Intrinsic impaired proplatelet formation and microtubule coil assembly of megakaryocytes in a mouse model of Bernard-Soulier syndrome

Abstract

Background: Giant platelets and thrombocytopenia are invariable defects in the Bernard-Soulier syndrome caused by deficiency of the GPIb-V-IX complex, a receptor for von Willebrand factor supporting platelet adhesion to the damaged arterial wall. Various properties of this receptor may be considered potential determinants of the macrothrombocytopenia.

Design and methods: To explore the underlying mechanisms of the disease, megakaryopoiesis was studied in a mouse model deficient in GPIbbeta. Megakaryocytes were initially characterized in situ in the bone marrow of adult mice, after which their capacity to differentiate into proplatelet-bearing cells was evaluated in cultured fetal liver cells.

Results: The number of megakaryocyte progenitors, their differentiation and progressive maturation into distinct classes and their level of endoreplication were normal in GPIbbeta(-/-) bone marrow. However, the more mature cells exhibited ultrastructural anomalies with a thicker peripheral zone and a less well developed demarcation membrane system. GPIbbeta(-/-) megakaryocytes could be differentiated in culture from Lin(-) fetal liver cells in normal amounts but the proportion of cells able to extend proplatelets was decreased by 41%. Moreover, the GPIbbeta(-/-) cells extending proplatelets displayed an abnormal morphology characterized by fewer pseudopodial extensions with thicker shaft sections and an increased diameter of the terminal coiled elements. GPIbbeta(-/-) released platelets were larger but retained a typical discoid shape. Proplatelet formation was similarly affected in bone marrow explants from adult mice examined by videomicroscopy. The marginal microtubular ring contained twice as many tubulin fibers in GPIbbeta(-/-) proplatelet buds in cultured and circulating platelets.

Conclusions: Altogether, these findings point to a role of the GPIb-V-IX complex intrinsic to megakaryocytes at the stage of proplatelet formation and suggest a functional link with the underlying microtubular cytoskeleton in platelet biogenesis.

Figure 1.

GPIbβ^−/− platelets have a normal life span. Mice were injected i.v. with N-hydroxy-succinomidyl biotin on day 0 and the percentage of biotinylated platelets was determined at the indicated times using flow cytometry and CD41 gating. Data are the mean values±SEM of quadruple determinations.

Figure 2.

GPIbβ^−/− bone marrow contains normal numbers of progenitors but increased numbers of identifiable megakaryocytes with a normal ploidy distribution. (A) Colony assays for CFU-Mk, CFU-GM, BFU-E and CFU-GEMM in bone marrow from GPIbβ^+/+ and GPIbβ^−/− mice. Results are the mean±SEM of eight independent experiments and no significant differences were found between the two groups. (B) Representative sections of GPIbβ^+/+ and GPIbβ^−/− mouse femora stained with H&E. (C) Morphologically recognizable megakaryocytes were counted and numbers expressed as the mean±SEM per mm² of surface in four or five fields from six mice in each group. Numbers of megakaryocytes were significantly increased in GPIbβ^−/− mice, ***p<0.006. (D) Ploidy analysis of CD41⁺ bone marrow cells from GPIbβ^+/+ and GPIbβ^−/− mice. Data are the mean±SEM of four experiments with two mice per experiment in each group. The ploidy distribution was not significantly different in GPIbβ^+/+ and GPIbβ^−/− cells.

Figure 3.

GPIbβ^−/− megakaryocytes display a normal distribution in different maturation stages but ultrastructural defects in stage III cells. (A) The distribution in the different maturation stages was established according to megakaryocyte morphology (see Methods) on TEM images and was not significantly different in GPIbβ^+/+ and GPIbβ^−/− mice. Results are the mean±SEM of the percentage of megakaryocytes in each stage. (B) Representative TEM images of stage III megakaryocytes in the bone marrow of GPIbβ^+/+ and GPIbβ^−/− mice. GPIbβ^−/− cells display a poorly developed demarcation membrane system and increased size of the cytoplasmic platelet territories. (C) Representative confocal microscopic images of stage III megakaryocytes labeled with phalloidin-TRITC (red) and a monoclonal antibody against CD41 (green). GPIbβ^−/− cells exhibit a wider actin-rich (red) and organelle-deficient peripheral zone (PZ) than GPIbβ^+/+ cells. (D) Detailed TEM images of the PZ in GPIbβ^+/+ and GPIbβ^−/− megakaryocytes.

