Actin-bundling protein L-plastin promotes megakaryocyte rigidity and dampens proplatelet formation

Normal hemostasis requires an adequate number of normally functioning platelets. A better understanding of platelet biogenesis may facilitate the expanding effort to manufacture platelets in vitro.¹ Platelets develop from mature bone marrow megakaryocytes (MK) that derive from megakaryocyte progenitor (MKP) cells.² In the classic model of bone marrow megakaryopoiesis, hematopoietic stem cells (HSC) in the proliferative niche give rise to MK-erythroid progenitors that differentiate into MKP during migration to the vascular nice.³ The cytoplasm of mature MK has an abundant invagi-nated membrane system (IMS) that is contiguous with the plasma membrane.^4,5 During the late stages of thrombopoiesis, the IMS begins extrude from the MK plasma membrane and form long extensions of propla-telets (PP) that eventually give rise to platelets released in the vasculature.^6,7 Cell migration and signaling requires alterations in the organization and thickness of the cell cortex, which is comprised of a network of overlapping, bundled and crosslinked actin filaments.⁸ Filamentous actin (F-actin) organization regulates MK shape, MKP migration, formation of the IMS, early stage of PP formation (PPF), and later stages of PP branching. Despite this understanding of various stages of MK differentiation, the molecular mechanisms that signal the MK to initiate PPF are not well understood.

Recently, our laboratory used an unbiased transcrip-tomic approach to identify an actin bundling protein, L-plastin, that decreased during MK differentiation, correlating with the onset of PPF.⁹ Using CRISPR/Cas to generate CD34⁺-derived human MK deficient in L-plas-tin, we found impaired MK migration in response to SDF-1α chemoattractant, increased podosome formation per MK, increased IMS and increased PPF. F-actin polymerization and depolymerization (i.e., “re-organization”) are dynamic reactions central to MK shape, migration, IMS formation, etc. F-actin also facilitates degradation of the extracellular matrix at sites of po-dosome formation.¹⁰ The MK cytoskeleton, comprised of a dense network of actin filaments and micro-tubules, is located at the periphery of the cell cytoplasm, below the plasma membrane. The manner by which the loose lipid-rich IMS is able to penetrate the dense actin cytoskeleton to initiate PPF has not been addressed. The prior work on MK L-plastin was limited by inter-individual variation affecting platelet number and artifacts of in vitro culturing.⁹ Because MK L-plastin has not been studied in vivo, and because L-plastin is expected to increase the density of the MK actin cortex via actin bundling, we used L-plastin-deficient mice¹¹ to assess the effects of L-plastin on in vivo hemostasis and thrombosis, determine the intracellular location of MK L-plastin, and assess MK membrane rigidity. We report that MK L-plastin colocalizes with actin in wild-type MK, and loss of MK L-plastin reduces MK membrane rigidity and increases MK PPF, providing a novel mechanism for regulating platelet formation.

We first confirmed L-plastin protein expression in platelets from wild-type mice and littermate knockout mice with a disrupted Lcp1 gene lacked platelet L-plas-tin protein (Online Supplementary Figure S1A). Initial experiments addressed the effect of in vivo L-plastin deficiency on screening assays for hemostasis and thrombosis. Tail bleeding times and survival times after a pulmonary embolism model were shorter in L-plastin expression mice (Figure 1A, B), suggesting a prothrom-botic phenotype in the knockout animals. Although this mouse strain is a global knockout, L-plastin express-ionis limited to hematopoietic cells, and prior studies have focused on L-plastin effects in leukocytes.¹² We, therefore, sought to investigate the role of L-plastin in thrombopoiesis using the L-plastin-deficient mice. Compared to wild-type mice, peripheral blood platelet counts in Lcp1^-/- mice were modestly but significantly higher, with no difference in mean platelet volume (Figure 1C, D). Under steady state conditions and compared to wild-type mice, Lcp1^-/- mice have normal red blood cell counts and modestly increased white blood cell counts (Online Supplementary Figure S1B, C), but no differences in body weight. Both male and female mice were studied and no effect of sex on the platelet count difference was observed. Note that the comparisons in this study were between inbred strains of Lcp1 wild-type and Lcp1^-/- littermates, so we would not expect environmental or other genetic confounding factors. These in vivo data support the use of this Lcp1^-/-mouse line for studies of L-plastin in the MK/platelet lineage.

