Direct and Continuous Inhibition of ADAM17 Using a Novel Selective Inhibitor Restores Functional Platelet Yield From Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2323-2323
Author(s):  
Shinji Hirata ◽  
Ryoko Jono-Ohnishi ◽  
Satoshi Nishimura ◽  
Naoya Takayama ◽  
Sou Nakamura ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Imaging System ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
P38 Map Kinase

Abstract Abstract 2323 Platelet transfusion is therapeutically important for patients with thrombocytopenia and/or bleeding disorders. Problems associated with a lack of donors and unknown infections in the blood have not been fully resolved, however. In that context, human induced pluripotent stem cells (hiPSCs) are a potentially abundant source of infection-free platelets. The pluripotent state of hiPSCs and their differentiation depend upon appropriate culture conditions defined in part by oxygen and temperature. We therefore initially examined whether temperatures at or below 24°C, which are required for preservation of platelet concentrates ex vivo, allow hiPSC differentiation to generate platelets. We found that only at 37°C were platelets generated. But at 37°C in vitro, platelets are subject to degradation exemplified by the shedding of GPIbα, a receptor for von Willebrand factor (vWF), which is caused by a disintegrin and metalloprotease (ADAM) 17. We therefore developed KP-457, a novel ADAM17 inhibitor that has a reverse hydroxamic acid structure and has been found safe in rats and dogs. Although inhibition of p38 MAP kinase, putatively upstream of ADAM17, reportedly inhibits GPIbα shedding in stored human platelets, even at 37°C, administration of the p38 inhibitor SB203580 induces cytotoxicity during differentiation, leading to a loss of platelet yield from hiPSCs. By contrast, KP-457 significantly protected GPIbα expression in platelets from hiPSCs and in aged human platelets in culture at 37°C. Moreover, iPSC-derived platelets generated in the presence of KP-457 displayed improved hemostatic function when studied using an imaging system that enables characterization of single-platelet kinetics during thrombus formation after laser-induced injury in vivo. We propose this new drug could markedly improve the maintenance of functional platelets generated in culture, particularly those derived from hiPSCs. Disclosures: No relevant conflicts of interest to declare.

Anticoagulant and antithrombotic properties of platelet protease nexin-1

Blood ◽  
2010 ◽  
Vol 115 (1) ◽  
pp. 97-106 ◽  
Author(s):  
Yacine Boulaftali ◽  
Frédéric Adam ◽  
Laurence Venisse ◽  
Véronique Ollivier ◽  
Benjamin Richard ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Protease Nexin ◽  
Deficient Mice

AbstractProtease nexin–1 (PN-1) is a serpin that inhibits plasminogen activators, plasmin, and thrombin. PN-1 is barely detectable in plasma but is expressed by platelets. Here, we studied platelet PN-1 in resting and activated conditions and its function in thrombosis. Studies on human platelets from healthy donors and from patients with a Gray platelet syndrome demonstrate that PN-1 is present both at the platelet surface and in α-granules. The role of PN-1 was investigated in vitro using human platelets incubated with a blocking antibody and using platelets from PN-1–deficient mice. Both approaches indicate that platelet PN-1 is active on thrombin and urokinase-type plasminogen activator. Blockade and deficiency of platelet PN-1 result in accelerated and increased tissue factor-induced thrombin generation as indicated by calibrated automated thrombography. Moreover, platelets from PN-1–deficient mice respond to subthreshold doses of thrombin, as assessed by P-selectin expression and platelet aggregation. Thrombus formation, induced ex vivo by collagen in blood flow conditions and in vivo by FeCl3-induced injury, is significantly increased in PN-1–deficient mice, demonstrating the antithrombotic properties of platelet PN-1. Platelet PN-1 is thus a key player in the thrombotic process, whose negative regulatory role has been, up to now, markedly underestimated.

Inhibitory Effect of Piracetam on Platelet-rich Thrombus Formation in an Animal Model

1998 ◽  
Vol 79 (01) ◽  
pp. 222-227 ◽  
Author(s):  
F. Stockmans ◽  
W. Deberdt ◽  
Å. Nyström ◽  
E. Nyström ◽  
J. M. Stassen ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Ex Vivo ◽  
Peak Plasma ◽  
Thrombus Formation ◽  
Plasma Concentrations ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Cell Deformability