Figure 4.

A decreased proportion of megakaryocytes extend pro-platelets in cultures of GPIbβ^−/− fetal liver cells. Lin⁻ selected fetal liver cells were cultured for 4 days in the presence of thrombopoietin and FBS and examined by confocal microscopy to determine the proportion of megakaryocytes bearing proplatelets. (A) Representative images of megakaryocytes stained for tubulin on day 4 showing a majority of proplatet-bearing GPIbβ^+/+ cells and an increased proportion of GPIbβ^−/− cells with a round morphology which did not reach the proplatelet stage. (B) The number of CD41⁺ cells forming proplatelets was counted from day 2 to day 4 (no proplatelets were formed at day 1) and expressed as the percentage of the total number of CD41⁺ cells. A significant percentage extended proplatelets at days 3 and 4 in both strains. This proportion was decreased in GPIbβ^−/−, representing 31.8±2.9% versus 54.8±3.3% in GPIbβ^+/+ at day 4. Values are the mean±SEM of six separate experiments, ***p<0.001.

Figure 5.

Proplatelet-bearing megakaryocytes and individual platelets have an abnormal morphology in cultures of GPIbβ^−/− fetal liver cells. (A) Representative confocal photomicrographs of proplatelet-bearing megakaryocytes labeled for β-tubulin showing an extensive network with thin shafts (arrow) and platelet-sized coils (arrowhead) in GPIbβ^+/+ cells and a decreased number of proplatelet extensions, thicker shafts and enlarged coils in GPIbβ^−/− cells. (B) The diameter of the coiled elements (in μm) and the shaft thickness (in nm) were measured on confocal photomicrographs using LAS acquisition software (AF version 1.62; Leica) and both parameters were increased in GPIbβ^−/− as compared to GPIbβ^+/+ cells. Values are the mean±SEM of three separate experiments in each group. (C) Scanning electron microscopy images of platelets in the culture suspension show a characteristic discoid morphology and a smooth cell surface with openings corresponding to entrances to the open canalicular system. GPIbβ^−/− platelets have a larger diameter than GPIbβ^+/+ cells but retain a disc-shaped morphology. (D) The mean diameter (±SEM) was measured on electron micrographs of 22 individual cultured platelets, ***p<0.001.

Figure 6.

Abnormal proplatelet formation in bone marrow explants from GPIbβ^−/− mice. Bone marrow sections (0.5 mm) from GPIbβ^+/+ and GPIbβ^−/− adult mice were incubated in serum-supplemented Tyrode’s buffer at 37°C and observed by time-lapse videomicroscopy. (A) Representative pictures of GPIbβ^+/+ megakaryocytes at the periphery of the bone marrow exhibiting a round immature morphology (a), a single thick protrusion (b), long fragmented pseudopodia (c). (B) Comparative photographs of GPIbβ^+/+ and GPIbβ^−/− megakaryocytes exhibiting decreased proplatelet number and size in GPIbβ^−/− (C) Quantification of the three representative morphologies of megakaryocytes (round cell, cell with protrusion and cell with proplatelets). The percentage of cells (±SEM) was measured in three separate experiments and represented 25–28 megakaryocytes in each strain.

Figure 7.

Proplatelets and platelets from GPIbβ^−/− mice display an abnormal microtubular ultrastructure. (A) Scannng electron microscopy images of proplatelets after removal of the membrane by treatment with Triton X-100 detergent revealed a cytoskeleton composed of an actin meshwork with bundles of microtubules along the shafts and in the coiled elements. Increased thickness of the microtubules was observed in GPIbβ^{− −} as compared to GPIbβ^+/+ proplatelets. (B) TEM images of cultured platelets in transverse section illustrating the increased number of marginal microtubular rings in GPIbβ^−/− as compared to GPIbβ^+/+ cells. Immunogold labeling of β-tubulin in circulating blood platelets also showed increased numbers of microtubules in the marginal band in GPIbβ^−/− cells on transverse (C) and equatorial ( D ) cryosections examined by TEM. (E) Quantification of the number of microtubule coils in the marginal band on TEM images of thin sections of circulating platelets. Values are the mean±SEM for 16 individual platelets in each group.