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Exposure of human megakaryocytes to high shear rates accelerates platelet production

Claire Dunois-Lardé, Claude Capron, Serge Fichelson, Thomas Bauer, Elisabeth Cramer-Bordé and Dominique Baruch

Data supplements

  • Supplemental materials for: Dunois-Lardé et al

    MKs were perfused at a shear rate of 1800 s−1 for 10 min on VWF-coated coverslips, followed by 10 min perfusion with culture medium to wash out non-adherent cells and to allow prolonged contact of adherent cells with the VWF surface. Digital images were recorded at 0.25 image/s and read at a velocity of 10 images/s (40-fold acceleration). Videos 1 and 2 are from the same cell deformation followed from 3 min to 31 min and separated in video 1 (3�13 min) and video 2 (13�31 min). Because of size frame limitations, the microscope stage was moved to keep track of MK displacement that always kept contact with the surface.

    Files in this Data Supplement:

    • Video 1. MK deformation, elongation and proplatelet formation (MOV, 1.47 MB) -
      At high shear rates, human MKs rolled on and irreversibly attached to the VWF-coated coverslip. The movie shows early deformations of MKs translocating on VWF, until full arrest. From an anchoring point located at one pole of the cell, a pseudopod is emitted which rapidly elongates, becomes filiform, and starts to exhibit swellings at its extremity and all along its shaft. Cytoplasmic fragments of different sizes remained attached to the cell nucleus until separating from the cell body. Cytoplasmic elongations growing from the MK cell body were usually mono or bi-directional along the flow. The cytoplasmic elongation is oriented along the flow, appearing as bipolar extensions. A filopodial extension appears at the front of the cell body, and a proplatelet displaying numerous swellings forms at the rear. The tail of the cell is immobilized, while the leading front considerably elongates and eventually detaches from the rear.

    • Video 2. Rupture of proplatelet and platelet release (MOV, 1.69 MB) -
      The reversible anchoring of the rear end detaches and the rest of the cell is removed by the flow while swellings form at both ends and along the proplatelet length. A stretched MK is anchored to the matrix by its rear end, while the front end elongates, becomes filiform and detaches to form a free proplatelet. The proplatelet is moved along by the flow and sheds one of its extremities to form a dumbbell proplatelet, and subsequently also sheds a platelet-sized extremity.

    • Video 3. Effect of Nocodazole addition after the elongation process had started (MOV, 1.35 MB) -
      The movie shows an elongating MK at 22 min. The microtubule inhibitor Nocodazole (10 µM) was added at 21 min and required five minutes to enter the chamber and interfere with cells (see also Fig. 4). At 25 min the elongation stops and the extended process starts to shrink, until the anchoring point and the nucleus almost come in contact (30 min), showing that Nocodazole could revert shear-induced elongation.

    • Video 4. Effect of αIIbβ3 inhibitor on MKs elongation (MOV, 351 KB) -
      In the presence of Abciximab, an inhibitor of αIIbβ3, most cells fail to elongate; when they do so, swellings appear along the bipolar cell but real elongations and filiform stretchings fail to occur and no detachment or rupture into platelets is seen.

    • Video 5. MKs slow translocation on mutated type 2B-rVWF (MOV, 1 MB) -
      Mutated type 2B-rVWF was purified and coated on the coverslip, and MKs were perfused at 1800 s−1

Article Figures & Data

Figures

  • Figure 1

    MK deformation on a VWF surface at high shear rate. Cells suspended in IMDM were perfused on VWF at 1800 s−1 for 10 minutes; then IMDM alone was perfused for 10 minutes. Adhesion led to major cell shape changes and to proplatelet formation. Microphotographs during perfusion indicate 4 different stages starting from undeformed MKs (stage 1) to early deformation characterized by loss of cell sphericity (stage 2); stage 3 includes later deformation of MKs with cytoskeletal reorganization at a cell pole or both ends, also a long thin filament of successive “beads on a thread”; stage 4 comprises fragmentation of proplatelets and cleavage from the nucleus, as well as platelet formation and release. Proportion of cells relative to the total number of cells counted in 10 fields was plotted as a function of perfusion time classified according to these 4 stages for cord blood MKs (bottom left panel) or bone marrow MKs (bottom right panel) perfused on VWF. Photographs are representative of 15 experiments, curves are mean (± SEM) of 8 experiments. The bar represents 10 μm. Black lines have been inserted to indicate repositioned images.

  • Figure 2

    Comparison of shear or static conditions of MK deformations on a VWF-containing surface. Coverslips were removed from the flow chamber and rinsed with PBS before fixation in ice-cold methanol and subsequent Romanovsky staining (A-B, magnification ×500): during static 20-minute adhesion, cells displayed a spherical shape, without any proplatelet extension (A), but after exposure to high shear rate for 20 minutes, MKs extended long filopods at their tip and beaded platelet-like spikes were formed along the shaft (B). In separate experiments, images were recorded on coverslips coated with VWF (C-D) or on stimulated HUVECs (E-F) during real-time perfusion. Panel C shows proplatelet formation during 16 hours on VWF-coated slides in static conditions extending in different directions (C). In shear conditions, after 20 minutes, proplatelet formation is bipolar and organized along the flow (D). Similar organization of proplatelet formation is seen on UL-VWF released by HUVECs stimulated by IBMX and forskolin (E-F). Bars on panels C through F represent 10 μm.

