Naohide Watanabe

Basic studies on the clinical applications of platelet-rich plasma.

Platelets, which contain many growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), are being used in clinical applications as platelet-rich plasma (PRP). Only a few studies, however, have been conducted on the growth factors present in PRP and on the clinical applications using the drug delivery system (DDS). For the purpose of clinical application, we first modified the PRP preparation method and assessed the amounts of growth factors contained in the human platelet concentrates. Furthermore, we assessed fibrin glue as a DDS of platelet concentrates. Platelet precipitations were made by twice centrifuging human whole blood. The precipitated platelet was resuspended to yield the platelet concentrates. The growth factor concentrations were measured. Fibrin glue sheets containing this platelet concentrate were implanted in rabbit pinna and samples were obtained for immunostaining (anti-PDGF antibody) to assess the use of PRP over time using the fibrin glue as the DDS. The mean concentration of growth factors present in the platelet concentrates was three times or greater than that of conventional PRP. Furthermore, the results indicated that when the platelet concentrate was used with fibrin glue as a carrier, the contents were released over a period of about 1 week. This raises the possibility that this system may be useful in clinical applications.

Basic studies on the bone formation ability by platelet rich plasma in rabbits

Purpose:Platelets, which contain many growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor-β (TGF-β), can be obtained in high concentrations by centrifugal separation and are being used as platelet-rich plasma (PRP) in clinical applications. The authors evaluated the bone formation ability by PRP in rabbits. Materials and Methods:In experiment 1, the authors made platelet precipitations from the whole blood obtained from rabbits. Each precipitated platelet was resuspended in fibrin glue. The glue was applied to a 1-mm wide slit defect of the same rabbit mandible. After 1 week, the authors observed the histologic appearances. In experiment 2, the fibrin glue containing the precipitated platelet was applied to a bicortical defect of a rabbit cranium with β-tricalcium phosphate (β-TCP) granules as artificial bone material. After 1 week, 1 month, and 2 months, the authors observed the radiologic and histologic appearances. Results and Conclusions:In both experiments, a prosperous bone formation was observed from the beginning more in the group treated with PRP than in the group untreated. In experiment 2, gradual appearances of the bone formation were observed in the bubble of β-TCP. In application with artificial bone materials, bone formations by PRP were suitably observed.

Impaired hematopoietic differentiation of RUNX1-mutated induced pluripotent stem cells derived from FPD/AML patients

Somatic mutation of RUNX1 is implicated in various hematological malignancies, including myelodysplastic syndrome and acute myeloid leukemia (AML), and previous studies using mouse models disclosed its critical roles in hematopoiesis. However, the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder (FPD)/AML is an autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. By in vitro differentiation assays, FPD-iPSCs were clearly defective in the emergence of hematopoietic progenitors and differentiation of megakaryocytes, and overexpression of wild-type (WT)-RUNX1 reversed most of these phenotypes. We further demonstrated that overexpression of mutant-RUNX1 in WT-iPSCs did not recapitulate the phenotype of FPD-iPSCs, showing that the mutations were of loss-of-function type. Taken together, this study demonstrated that haploinsufficient RUNX1 allele imposed cell-intrinsic defects on hematopoietic differentiation in human experimental settings and revealed differential impacts of RUNX1 dosage on human and murine megakaryopoiesis. FPD-iPSCs will be a useful tool to investigate mutant RUNX1-mediated molecular processes in hematopoiesis and leukemogenesis.