SummaryIntravenous administration of piracetam to hamsters reduced the formation of a platelet-rich venous thrombus induced by a standardised crush injury, in a dose-dependent fashion with an IC50 of 68 ± 8 mg/kg. 200 mg/kg piracetam also significantly reduced in vivo thrombus formation in rats. However, in vitro aggregation of rat platelets was only inhibited with piracetam-concentrations at least 10-fold higher than plasma concentrations (6.2 ± 1.1 mM) obtained in the treated animals. No effects were seen on clotting tests.In vitro human platelet aggregation, induced by a variety of agonists, was inhibited by piracetam, with IC50’s of 25-60 mM. The broad inhibition spectrum could be explained by the capacity of piracetam to prevent fibrinogen binding to activated human platelets. Ex vivo aggregations and bleeding times were only minimally affected after administration of 400 mg/kg piracetam i.v. to healthy male volunteers, resulting in peak plasma levels of 5.8 ± 0.3 mM.A possible antiplatelet effect of piracetam could be due to the documented beneficial effect on red blood cell deformability leading to a putative reduction of ADP release by damaged erythrocytes. However similarly high concentrations were needed to prevent stirring-induced “spontaneous” platelet aggregation in human whole blood.It is concluded that the observed antithrombotic action of piracetam cannot satisfactorily be explained by an isolated direct effect on platelets. An additional influence of piracetam on the rheology of the circulating blood and/or on the vessel wall itself must therefore be taken into consideration.

A Dendritic Cell Based Vaccination Strategy Geared towards Eradication of Leukemic Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2046-2046
Author(s):  
Hetty J Bontkes ◽  
Jurjen Ruben ◽  
Willemijn van den Ancker ◽  
Theresia M Westers ◽  
G. Ossenkoppele ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Tumor Antigens ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Vaccination Strategy ◽  
Leukemic Stem Cells ◽  
Cell Immunity

Abstract Abstract 2046 Poster Board II-23 Introduction: In the majority of cases, initial remission of acute myeloid leukemia (AML) is reached but unfortunately relapse rates remain high and therefore novel treatments are needed. It is thought that recurrent AML originates from chemotherapy resistant quiescent leukemic stem cells (LSC). The application of immunotherapeutic approaches to eradicate LSC remaining after first line chemotherapy may contribute to improved disease outcome. Vaccination strategies have often used dendritic cells (DC) ex vivo pulsed with tumor-derived whole lysates or peptides as modalities to present a broad range of tumor antigens to T cells to stimulate effective anti-tumor T-cell immunity in vivo. It is likely that certain proteins expressed by LSC have a distinct antigenicity as compared to more mature AML blasts and thus provide targets for specific T-cells. Even without identification of specific antigens, LSC can be a useful source of tumor antigens in DC vaccination-based immunotherapy. CD34+CD38- LSC can be identified using malignant stem cell associated cell surface markers including CLL-1 and lineage markers such as CD7, CD19 and CD56. However, the low frequency of these cells precludes the use of LSC derived apoptotic cells or lysates for DC loading. Alternatively, mRNA isolated from LSC can be amplified and subsequently transfected into DC. Materials and Methods: We have made use of the CD38- AML derived cell line MUTZ-3 which contains a subpopulation of CD34+CLL1+ cells which resembles the phenotype of a putative LSC. CLL1+CD34+ and CLL1-CD34- cells were isolated by FACS sorting and total RNA was isolated. mRNA was converted to cDNA and amplified by PCR using the SMART system. Subsequently, mRNA was in vitro transcribed from the amplified cDNA. Mature monocyte derived DC (MoDC) were generated from healthy donor blood and transfected with amplified CLL1+CD34+ derived mRNA and used to stimulate autologous CD8β+ T-cells. After three weekly re-stimulations with CLL1+CD34+ mRNA transfected DC, specificity of the T-cells was analyzed by intracellular IFNγ staining upon 5 hour stimulation with autologous immature MoDC transfected with GFP mRNA, mRNA amplified from unsorted, CLL1+CD34+ or CLL1-CD34- MUTZ-3 subpopulations. Results: Amplification of CLL1 and survivin (also expressed by MUTZ-3) transcripts was confirmed by RT-PCR. After 3 weekly re-stimulations with CLL1+CD34+ amplified RNA transfected DC, 0.04% (range 0.01-0.12%) of the T-cells were positive for IFNγ upon a 5 hr re-stimulation with GFP transfected DC. 0.44% (range 0.04-0.69%) of the T-cells responded to DC transfected with unsorted MUTZ-3 amplified mRNA (p<0.00005 versus GFP control, 2-sided student's T-test), 0.51% (range 0.24-1.35%) responded to DC transfected with CLL1+CD34+ amplified mRNA (p<0.005 versus GFP control) and 0.46% (range 0.24-0.94%) responded to DC transfected with CLL1-CD34- amplified mRNA (p<0.0001 versus GFP control). Conclusion: We show that MoDC transfected with RNA amplified from one MUTZ-3 sub-population resembling the phenotype of LCS cells are capable of inducing T-cells which recognize both cells transfected with mRNA from the LSC resembling MUTZ-3 subset as well as the CLL1-CD34- subset. We are currently testing the efficacy and feasibility of this approach in an autologous setting in vitro. CD8β+ T-cells are stimulated with autologous MoDC from AML patients transfected with amplified mRNA isolated from their own LSC enriched populations. The capacity of these T-cells to kill autologous AML blasts and LSC is subsequently analysed in a 6-colour FACS based cytotoxicity assay. Disclosures: No relevant conflicts of interest to declare.

