Increased RhoA pathway activation downstream of αIIbβ3/SRC contributes to heterozygous Bernard Soulier syndrome

Abstract

Bernard Soulier syndrome (BSS) is a severe bleeding disorder with moderate to severe thrombocytopenia, giant platelets, and platelet dysfunction, caused by biallelic mutations in GP1BA, GP1BB, or GP9 genes. We generated induced pluripotent stem cells (iPSC) from a BSS patient with a novel heterozygous GP1BA p.N103D mutation, resulting in moderate macrothrombocytopenia. The mutation does not affect megakaryocyte (MK) differentiation or GPIb-GPIX complex expression but reduces affinity to von Willebrand factor (VWF). It induces increased signaling independent of VWF and αIIbβ3-mediated outside-in signaling, causing a profound defect in proplatelet formation after adhesion on fibrinogen. Pre-activation of αIIbβ3 integrin and heightened stress fiber formation linked to RhoA pathway overactivation were observed, likely due to increased phosphorylation of SRC at Y419 downstream of GPIbα. Dasatinib, a SRC inhibitor, restored stress fiber formation. Using a 3D bone marrow model to mimic platelet release under flow, we demonstrated that the ROCK1/2 inhibitor Y27632 increased platelet number and restored platelet size in GPIbαN103D MK, as well as in MK from two other patients with heterozygous GP1BA mutations (p.L160P and p.N150S). However, Y27632 had no additional effect on platelet generation from MK of two patients with biallelic BSS, suggesting a distinct molecular mechanism in biallelic cases.

Figure 1.

GPIbα^N103D mutant does not affect megakaryocyte differentiation from iPSC. (A) Flow cytometry plots showing expression of CD41, CD42a and CD42b on control (CNT1, CNT2) and GPIbα^N103D (2 different clones were used) megakaryocytes (MK). (B) Representative western blot of GPIb-GPIX complex (GPIbα, GPIbβ, GPIX) on mature control (CNT) and GPIbα^N103D MK. GAPDH, HSC70 and actin were used as loading controls. Quantification is shown in Online Supplementary Figure S3A. (C) 3D structure predicted by using AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/entry/P07359). hGPIbα wild-type and hGPIbα^N103D mutants aligned, focus on N/ D103. (D) Representative immunofluorescence staining pictures of mature control (CNT) an GPIbα^N103D MK: GPIbα (CD42b, green), F-actin (gray), filamin A (red), DAPI (nuclei, blue). Scale bar: 2 mm, z-stack middle frame is shown. (E) Co-immunoprecipitation assay performed on control (CNT) and GPIbα^N103D MK confirming the interaction between GPIbα and filamin A (FNLa). Mouse anti-GPIbα was used for immunoprecipitation and both anti-GPIbα and rabbit anti-FLNa antibodies were used for western blot assay. The quantification revealed no defect in the GPIbα-FLNa interaction in presence of GPIbα^N103D mutant. (F) Flow cytometry analysis of P-selectin at cell surface after activation of MK with indicated concentration of thrombin for 5 minutes. Anti-P-selectin antibody conjugated with PE was used. Data represent the mean ± standard error of the mean, N=3; *P<0.05, paired t test.

Figure 2.

GPIbα^N103D mutant alters proplatelet formation and platelet generation. (A) Frequency of proplatelet forming control (CNT) and GPIbα^N103D mutant megakaryocytes (MK) cultured in liquid medium in presence of thrombopoietin (TPO) and stem cell factor (SCF). Data are expressed as mean ± standard error of the mean (SEM) (CNT N=7, GPIbα^N103D N=10). (B) Frequency of proplatelet forming control (CNT) and GPIbα^N103D mutant MK on after adhesion of slides coated with 20 mg/mL of fibrinogen. Data are expressed as mean ± SEM (CNT N=6, GPIbα^N103D N=3); ***P<0.001, unpaired t test. (C) Schematic representation of ex vivo platelet collection from MK cultured into the silk bone marrow model functionalized with fibrinogen (50 µg/mL); 1.5x10⁵ control (CNT) and GPIbα^N103D mutant MK were seeded into scaffolds. Samples were perfused with culture medium, into a multi-well flow chamber, for 6 hours. Released platelets were collected into gas-permeable bags. Samples were mixed with counting beads to quantify the number of platelets that were identified as CD41⁺CD42a⁺ events. (D) The number of recovered platelets is shown. Data are expressed as mean ± SEM (CNT N=3, GPIbα^N103D N=3); *P<0.05, unpaired t test. (E) Platelet diameters (mm) of ex vivo-released platelets were measured by Arivis Vision 4D (Zeiss). Data are expressed as mean ± minimum to maximum, CNT N=127, GPIbα^N103D N=136; ****P<0.0001, unpaired t test. (F) Transmission electron microscopy of ex vivo produced platelets (scale bar: 2 µm).

