Aae Suzuki

RhoA Is Essential for Maintaining Normal Megakaryocyte Ploidy and Platelet Generation

RhoA plays a multifaceted role in platelet biology. During platelet development, RhoA has been proposed to regulate endomitosis, proplatelet formation, and platelet release, in addition to having a role in platelet activation. These processes were previously studied using pharmacological inhibitors in vitro, which have potential drawbacks, such as non-specific inhibition or incomplete disruption of the intended target proteins. Therefore, we developed a conditional knockout mouse model utilizing the CRE-LOX strategy to ablate RhoA, specifically in megakaryocytes and in platelets to determine its role in platelet development. We demonstrated that deleting RhoA in megakaryocytes in vivo resulted in significant macrothrombocytopenia. RhoA-null megakaryocytes were larger, had higher mean ploidy, and exhibited stiff membranes with micropipette aspiration. However, in contrast to the results observed in experiments relying upon pharmacologic inhibitors, we did not observe any defects in proplatelet formation in megakaryocytes lacking RhoA. Infused RhoA-null megakaryocytes rapidly released platelets, but platelet levels rapidly plummeted within several hours. Our evidence supports the hypothesis that changes in membrane rheology caused infused RhoA-null megakaryocytes to prematurely release aberrant platelets that were unstable. These platelets were cleared quickly from circulation, which led to the macrothrombocytopenia. These observations demonstrate that RhoA is critical for maintaining normal megakaryocyte development and the production of normal platelets.

Loss of PIKfyve in platelets causes a lysosomal disease leading to inflammation and thrombosis in mice

PIKfyve is essential for the synthesis of phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and for the regulation of endolysosomal membrane dynamics in mammals. PtdIns(3,5)P2 deficiency causes neurodegeneration in mice and humans, but the role of PtdIns(3,5)P2 in non-neural tissues is poorly understood. Here we show that platelet-specific ablation of PIKfyve in mice leads to accelerated arterial thrombosis, and, unexpectedly, also to inappropriate inflammatory responses characterized by macrophage accumulation in multiple tissues. These multiorgan defects are attenuated by platelet depletion in vivo, confirming that they reflect a platelet-specific process. PIKfyve ablation in platelets induces defective maturation and excessive storage of lysosomal enzymes that are released upon platelet activation. Impairing lysosome secretion from PIKfyve-null platelets in vivo markedly attenuates the multiorgan defects, suggesting that platelet lysosome secretion contributes to pathogenesis. Our findings identify PIKfyve as an essential regulator for platelet lysosome homeostasis, and demonstrate the contributions of platelet lysosomes to inflammation, arterial thrombosis and macrophage biology.

Platelets lacking PIP5KIγ have normal integrin activation but impaired cytoskeletal-membrane integrity and adhesion.

Three isoforms of phosphatidylinositol-4-phosphate 5-kinase (PIP5KIα, PIP5KIβ, and PIP5KIγ) can each catalyze the final step in the synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2), which in turn can be either converted to second messengers or bind directly to and thereby regulate proteins such as talin. A widely quoted model speculates that only p90, a longer splice form of platelet-specific PIP5KIγ, but not the shorter p87 PIP5KIγ, regulates the ligand-binding activity of integrins via talin. However, when we used mice genetically engineered to lack only p90 PIP5KIγ, we found that p90 PIP5KIγ is not critical for integrin activation or platelet adhesion on collagen. However, p90 PIP5KIγ-null platelets do have impaired anchoring of their integrins to the underlying cytoskeleton. Platelets lacking both the p90 and p87 PIP5KIγ isoforms had normal integrin activation and actin dynamics, but impaired anchoring of their integrins to the cytoskeleton. Most importantly, they formed weak shear-resistant adhesions ex vivo and unstable vascular occlusions in vivo. Together, our studies demonstrate that, although PIP5KIγ is essential for normal platelet function, individual isoforms of PIP5KIγ fulfill unique roles for the integrin-dependent integrity of the membrane cytoskeleton and for the stabilization of platelet adhesion.

Loss of ATE1-mediated arginylation leads to impaired platelet myosin phosphorylation, clot retraction, and in vivo thrombosis formation

Protein arginylation by arginyl–transfer RNA protein transferase (ATE1) is emerging as a regulator protein function that is reminiscent of phosphorylation. For example, arginylation of β-actin has been found to regulate lamellipodial formation at the leading edge in fibroblasts. This finding suggests that similar functions of β-actin in other cell types may also require arginylation. Here, we have tested the hypothesis that ATE1 regulates the cytoskeletal dynamics essential for in vivo platelet adhesion and thrombus formation. To test this hypothesis, we generated conditional knockout mice specifically lacking ATE1 in their platelets and in their megakaryocytes and analyzed the role of arginylation during platelet activation. Surprisingly, rather than finding an impairment of the actin cytoskeleton structure and its rearrangement during platelet activation, we observed that the platelet-specific ATE1 knockout led to enhanced clot retraction and in vivo thrombus formation. This effect might be regulated by myosin II contractility since it was accompanied by enhanced phosphorylation of the myosin regulatory light chain on Ser19, which is an event that activates myosin in vivo. Furthermore, ATE1 and myosin co-immunoprecipitate from platelet lysates. This finding suggests that these proteins directly interact within platelets. These results provide the first evidence that arginylation is involved in phosphorylation-dependent protein regulation, and that arginylation affects myosin function in platelets during clot retraction.