No difference was observed between the number of acetylcholinesterase-positive MK in wild-type and Lcp1^-/- mice (Online Supplementary Figure S2), suggesting L-plastin may not affect megakaryopoiesis. However, compared to wild-type mice, Lcp1^-/-mice had significantly more PP-forming MK (Figure 2A, B), consistent with an inhibitory role of L-plastin in platelet produc-tion.⁹ We examined the subcellular distribution of L-plastin in MK using immunofluorescent confocal microscopy. Figure 2C demonstrates co-localization between L-plastin and F-actin in MK adherent to both poly-L-lysine and fibrinogen. A prominent distribution of L-plastin was observed below the plasma membrane of MK. Similar results were obtained with human CD34⁺-derived MK (Online Supplementary Figure S3). Taken together, these data suggest a hypothesis whereby L-plastin-mediated F-actin bundling results in a denser MK actin cortex that impedes the IMS protrusion necessary to initiate PPF. However, we cannot exclude an increase in lifespan as an additional contributor to the increased platelet count in the Lcp1^-/- mice.

Figure 1.In vivo hemostasis and thrombosis models. (A) Tail-bleeding time for wild-type (WT) Lcp1^+/+ and Lcp1^-/- littermate mice (Lcp1^+/+ N=10, Lcp1^-/- N=10). (B) Pulmonary thromboembolism was induced by injecting Lcp1^+/+ or Lcp1^-/- mice with collagen and epinephrine. Time to death was monitored and plotted (Lcp1^+/+ N=10, Lcp1^-/- N=9). (C) Platelet count and (D) mean platelet volume (MPV) in the peripheral blood from littermate Lcp1^+/+ and Lcp1^-/- mice of both sexes. Each dot/circle is an individual mouse (N=19-20).

We have previously shown that L-plastin expression decreases with MK maturation.⁹ A corollary of the above hypothesis is that lower levels of L-plastin in mature MK cause reduced actin bundling and hence, a less stiff MK actin cytoskeleton. Because integral membrane proteins directly or indirectly attach the plasma membrane to the outermost layer of actin filaments of the cortical cytoskeleton,¹³ we tested this corollary by measuring MK cytoskeleton stiffness using the Bio-membrane Force Probe (BFP) tether assay (illustrated in Figure 3A, B).^14,15 Compared to wild-type MK, L-plastin-deficient MK were significantly less rigid (Figure 3C). Lastly, we used CRISPR to knockdown expression in cultured human MK and observed a similar relationship between L-plastin and membrane rigidity (Figure 3D).

The molecular switches that tell the mature MK to begin PPF are incompletely understood. Actin, myosin heavy and light chains, tubulin, kinases, and phospha-tases are all critical for this process. Bury et al. have reviewed inherited causes of thrombocytopenia, and listed 11 genes that regulate PPF.¹⁶ Based on the rationale that changes in gene expression during MK differentiation might provide clues to genes regulating normal PPF, we previously performed RNA sequencing on biologic replicates of day 6, day 9 and day 13 cord blood-differentiated Mk,⁹ and found numerous candidate genes, most of which increased as MK began to form PP. Like ROCK1,¹⁷ L-plastin is one of the few inhibitors of PPF described to date.

In summary, we measured MK cytoskeleton stiffness for the first time and found an important role of the actin cytoskeleton stiffness in PPF. We showed that i) mice deficient in the actin-bundling protein L-plastin have an increased MK PPF and higher platelet counts, and ii) mouse and human MK L-plastin levels strongly correlate with MK membrane rigidity. Since higher MK membrane rigidity likely correlates with actin cytoskel-etal density, higher levels of L-plastin in immature MK could prevent premature release of platelets in the bone marrow. At the mature stage of MK differentiation, L-plastin levels have decreased, which is expected to reduce actin bundling and actin cytoskeletal rigidity, and facilitate initiation of membrane PP protrusions (Figure 3E). Bisaria et al. recently showed that newly forming lamellipodial protrusions form in areas of low membrane proximal F-actin density,¹⁸ and it will be of interest for future studies to assess the effect of L-plastin-mediated actin bundling on the ability of cofilin to depolymerize F-actin.

