Platelet functional alterations in a Bernard-Soulier syndrome patient with filamin A mutation

Filamin A (FLNA), a dimeric actin crosslinking protein, anchors the platelet adhesion glycoprotein (GP)Ib-IX-V receptor to actin cytoskeleton. The GP Ibα-FLNA interaction is essential for the platelet adhesion to von Willebrand factor that binds the GP Ib-IX-V receptor, for normal signal transduction reactions involved in platelet activation, and for maintaining normal platelet shape and integrity [1]. The absence of or mutations in the GP Ib-IX-V receptor or FLNA gene is responsible for Bernard-Soulier syndrome (BSS) or macrothrombocytopenia [2], respectively. We described here a female BSS case with FLNA mutation, showing her platelet functional defects in the platelet aggregation, secretion, and GP VI signaling pathways.

Patient’s platelet counts ranged between 20 and 30 × 10⁹/L, and enlarged platelets were observed in her blood film. Platelet aggregation on ristocetin was null. Collagen was decreased to induce platelet aggregation (0.5 μg/ml, 17 %; 1 μg/ml, 37 % versus 45 %, 70 % of controls). The GPVI-specific agonist, convulxin, was also decreased (400 PM, 10 %; 800 PM, 40 % versus 55 %, 70 % of controls). Platelets released 33–86 % less ATP than control platelets (Fig. 1a). The GP Ibα expression was practically undetectable on the platelet surface. DNA sequencing analysis revealed the presence of a new GP Ibα c.987G > A mutation in a homozygous state and a FLNA c.1582 G > A mutation in a heterozygous state (Fig. 1b).

To establish that GP Ibα c.987G > A mutation was responsible for the lack of GP Ibα surface expression in the patient’s platelets, we expressed in Chinese hamster ovary (CHO) cells the mutant or normal GP Ibα cDNA together with normal human cDNAs of GP Ibβ and GPIX. Only trace amounts of GP Ibα and GPIX was detected on CHO cells harboring the mutation, but truncated GP Ibα and normal GPIX was present in the CHO cell lysates (Fig. 1c). Together, these data could suggest shorten GP Ibα and GPIX were synthesized, but failed to be anchored and inserted into the plasma membrane.

A question raised by our observation was that patient’s platelet responses to collagen were impaired, despite a normal amount of platelet GPVI and FcRγ chain in her platelets. Several lines of evidence demonstrated that FLNA acted as a signaling scaffold for GPVI through interaction with tyrosine kinase Syk [3]. In our study, the normal level and regular distribution of FLNA was detected in the patient’s platelets (Fig. 1d). However, the phosphorylation of Syk was low (41 % of control) in the platelet signaling pathway of GPVI induced by Cvx (400 pmol/L). Similarly, the phosphorylation of LAT, a direct substrate of Syk, was decreased (45 % of control) (Fig. 1e). FLNA could bind to the tyrosine kinase Syk through its immunoglobulin-like repeat 3–5 in platelets [4]. In our patient, the identified FLNA c.1582G > A, an Ig repeat 3 mutation, interfered with the Ig repeats engaged in signaling of GPVI–collagen interaction. In turn, platelet aggregation and ATP secretion on collagen was interrupted. We thus conclude that abnormal responses to collagen were associated with FLNA c.1582G > A mutation.

To the best of our knowledge, this was the first report of a FLNA mutation causing abnormal response to collagen in a BSS patient. According to our results, we demonstrated that the synergistic effect of both GP Ibα c.987G > A mutation and FLNA c.1582G > A mutation could lead to the platelet functional alteration, including aggregation, secretion, and the GPVI signaling pathway. The relationship between GP Ibα and FLNA should constitute an area of interest for future studies.