Phosphatidylinositol transfer protein-α in platelets is inconsequential for thrombosis yet is utilized for tumor metastasis

Platelets are increasingly recognized for their contributions to tumor metastasis. Here, we show that the phosphoinositide signaling modulated by phosphatidylinositol transfer protein type α (PITPα), a protein which shuttles phosphatidylinositol between organelles, is essential for platelet-mediated tumor metastasis. PITPα-deficient platelets have reduced intracellular pools of phosphoinositides and an 80% reduction in IP3 generation upon platelet activation. Unexpectedly, mice lacking platelet PITPα form thrombi normally at sites of intravascular injuries. However, following intravenous injection of tumor cells, mice lacking PITPα develop fewer lung metastases due to a reduction of fibrin formation surrounding the tumor cells, rendering the metastases susceptible to mucosal immunity. These findings demonstrate that platelet PITPα-mediated phosphoinositide signaling is inconsequential for in vivo hemostasis, yet is critical for in vivo dissemination. Moreover, this demonstrates that signaling pathways within platelets may be segregated into pathways that are essential for thrombosis formation and pathways that are important for non-hemostatic functions.Platelets support tumor metastasis formation. Here, the authors show that the phosphoinositide signaling modulated by PITPα is essential for platelet-mediated tumor metastasis signaling, but surprisingly it is dispensable in hemostasis in mice.

Rattus norvegicus melanocortin 3 receptor: A corrected sequence

Examination of the Rattus norvegicus genome reveals differences in the melanocortin 3 receptor (MC3R) compared with the published sequence (accession X70667). To clarify these differences, we used RT-PCR to clone MC3R from Sprague Dawley rats. These efforts revealed a sequence for the rat MC3R consistent with that predicted by the rat genome, but different from the published receptor by three amino acids, all of which were located in the predicted second transmembrane domain (TM2). Analysis of these residues revealed that TM2 of the rat MC3R is more homologous with other species than previously considered. The presently described sequence maps onto chromosome 3 of the rat genome, which shows highly conserved synteny with the mouse chromosome 2 and the human chromosome 20. Transient expression revealed high affinity binding of [125I]-NDP-MSH and a concentration-dependent cAMP response to the synthetic agonist MTII. These data both clarify the sequence of the MC3R and demonstrate the great utility of genomic information recently made available.

Phosphatidylinositol transfer proteins regulate megakaryocyte TGF-β1 secretion and hematopoiesis in mice

We hypothesized that megakaryocyte (MK) phosphoinositide signaling mediated by phosphatidylinositol transfer proteins (PITPs) contributes to hematopoietic stem cell (HSC) and hematopoietic progenitor cell (HPC) regulation. Conditional knockout mice lacking PITPs specifically in MKs and platelets (pitpα-/- and pitpα-/-/β-/-) bone marrow (BM) manifested decreased numbers of HSCs, MK-erythrocyte progenitors, and cycling HPCs. Further, pitpα-/-/β-/- BM had significantly reduced engrafting capability in competitive transplantation and limiting dilution analysis. Conditioned media (CM) from cultured pitpα-/- and pitpα-/-/β-/- BM MKs contained higher levels of transforming growth factor β1 (TGF-β1) and interleukin-4 (IL-4), among other myelosuppressive cytokines, than wild-type BM MKs. Correspondingly, BM flush fluid from pitpα-/- and pitpα-/-/β-/- mice had higher concentrations of TGF-β1. CM from pitpα-/- and pitpα-/-/β-/- MKs significantly suppressed HPC colony formation, which was completely extinguished in vitro by neutralizing anti-TGF-β antibody, and treatment of pitpα-/-/β-/- mice in vivo with anti-TGF-β antibodies completely reverted their defects in BM HSC and HPC numbers. TGF-β and IL-4 synergized to inhibit HPC colony formation in vitro. Electron microscopy analysis of pitpα-/-/β-/- MKs revealed ultrastructural defects with depleted α-granules and large, misshaped multivesicular bodies. Von Willebrand factor and thrombospondin-1, like TGF-β, are stored in MK α-granules and were also elevated in CM of cultured pitpα-/-/β-/- MKs. Altogether, these data show that ablating PITPs in MKs indirectly dysregulates hematopoiesis in the BM by disrupting α-granule physiology and secretion of TGF-β1.