Figure 3.

GPIbα^N103D mutant induces an increase in RhoA activity downstream aIIbβ3. (A) Flow cytometry analysis fibrinogen (Fb) binding to aIIbβ3 receptor on control (CNT) and GPIbα^N103D mutant megakaryocytes (MK) before and after its activation by thrombin (Thr). MK were stimulated or not for 5 minutes with thrombin (1 U/mL) and then incubated with Alexa 488-conjugated fibrinogen. Representative picture of plots is shown on the left panel. The histogram presenting the mean fluorescence intensity (MFI) of fibrinogen (Fb) staining at basal state is shown on the middle panel and after stimulation by thrombin on the right panel. The MFI after stimulation was normalized to the corresponding condition without stimulation. The histograms present the MFI of Fb normalized to 1 control in each experiment. Three independent experiments are shown. Data are expressed as mean ± standard error of the mean (SEM) (CNT N=5, GPIbα^N103D N=5); *P<0.05, Mann-Whitney test. (B) Spreading assay of mature control (CNT) and GPIbα^N103D mutant MK plated on different substrates. Representative picture of immunofluorescence staining of MK plated on fibrinogen-coated surface and stained for F-actin (green) and DAPI (blue) is shown on the left panel, single z-stack frame at the glass adhesion site is shown. The histogram presenting the frequency (%) of stress fiber forming MK plated on fibrinogen-, von Willebrand factor (VWF)- or fibronectin-coated surface is shown on the right panel. Data are expressed as mean ± SEM, N=4; **P<0.01, paired t test. (C) FRET analysis for RhoA or Cdc42 activation on fibrinogen-coated surface. At least 15 cells per condition were analyzed in each experiment. The histograms represent the average of 4-5 independent experiments. Data are normalized to control condition (CNT) for each experiment and are expressed in arbitrary units (a.u.) as mean ± SEM, N=5 for RhoA, N=4 for CDC42; **P<0.01, Mann-Whitney test. (D, E) Immunofluorescence analysis of stress fibers formation by mature control (CNT) and GPIbα^N103D mutant MK. Mature MK were plated on fibrinogen-coated surface in presence or absence of ROCK1/2 inhibitor Y27632 for 30 minutes and stained for F-actin (green) and DAPI (blue). (D) Representative pictures of immunofluorescence staining. Scale bar: 30 µm, single z-stack frame at the glass adhesion site is shown. (E) The histogram presenting the frequency (%) of stress fiber forming MK plated on fibrinogen-coated surface. Data are expressed as mean ± SEM, N=4; *P<0.05; **P<0.01, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison method was used. (F) Immunofluorescence analysis of proplatelet formation by mature control (CNT) and GPIbα^N103D mutant MK. The histogram presenting the frequency (%) of proplatelet forming MK plated on the fibrinogen-coated surface in the presence or absence of ROCK1/2 inhibitor Y27632, for 24 hours. Data are expressed as mean ± SEM, CNT N=5, GPIbα^N103D N=3; *P<0.05; ***P<0.001, one-way ANOVA with Tukey’s multiple comparison method was used. Representative pictures of immunofluorescence staining are shown in SF 7B. (G, H) 1.5x10⁵ MK were seeded in the silk scaffolds for 48 hours with or without the ROCK1/2 inhibitor Y27632, perfused with culture media for 6 hours, and released platelets were collected into gas-permeable bags. (G) The histogram represents the number of ex vivo-released platelets. Samples were mixed with counting beads to quantify the number of platelets identified as CD41⁺CD42a⁺ events. Data are expressed as mean ± SEM, N=3; *P<0.05; **P<0.01, paired t test. (H) Platelet diameters (mm) of ex vivo-released platelets with or without ROCK1/2 inhibitor Y27632 were measured by Arivis Vision 4D (Zeiss). Data are expressed as mean ± min to max, CNT N=127, CNT+Y27632 N=133, GPIbα^N103D N=136, GPIbα^N103D +Y27632 N=127; ****P<0.0001, one-way ANOVA with Tukey’s multiple comparison method was used. Representative pictures of ex vivo-released platelets with or without ROCK1/2 inhibitor Y27632 are shown in Online Supplementary Figure S7C.

Figure 4.

