received: 21 November 2025

Accepted: 28 January 2026

Article published online:
24 February 2026

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