ALBERT

Description: Platelets are specialized anucleate cells that circulate in the blood and serve to prevent bleeding and minimize blood vessel injury. In addition to their hemostatic functions, platelets participate in wound healing, angiogenesis, inflammation, and immunity, and are therefore central players in both maintaining normal physiology and in disease pathogenesis. Platelets are derived from their precursor cells, megakaryocytes (MKs), that reside principally in the bone marrow. During maturation, MKs undergo an altered cell cycle called endomitosis in which they replicate their DNA but avoid cell division, resulting in polyploid MKs with amplified microtubule (MT)-organizing centers called centrosomes. Subsequently, MT-dependent forces are responsible for extending long cytoplasmic protrusions called proplatelets into sinusoidal blood vessels, eventually giving rise to circulating platelets. Despite progress in elucidating key steps of platelet production, there is a conspicuous lack of understanding of what triggers mature, polyploid MKs to undergo the MT rearrangements required for proplatelet production. Using live cell imaging of mouse fetal liver-derived MKs expressing fluorescent b1-tubulin, we have identified a novel MT-based structure in MKs termed a monospindle. Our data suggest that monospindles result from polyploid MKs clustering multiple centrosomes into a centralized MT-organizing center during mitosis, leading to an enlarged array of MTs oriented towards the cell cortex. These structures were also apparent in mouse bone marrow- and human cord blood-derived MKs, suggesting that monospindle formation is a general phenomenon in MKs. Interestingly, a higher percentage of MKs contained monospindles at a timepoint directly prior to proplatelet production (50%) compared to when proplatelets were actively being produced (22%), indicating a possible role in initiating proplatelet formation. Centrosome clustering in cancer cells is mediated by the MT-based mitotic motor protein, KIFC1. Consistently, we found that small molecule inhibition of KIFC1 decreased the percentage of MKs containing monospindles (55% ctrl vs. 6% KIFC1 inhibitor). Strikingly, KIFC1 inhibitor treatment also drastically reduced the percentage of MKs producing proplatelets (peak proplatelet formation: 40% ctrl vs. 5% KIFC1 inhibitor), suggesting that KIFC1-mediated centrosome clustering into monospindles is important for proplatelet production. To test how KIFC1 contributes to these phenotypes, we assessed its expression at different timepoints by Western blot and detected increased KIFC1 levels in more mature MKs preceding proplatelet formation. Cell sorting of MKs into distinct ploidy populations followed by Western blot showed that KIFC1 expression increased with higher ploidy. Thus, our results lead us to suggest a working model in which elevated KIFC1 levels in mature MKs drive monospindle formation to trigger proplatelet formation. Investigating the role of KIFC1-mediated monospindle formation for initiating proplatelet formation could yield a coherent, molecular understanding of how mature, polyploid MKs reorganize MTs for proplatelet production. In addition, these data will help inform basic cell biology, as there are important parallels between centrosome clustering in MKs and cancer cells. Finally, our findings could yield novel therapeutic strategies for treating patients with thrombocytopenia (low platelet counts) by directly stimulating platelet production from mature MKs residing in the bone marrow. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding.

Description: von Willebrand factor (vWF) is an essential hemostatic protein that is synthesized in endothelial cells and stored in Weibel-Palade bodies (WPBs). Understanding the mechanisms underlying WPB biogenesis and exocytosis could enable therapeutic modulation of endogenous vWF, yet optimal targets for modulating vWF release have not been established. Since biogenesis of lysosomal related organelle-2 (BLOC-2) functions in the biogenesis of platelet dense granules and melanosomes, which like WPBs are lysosome-related organelles (LROs), we hypothesized that BLOC-2-dependent endolysosomal trafficking is essential for WPB biogenesis and sought to identify BLOC-2 interacting proteins. Depletion of BLOC-2 caused misdirection of cargo-carrying transport tubules from endosomes, resulting in immature WPBs that lack endosomal input. Immunoprecipitation of BLOC-2 identified the exocyst complex as a binding partner. Depletion of the exocyst complex phenocopied BLOC-2 depletion, resulting in immature WPBs. Furthermore, releasates of immature WPBs from either BLOC-2 or exocyst-depleted endothelial cells lacked high-molecular weight forms of vWF, demonstrating the importance of BLOC-2/exocyst-mediated endosomal input during vWF maturation. Yet BLOC-2 and exocyst showed very different effects on vWF release. While BLOC-2 depletion impaired exocytosis, exocyst depletion augmented WPB exocytosis, indicating that it acts as a clamp. Exposure of endothelial cells to a small molecule inhibitor of exocyst, Endosidin2, reversibly augmented secretion of mature WPBs containing high molecular weight forms of vWF. These studies show that while BLOC-2 and exocyst cooperate in WPB formation, only exocyst serves to clamp WPB release. Exocyst function in vWF maturation and release are separable, a feature that can be exploited to enhance vWF release.