N-terminal region of CCAAT/enhancer-binding protein ∈ is critical for cell cycle arrest, apoptosis, and functional maturation during myeloid differentiation

CCAAT/enhancer-binding protein ϵ (C/EBPϵ) plays a critical role in terminal myeloid differentiation. Differentiation is an integrated process of cell cycle arrest, morphological change, functional maturation, and apoptosis. However, the molecular networks underlying these events in C/EBPϵ-induced differentiation remain poorly understood. To reveal these mechanisms, we performed a detailed molecular analysis of C/EBPϵ-induced differentiation using an inducible form of C/EBPϵ. The activation of C/EBPϵ induced growth arrest, morphological differentiation, the expression of CD11b and secondary granule proteins, and apoptosis in myeloid cell lines. Unlike C/EBPα, C/EBPϵ dramatically up-regulated p27 with a concomitant down-regulation of cdk4/6 and cyclin D2/A/E. Moreover, the anti-apoptotic proteins Bcl-2 and Bcl-x were down-regulated, whereas pro-apoptotic protein Bax remained unchanged. Using a variety of mutants, we revealed that these events were all regulated by the N-terminal activation domain of C/EBPϵ. Interestingly, some of the differentiation processes such as the induction of secondary granule protein genes were clearly inhibited by c-Myc; however, inhibition of apoptosis by Bcl-x did not affect the entire differentiation processes. These data indicate the N terminus of C/EBPϵ to be solely responsible for most aspects of myeloid differentiation, and these events were differentially affected by c-Myc.

Generation of megakaryocytes and platelets from human subcutaneous adipose tissues.

Recent advances in regenerative medicine have created a broad spectrum of stem cell research. Among them, tissue stem cell regulations are important issues to clarify the molecular mechanism of differentiation. Adipose tissues have been shown to contain abundant preadipocytes, which are multipotent to differentiate into cells including adipocytes, chondrocytes, and osteoblasts. In this study, we have first shown that megakaryocytes and platelets can be generated from adipocyte precursor cells. Human adipocyte precursor cells were cultured in conditioned media for 12 days to differentiate adipocytes, followed by 12 days of culture in media containing thrombopoietin. The ultrastructures of adipocyte precursor cell- and bone marrow CD34-positive cell-derived megakaryocytes and platelets were similar. In addition, adipocyte precursor cell-derived platelets exhibited surface expression of P-selectin and bound fibrinogen upon stimulation with platelet agonists, suggesting that these platelets were functional. This is the first demonstration that human subcutaneous adipocyte precursor cells can generate megakaryocyte and functional platelets in an in vitro culture system.

Decoration of fibrinogen γ-chain peptide on adenosine diphosphate-encapsulated liposomes enhances binding of the liposomes to activated platelets.

For the purpose of efficient hemostasis, we previously developed ADP-encapsulated liposomes modified with a dodecapeptide (HHLGGAKQAGDV, H12), H12-(ADP)Lipo. This liposome actually enhanced platelet aggregation in vitro, and showed significant hemostatic effect in vivo. Since fibrinogen (Fbg) is abundant in the bloodstream, it is unclear why this liposome binds platelets so efficiently, overcoming the competition with Fbg. Therefore, we investigated the relationship between H12 density on the liposome and the binding ability to platelets, and evaluated the inhibitory effect of Fbg on the binding of H12-(ADP)Lipo to platelets. As a result, the binding ability to platelets steeply increased depending on H12 density until it reached about 3×10(15) H12 molecules/m(2). The 50% inhibition concentration of Fbg on the binding of H12-(ADP)Lipo to platelets was about 25-fold over the concentration of H12 molecules on the liposome. Moreover, almost no inhibition by Fbg was observed at the physiological concentration of it. This result suggests that the ability of H12 to bind to GPIIb/IIIa increased overwhelmingly by the anchoring to the liposome that enabled the cooperative binding of H12 peptides to the platelets.