Mechanism of RhoA pathway activation. (A) Phosphoproteome analysis of control (CNT) and GPIbα^N103D mutant megakaryocytes (MK). Mature MK were seeded on fibrinogen-coated surface for 30 minutes (min), lysed and were analyzed using membrane-based human phospho-kinase antibody array. One of 2 experiments is shown. (B) Western blot analysis of P-SRC Y419 and P-STAT3 Y705 in control (CNT) and GPIbα^N103D mutant MK after 30 min incubation on fibrinogen-coated surface. Immunoblots were probed with monoclonal antibodies directed against P-STAT3 Y705, P-SRC Y419, STAT3, SRC and actin as loading control. Representative picture of immunoblot is shown on the left. Quantification of P-STAT3 Y705/total STAT3 is shown in the middle panel and that of P-SRC Y419/total SRC on the right panel. The ratios for mutant GPIbα^N103D MK were normalized to control MK. Data are expressed as mean ± minimum to maximum, P-STAT3 N=7, P-SRC N=8; *P<0.05, unpaired t test. (C-E) Effect of dasatinib on MK spreading. Control (CNT) and GPIbα^N103D mutant MK were incubated in presence of dasatinib (10 µM) for 15 min and plated on fibrinogen-coated surface for 30 min. (C) Western blot on MK showing a decrease in P-SRC Y419 in CNT and GPIbα^N103D mutant MK incubated with dasatinib. (D) Representative pictures of stress fiber forming MK stained with F-actin (green) and DAPI (blue). Scale bar: 30 µm, single z-stack frame at the glass adhesion site is shown. (E) Frequency of stress fiber forming MK. Data are expressed as mean ± standard error of the mean, CNT N=3, GPIbα^N103D N=4; *P<0.05; **P<0.01, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison method was used.

Figure 5.

RhoA inhibition restores the platelet generation in heterozygous Bernard Soulier syndrome. Hematopoietic progenitors were isolated from peripheral blood of 3 healthy controls and 1 patient carrying heterozygous GP1BA p.L160P (P2) mutation, and cultured in presence of thrombopoietin (TPO) and stem cell factor (SCF) for 10 days. (A, B) Flow cytometry analysis of control (CNT1, CNT2) and patient 2 (P2) megakaryocytes (MK). (A) Percent of mature CD41⁺CD42⁺ MK. (B) CD41 and CD42 expression level. (C-E) MK were seeded into the silk sponge with or without ROCK1/2 inhibitor Y27632. (C) Representative picture of control (CNT2) and P2 proplatelet forming MK. MK and platelets are stained with anti-CD61 the antibody (green) and the silk sponge is in blue. Scale bar: 50 μm, full 3D volume is shown. (D) Silk scaffolds were perfused with culture media for 6 hours, and released platelets were collected into gas-permeable bags. Samples were mixed with counting beads to quantify the number of platelets, identified as CD41⁺CD42a⁺ events. The number of recovered platelets is shown. A 1.48-fold increase in platelet number was detected for controls after incubation with ROCK1/2 inhibitor (N=3, data are presented as mean ± standard deviation) and 2.12-fold increase for P2 after incubation with ROCK1/2 inhibitor. (E) Platelet diameters (mm) of ex vivo-released platelets with or without ROCK1/2 inhibitor Y27632 were measured by Arivis Vision 4D (Zeiss). Data are expressed as mean ± minimum to maximum, N=50; ****P<0.0001, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison method was used.

Figure 6.

RhoA inhibition restores the platelet generation in heterozygous Bernard Soulier syndrome. Hematopoietic progenitors were isolated from peripheral blood of 3 healthy controls and 1 patient carrying heterozygous GP1BA p.N150S (P3) mutation respectively, and cultured in presence of thrombopoietin (TPO) and stem cell factor (SCF) for 10 days. (A-C) Flow cytometry analysis of control (CNT3) and patient 3 (P3) megakaryocytes (MK). (A) Percentage of mature CD41⁺CD42⁺ MK. (B) CD41 and CD42 expression level. (C) Ploidy level. CD41⁺CD42⁺ cells were incubated with Hoechst for 2 hours to label the nucleus. N represents a mean ploidy level. (D) MK were seeded into the silk scaffold with or without ROCK1/2 inhibitor Y27632. Silk scaffolds were perfused with culture media for 6 hours, and released platelets were collected into gas-permeable bags. Samples were mixed with counting beads to quantify the number of platelets, identified as CD41⁺CD42a⁺ events. The number of recovered platelets is shown. A 4.67-fold increase was detected for P3 after incubation with ROCK1/2 inhibitor. (E) Platelet diameters (mm) of ex vivo-released platelets with or without ROCK1/2 inhibitor Y27632 were measured by Arivis Vision 4D (Zeiss). Data are expressed as mean ± minimum to maximum, CNT N=57, CNT+Y27632 N=102, P3 N=57, P3+Y27632 N=57; ****P<0.0001, one-way analysis of variance (ANOVA) with Tukey’s multiple comparison method was used.