Anti-Human VWF Monoclonal Antibody SZ-123 Prevents Arterial Thrombus Formation by Inhibiting VWF–collagen and VWF-Platelet Interactions in Rhesus Monkeys

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5194-5194
Author(s):  
Yiming Zhao ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Significant Prolongation ◽  
Arterial Thrombus

Abstract Abstract 5194 Objective: To investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123, and its potential underlying mechanism. Methods and Results: Cyclic flow reductions (CFRs) were measured in the femoral artery of monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet count and template bleeding time were performed as measurements of antithrombotic activity. In addition, plasma VWF, SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0. 1, 0. 3, and 0. 6 mg/kg SZ-123 resulted in 45. 3%, 78. 2%, and 100% reduction in CFRs, respectively. When 0. 3 and 0. 6 mg/kg SZ-123 were administrated, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60–95% inhibition of platelet aggregation were observed from 15 min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiment also revealed that SZ-123 not only blocks collagen-VWF A3 interaction but also inhibits indirectly VWF A1 binding to GPIba induced by ristocetin. Conclusions: SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1-platelet interactions and does not prolong bleeding time. Disclosures: No relevant conflicts of interest to declare.

Strategies to Protect Hematopoietic Stem Cells from Culture-Induced Stress Conditions

2020 ◽  
Vol 15 ◽  
Author(s):  
Fatima Aerts-Kaya
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Ex Vivo ◽  
Stress Conditions ◽  
Stress Factors ◽  
Hematopoietic Stem ◽  
Induced Stress

: In contrast to their almost unlimited potential for expansion in vivo and despite years of dedicated research and optimization of expansion protocols, the expansion of Hematopoietic Stem Cells (HSCs) in vitro remains remarkably limited. Increased understanding of the mechanisms that are involved in maintenance, expansion and differentiation of HSCs will enable the development of better protocols for expansion of HSCs. This will allow procurement of HSCs with long-term engraftment potential and a better understanding of the effects of the external influences in and on the hematopoietic niche that may affect HSC function. During collection and culture of HSCs, the cells are exposed to suboptimal conditions that may induce different levels of stress and ultimately affect their self-renewal, differentiation and long-term engraftment potential. Some of these stress factors include normoxia, oxidative stress, extra-physiologic oxygen shock/stress (EPHOSS), endoplasmic reticulum (ER) stress, replicative stress, and stress related to DNA damage. Coping with these stress factors may help reduce the negative effects of cell culture on HSC potential, provide a better understanding of the true impact of certain treatments in the absence of confounding stress factors. This may facilitate the development of better ex vivo expansion protocols of HSCs with long-term engraftment potential without induction of stem cell exhaustion by cellular senescence or loss of cell viability. This review summarizes some of available strategies that may be used to protect HSCs from culture-induced stress conditions.

scholarly journals A Tumbling Magnetic Microrobot System for Biomedical Applications

Micromachines ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 861
Author(s):  
Elizabeth E. Niedert ◽  
Chenghao Bi ◽  
Georges Adam ◽  
Elly Lambert ◽  
Luis Solorio ◽  
...  
Keyword(s):  
Ultrasound Imaging ◽  
Biomedical Applications ◽  
Imaging System ◽  
Ex Vivo ◽  
Negative Control ◽  
Circular Path ◽  
Rotational Axis ◽  
Significant Difference