Shear-dependent suppression of platelet thrombus formation by phosphodiesterase 3 inhibition requires low levels of concomitant Gs-coupled receptor stimulation

Phosphodiesterase (PDE)3 inhibitors exert potent antiplatelet effects through maintaining elevated intracellular cyclic adenosine monophosphate levels, but do not prolong bleeding time. To resolve this discrepancy, we hypothesised that PDE3 inhibitors effectively suppress shear-induced platelet thrombus formation initiated by the interaction of the platelet receptor GPIb/V/IX with its ligand, von Willebrand factor (VWF), since arterial thrombosis is more dependent on shear stress as compared with haemostatic plug formation. To test the hypothesis, we compared the in vitro effects of K-134 (a PDE3 inhibitor), tirofiban (a GPIIb/IIIa inhibitor) and acetylsalicylic acid (ASA) on ristocetin-induced platelet aggregation and platelet thrombus formation on VWF or collagen surfaces under flow conditions. K-134 inhibited GPIIb/IIIa-dependent platelet aggregation to the same extent as tirofiban and more potently than ASA. Likewise, K-134 and tirofiban effectively inhibited stable platelet thrombus formation (platelet firm adhesion and subsequent aggregation) on the VWF or collagen surface under high shear, but ASA only inhibited aggregation. Notably, inhibition by K-134 became evident only when a low concentration of PGE1 was present. These inhibitors did not block shear-induced initial platelet contact with VWF via GPIb/V/IX. In contrast, under low shear, the inhibitory effects of K-134 on platelet aggregation on the collagen surface were lower than tirofiban or ASA. The observed shear-dependent suppression of platelet thrombus formation by PDE3 inhibitor in the presence of low levels of adenylate cyclase stimulator may contribute to high therapeutic benefit with low risk of bleeding.

SHD1 is a novel cytokine-inducible, negative feedback regulator of STAT5-dependent transcription

STAT5 is a critical mediator of a variety of cytokine signaling whose transcriptional activity is regulated by associating with various proteins. During a search for STAT5-interacting proteins, we identified SHD1, a mammalian homologue of yeast gene Sac3, as a potential interacter. SHD1 was localized in the nucleus, and induced by cytokines that activate STAT5, such as erythropoietin, interleukin-2 (IL-2), or IL-3. SHD1 interacted specifically with STAT5A and STAT5B, and interestingly, it specifically repressed STAT5-dependent transcription in vitro without affecting the stability or phosphorylation of STAT5 protein. Gene disruption study revealed that T, B, or bone marrow cells from mice lacking SHD1 were hyperresponsive to T-cell-receptor engagement, or stimulation with various STAT5-activating cytokines. These results suggest that SHD1 is a novel cytokine-inducible negative feedback regulator of STAT5.

Refined methods to evaluate the in vivo hemostatic function and viability of transfused human platelets in rabbit models

To bridge the gap between in vitro function and clinical efficacy of platelet (PLT) transfusion products, reliable in vivo PLT functional assays for hemostasis and survival in animal models are required. However, there are no standardized methods for assessing the in vivo quality of transfused human PLTs.

Ability of fibrinogen γ-derived dodecapeptides with different sequences to bind to rat platelets

A dodecapeptide (γ400-411) derived from a fibrinogen γ-chain carboxyl-terminal sequence recognizes specifically the active form of GPIIb/IIIa on the surface of activated platelets. For the purpose of efficient hemostasis, we previously developed ADP-encapsulated liposomes modified with human-dodecapeptide (HHLGGAKQAGDV, human-H12). On the other hand, the amino-acid sequence of H12 from rats is HHMGGSKQVGDM, having only 67% homology to that from humans. Here, we investigated the ability of rat-H12 in comparison with human-H12 to bind to platelets. Firstly, rat platelets were activated with phorbol-12-myristate-13-acetate (PMA), and the activation was confirmed by flow cytometry. Next, we evaluated the dissociation constant (K(d)) of human-H12 and rat-H12 for dissociation from rat platelets by using FACS. As a result, the K(d) of human-H12 and rat-H12 with respect to rat platelets was 2.78 ± 0.21 and 2.91 ± 0.22 μM, respectively. Furthermore, H12 from both species inhibited quite similarly the aggregation of rat platelets in platelet-rich plasma (PRP). These results suggest that H12 from different species with different amino acid sequences interacts similarly with GPIIb/IIIa on platelets.