  • Figure 3

    Distribution of α-granule proteins and tubulin in shear-induced proplatelets and platelets. Confocal microscope analysis of immunofluorescence showing labeling (left panels) with anti–P-selectin, anti-tubulin antibodies, and overlay of (from top to bottom) an MK extending a thick proplatelet, a long and thin proplatelet, a dumbbell-shaped proplatelet with its central narrowing, and 2 platelets. P-selectin staining is seen in organelles in forming proplatelets and in platelets in different spots, whereas tubulin stains the entire microtubule shaft of the proplatelet and the periphery of platelets. Right panels: labeling with phalloidin, anti-tubulin antibodies, and overlay of (from top to bottom) an MK extending proplatelet, a long and thin proplatelet with its shaft as well as its tip, labeled for tubulin, a dumbbell-shaped proplatelet with its central narrowing, and a platelet exhibiting a circular labeling pattern. No colocalization between actin and tubulin is visible. The bars represent 10 μm.

  • Figure 4

    Reversal of proplatelet elongation by nocodazole addition during proplatelet elongation. Frames from experiment shown in supplemental Video 3 were selected from (top to bottom) before (13 minutes) and during elongation (21 minutes), after addition of nocodazole (at 21 minutes and 20 seconds), during effect of nocodazole (25 minutes), and at the start (28 minutes), during the progress (31 minutes), and at the completion (38 minutes) of elongation reversal. Notice the modifications between the cell body and its anchorage point (white arrow) during different steps. Confocal microscope analysis of indirect immunofluorescence labeling with anti-tubulin antibody of an MK extending proplatelet before and after nocodazole treatment. MKs exhibit diffuse staining after action of nocodazole, indicating tubulin depolymerization. Bars represent 10 μm.

  • Figure 5

    Ultrastructure of MKs exposed to high shear rates leading to platelet formation. Effluent cell suspensions (A-E) or alternatively cells present on the coverslips (F) were processed for electron microscopy as described in “Electron microscopy.” Mature MKs were elongated, extending an often unique long cytoplasmic filopod enclosing parallel longitudinal microtubules (inset). These proplatelets exhibited regular swellings containing cytoplasmic organelles. A large spherical cytoplasmic fragment, probably a detached proplatelet (pp) was located nearby (A). The nuclear lobes (N) containing dense chromatin were elongated and located at one pole of the cell. Naked nuclei with an oval shape and compact nuclear lobes containing dense chromatin, which are normally absent from MK cultures, were retrieved in the effluents (B). Proplatelets (pp) filled with cytoplasmic organelles appeared as large cytoplasmic fragments, devoid of nuclei, roughly spherical, dumbbell-shaped, or elongated with slender extremities (C-D). Several isolated platelet-sized fragments (P) were observed (E). Panel F shows the section of the cell monolayer present on a VWF-coated coverslip removed from the chamber after exposition to shear. It shows 2 platelet-sized fragments (P) located close to an adhering MK, displaying α granules and surface-connected canalicular system characteristic of platelets, and whose distribution pattern resembles that of the putative mother cell (MK). Scale bar represents 2 μm.

  • Figure 6

    Platelets obtained from MK exposure to high shear rates can be activated by thrombin. Cell effluents in the flow-through of MKs exposed to high shear rates were analyzed in a fibrinogen adhesion assay in static conditions, followed by confocal microscopy, with cell staining with phalloidin–Alexa 546 to visualize actin and Alexa 488 to visualize αIIbβ3. Washed blood platelets (A) and MK-derived platelets generated by shear exposure (B) adhered to fibrinogen in the absence (left panels) or in the presence (right panels) of thrombin. Nonactivated cells display diffuse actin staining and αIIbβ3 membrane localization. After thrombin activation, actin filaments are organized as stress fibers. They display a similar cytoskeletal organization to washed blood platelets. The bars represent 10 μm. Flow cytometry assay (C): Samples were labeled with anti–CD62P-FITC (FL1, P-selectin) and anti–CD41-PE (FL2, αIIb). Settings of FSC-SSC profiles of washed blood platelets were used to analyze flow-through cells. Histogram plot of activated platelets in the flow-through, in the absence or presence of thrombin, after labeling with nonimmune IgG (thin line, gray background), anti-CD62P, or anti-CD41a (thick line). Electron microscopy: (D) in the presence of thrombin, the platelet-sized fragment displayed morphologic changes characteristic of activated platelets, namely a spherical shape, surface pseudopods (p), dense material within dilated cisternae of SCCS, no granulation in the cytoplasm, and a central bundle of microfilaments, reminiscent of activated blood platelets. The bar represents 2 μm.

  • Figure 7

    MK deformations at high shear rate on different surfaces coated with matrix proteins and effect of VWF inhibitors. Cord blood MKs suspended in IMDM were perfused on VWF (in the absence or presence of inhibitors), fibrinogen, collagen, or fibronectin at 1800 s−1 for 10 minutes; then IMDM alone was perfused for 10 minutes. Cell numbers were counted in 10 fields at the end of perfusion (20 minutes) and classified according to the 4 stages defined in Figure 1. Intense cell deformation and proplatelet formation occur on VWF. Cell perfusion on VWF in the presence of GPIb-VWF interaction inhibitors, either a blocking antibody to glycocalicin or an anti-VWF MoAb directed against VWF binding site to GPIb, indicated a major inhibition of MK adhesion to VWF and of subsequent steps, thus abolishing proplatelet formation. Abciximab, blocking the interaction of αIIbβ3 with VWF, prevented proplatelet and platelet formation. MK deformations and proplatelet formation on non-VWF surfaces were minimal in shear conditions as shown for fibrinogen, fibronectin, and collagen.