A microrobot system comprising an untethered tumbling magnetic microrobot, a two-degree-of-freedom rotating permanent magnet, and an ultrasound imaging system has been developed for in vitro and in vivo biomedical applications. The microrobot tumbles end-over-end in a net forward motion due to applied magnetic torque from the rotating magnet. By turning the rotational axis of the magnet, two-dimensional directional control is possible and the microrobot was steered along various trajectories, including a circular path and P-shaped path. The microrobot is capable of moving over the unstructured terrain within a murine colon in in vitro, in situ, and in vivo conditions, as well as a porcine colon in ex vivo conditions. High-frequency ultrasound imaging allows for real-time determination of the microrobot’s position while it is optically occluded by animal tissue. When coated with a fluorescein payload, the microrobot was shown to release the majority of the payload over a 1-h time period in phosphate-buffered saline. Cytotoxicity tests demonstrated that the microrobot’s constituent materials, SU-8 and polydimethylsiloxane (PDMS), did not show a statistically significant difference in toxicity to murine fibroblasts from the negative control, even when the materials were doped with magnetic neodymium microparticles. The microrobot system’s capabilities make it promising for targeted drug delivery and other in vivo biomedical applications.

scholarly journals Mesenchymal stem cells accelerated growth and metastasis of neuroblastoma and preferentially homed towards both primary and metastatic loci in orthotopic neuroblastoma model

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Jiao-Le Yu ◽  
Shing Chan ◽  
Marcus Kwong-Lam Fung ◽  
Godfrey Chi-Fung Chan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Primary Tumor ◽  
Imaging System ◽  
Ex Vivo ◽  
Tumor Formation ◽  
Human Neuroblastoma Cell ◽  
Fluorescence Signal ◽  
Human Neuroblastoma

Abstract Background Majority of neuroblastoma patients develop metastatic disease at diagnosis and their prognosis is poor with current therapeutic approach. Major challenges are how to tackle the mechanisms responsible for tumorigenesis and metastasis. Human mesenchymal stem cells (hMSCs) may be actively involved in the constitution of cancer microenvironment. Methods An orthotopic neuroblastoma murine model was utilized to mimic the clinical scenario. Human neuroblastoma cell line SK-N-LP was transfected with luciferase gene, which were inoculated with/without hMSCs into the adrenal area of SCID-beige mice. The growth and metastasis of neuroblastoma was observed by using Xenogen IVIS 100 in vivo imaging and evaluating gross tumors ex vivo. The homing of hMSCs towards tumor was analyzed by tracing fluorescence signal tagged on hMSCs using CRI Maestro™ imaging system. Results hMSCs mixed with neuroblastoma cells significantly accelerated tumor growth and apparently enhanced metastasis of neuroblastoma in vivo. hMSCs could be recruited by primary tumor and also become part of the tumor microenvironment in the metastatic lesion. The metastatic potential was consistently reduced in lung and tumor when hMSCs were pre-treated with stromal cell derived factor-1 (SDF-1) blocker, AMD3100, suggesting that the SDF-1/CXCR4 axis was one of the prime movers in the metastatic process. Conclusions hMSCs accelerated and facilitated tumor formation, growth and metastasis. Furthermore, the homing propensity of hMSCs towards both primary tumor and metastatic loci can also provide new therapeutic insights in utilizing bio-engineered hMSCs as vehicles for targeted anti-cancer therapy.

Abstract 13598: The G-protein BetaGamma Subunits Regulate Platelet Function

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Ahmed Alarabi ◽  
Zubair Karim ◽  
Victoria Hinojos ◽  
Patricia A Lozano ◽  
Keziah Hernandez ◽  
...  
Keyword(s):  
G Protein ◽  
Platelet Function ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Inhibitory Effects ◽  
Clot Retraction ◽  
Betagamma Subunits

Platelet activation involves tightly regulated processes to ensure a proper hemostasis response, but when unbalanced, can lead to pathological consequences such as thrombus formation. G-protein coupled receptors (GPCRs) regulate platelet function by interacting with and mediating the response to various physiological agonists. To this end, an essential mediator of GPCR signaling is the G protein Gαβγ heterotrimers, in which the βγ subunits are central players in downstream signaling pathways. While much is known regarding the role of the Gα subunit in platelet function, that of the βγ remains poorly understood. Therefore, we investigated the role of Gβγ subunits in platelet function using a Gβγ (small molecule) inhibitor, namely gallein. We observed that gallein inhibits platelet aggregation and secretion in response to agonist stimulation, in both mouse and human platelets. Furthermore, gallein also exerted inhibitory effects on integrin αIIbβ3 activation and clot retraction. Finally, gallein’s inhibitory effects manifested in vivo , as documented by its ability to modulate physiological hemostasis and delay thrombus formation. Taken together, our findings demonstrate, for the first time, that Gβγ directly regulates GPCR-dependent platelet function, in vitro and in vivo . Moreover, these data highlight Gβγ as a novel therapeutic target for managing thrombotic disorders.

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