Figure 2.L-plastin inhibits megakaryocyte proplatelet formation and localizes with F-actin. (A) Representative images of mega-karyocyte (MK)-forming proplatelets (PP) from both Lcp1^+/+ and Lcp1^-/- mice. Mouse bone marrow cells were stimulated with 50 ng/mL thrombopoietin for 3-4 days. MK were enriched using bovine serum albumin gradient and cultured for another 16-24 hours and examined by light transmission (LT) microscopy. (B) Quantification of PP-forming MK. PP-forming MK were defined by at least 1 filamentous pseudopod observed by light microscopy with a 20x objective of day 3-4 cultured cells. Scoring was blinded as to Lcp1 genotype, and the percent of total MK number plotted. Three or more images from each of 3 pairs of littermates Lcp1^+/+ and Lcp1^-/- mice were counted and the average percentage per mouse was plotted. (C) Representative immunofluorescent confocal images of C57BL/6 MK stained for DNA (DAPI), F-actin (magenta) and L-plastin (green). F-actin/L-plastin co-localization is white. MK seeded on poly-L-lysine (PLL) or fibrinogen (FGN). PPF: proplatelet formation.

Figure 3.L-plastin maintains megakaryocyte membrane stiffness. (A) Illustration of the Biomembrane Force Probe (BFP) tether assay for the measurement of cell stiffness. Borosilicate beads labeled with mouse anti-CD41 antibody were attached to the apex of red blood cell (RBC) at the tip of a micropipette (left). A single megakaryocyte (MK) held by a micropipette (right) approaches the antibody-coated beads and is allowed to attach. Once attached, the MK pipette is pulled back under a constant speed. The cell stiffness is calculated using the pulling force required (= the elastic deformation phase). (B) Representative BFP tether assay readout plot. During the initial approaching stage, no force was detected. During the contact phase, the MK squeezes the bead and shows negative force. During the retraction phase, force is rapidly generated in the linear elastic deformation phase. Cell stiffness was calculated from the elastic deformation phase using the customized package run by LabVIEW (National Instruments). The experiment terminates when the MK detaches from the anti-CD41 coated bead. (C) The BFP tether assay MK membrane stiffness in pico-Newtons (pN) per nanometer (nm) for 3 pairs of mice (wild-type [WT] 1-3 and Lcp1^-/- 1-3). Each circle represents a single MK. (D) Similar to panel (C), the BFP tether assay was used to assess human MK membrane rigidity in CD34⁺-derived MK treated without (control 1-3) and with (knockout [KD] 1-3) CRISPR-mediated knockdown of L-plastin. N=3 pairs of different cord blood donor cells.

Footnotes

• Received February 24, 2023
• Accepted July 6, 2023

Correspondence

^#SB and PFB contributed equally as senior authors

P. F. BRAY - Paul.bray@hsc.utah.edu

S. BHATLEKAR - saseems@gmail.com

Disclosures

No conflicts of interest to disclose.

Contributions

LG, SJ, BKM, EKM, SG, XW, DM, EAT, IP, YK, CB and SB performed research. LG, SB and PFB wrote the manuscript. MTR, BE and CM provided scientific expertise.

Data-sharing statement

The data and experimental protocol are available upon request.

Funding

Acknowledgments

The authors wish to acknowledge experiment technical support from Jesse W. Rowley, Frederik Denorme, Mark J. Cody, Neal D. Tolley, Marina Tristao and Meenakshi Banerjee, editorial help from Neal D. Tolley, and figure preparation expertise from Diana Lim and Nikita Abraham, Molecular Medicine Program, University of Utah, as well as the technical support from Xue Lin, Washington University in St. Louis.