Description: von Willebrand factor (vWF) is an essential plasma hemostatic factor that also participates in pathophysiologic processes such as thrombosis, angiogenesis and tumor metastasis. vWF is synthesized in endothelial cells and stored in specialized granules, Weibel-Palade bodies (WPBs), which exhibit characteristics of secretory granules termed lysosomal-related organelles (LROs). Understanding the mechanisms underlying WPB biogenesis and vWF exocytosis could enable therapeutic modulation of endogenous vWF; however, fundamental aspects of vWF trafficking mechanisms remain unknown. Since biogenesis of lysosomal related organelle 2 (BLOC-2), a complex of HPS3, HPS5 and HPS6 proteins, functions in biogenesis of other LROs such as platelet dense granules and melanosomes, we hypothesized that BLOC-2-dependent endolysosomal trafficking was essential for WPB biogenesis. BLOC-2 was depleted in human umbilical vein endothelial cells (HUVECs) by transduction of cells with lentiviral particles containing HPS6 shRNA. Evaluation of WPBs using multicolor immunofluorescence microscopy showed that the WPBs were immature, granular and abnormally localized to the perinuclear space, as compared to mature, linear-shaped WPBs, distributed peripherally in control cells (number of WPBs 〉 1.5 µm per cell, HPS6 3±2 vs. control 16±6). Immature WPBs in BLOC-2-depleted HUVECs lacked expression of endosome-derived cargo CD63, but not P-selectin, a synthetic cargo that enters WPBs at the trans-Golgi network. Live cell imaging showed that when BLOC-2 was depleted from HUVECs expressing CD63-GFP, CD63-GFP no longer trafficked to WPBs. Instead, endosome-derived transport tubules were mistargeted to the core of the cell. In comparison, CD63-GFP labeled WPBs intensely in control cells, where direct interaction between endosome-derived transport tubules and WPBs was evident. To identify binding partners of BLOC-2 that facilitate targeting of endosome-derived transport tubules to maturing WPBs, we performed immunoprecipitation of endogenous BLOC-2 in HUVEC lysates and evaluated the captured proteins using mass spectrometry. Several components of the exocyst complex, namely EXOC2, EXOC4 and EXO70, were enriched in BLOC-2 immunoprecipitates. This interaction was confirmed by reciprocal co-immunoprecipitation experiments with HPS6 and EXOC4. Evaluation by immunogold electron microscopy showed that EXOC4 colocalized with HPS6 in HUVECs. EXOC4 depletion using targeted siRNA resulted in WPBs that were immature, granular-appearing and perinuclearly localized, similar to BLOC-2-depleted HUVECs (number of WPBs 〉 1.5 µm per cell, EXOC4 3±2.5 vs. control 16±5). Overall, these observations indicate that both BLOC-2 and the exocyst complex are critical for endolysosomal trafficking involved in WPB maturation. We next evaluated the roles of BLOC-2 and the exocyst complex in vWF exocytosis. Depletion of BLOC-2 from HUVECs resulted in a 63±3% decrease in thrombin-induced vWF exocytosis and a 41±4% decrease in constitutive vWF exocytosis. Multimer analysis showed loss of high-molecular weight vWF multimers. In vivo studies showed that vWF exocytosis following systemic epinephrine infusion was 158±12% of basal levels in WT mice compared 100±20% in HPS6-/-mice, indicating lack of agonist-induced vWF release in BLOC-2 depleted mice. To evaluate the role of the exocyst complex in vWF release, both EXOC4 and EXO70 were depleted from HUVECS using siRNA. Unexpectedly, depletion of either exocyst complex component augmented thrombin-induced vWF exocytosis (EXOC4 105±21%; EXO70 99±22%). Similarly, endosidin 2, a small molecule inhibitor of EXO70, also augmented vWF exocytosis by 122±26%. These studies demonstrate that both BLOC-2 and the exocyst complex contribute to WPB biogenesis, perhaps working together based on their binding and colocalization on WBPs. However, these two complexes have opposing effects on vWF secretion. While BLOC-2 is necessary for vWF secretion, the exocyst suppresses release. Disclosures Italiano: Platelet Biogenesis: Employment, Equity Ownership; Ionis Research Funding: Research Funding. Flaumenhaft:PlateletDiagnostics: Consultancy, Other: Founder; Relay Therapeutics: Consultancy.