bluuebunny/crossref_metadata_2025_split huggingface dataset:
Megakaryocyte-Derived Microparticles: Mechanism of Formation and Circulation in Plasma.
Authors: Flaumenhaft Robert , Dilks James R. , Patel Sunita R. , Klement Giannoula , Italiano Joseph E. 
Published: October 2019

Abstract
Platelet microparticles are submicron membrane vesicles expressing platelet markers and are a normal constituent of circulating blood. Several studies have demonstrated positive correlations between thrombotic states and increased platelet microparticle levels. Yet the physiology of these microparticles is poorly understood. Specifically, the origin of circulating platelet microparticles is not known. Platelet microparticles are routinely formed in vitro following exposure of platelets to pharmacologic concentrations of platelet agonists such as the combination of thrombin and collagen. This observation has lead to the widely held assumption that circulating platelet microparticles are derived from activated platelets. We now have identified and characterized platelet microparticles derived directly from megakaryocytes. Videomicroscopy of microparticle production by mouse megakaryocytes demonstrated an active process in which microparticles form as submicron beads along the lengths of thin, extended pseudopods. These pseudopods were morphologically distinct from the broad, branching cytoplasmic extensions from which proplatelets form. Studies using cytoskeletal inhibitors confirmed that megakaryocytes produce microparticles and platelets by two distinct mechanisms. The microtubule inhibitor nocodazole (5 μM) totally abolished proplatelet formation, but failed to affect microparticle formation. Latrunculin A (5 μM), which depolymerizes F-actin, inhibited proplatelet branching, but resulted in a 2-fold increase in megakaryocyte-derived microparticle formation. Megakaryocyte-derived microparticles had an average diameter of 0.5 μm. These microparticles were CD41+ and bound annexin V, indicated surface phosphatidylserine exposure. To determine whether plasma microparticles in wild-type mice are derived primarily from activated platelets or generated from megakaryocytes, we identified markers that distinguish between these two populations. CD62 and CD63 were found only on microparticles generated by activated platelets. In contrast, full-length filamin A was found in megakaryocyte-derived microparticles, but not in microparticles from activated platelets. Circulating microparticles isolated from mice were CD62−,CD63−, and expressed full-length filamin A indicating a megakaryocytic origin. Similarly, circulating microparticles isolated from healthy volunteers were CD62−,CD63−, and expressed full-length filamin A. These results indicate that direct production by megakaryocytes represents a physiologic means to generate circulating platelet microparticles. We propose that circulating platelet microparticles can be derived from two sources. One source is the mature platelet, from which microparticles are generated following platelet activation in pathological conditions. The second source is megakaryocytes, which produce microparticles in a constitutive manner.
Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.
Authors: Jobe Shawn M. , Wilson Katina M. , Leo Lori , Molkentin Jeffery D. , Lentz Steven R. , Di Paola Jorge 
Published: October 2019

Abstract
Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation includeincreased surface retention of procoagulant alpha granule proteins,high-level phosphatidylserine (PS) externalization, andmodulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A.
Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨm), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H2O2 (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05).
Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses.
We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.
PKCδ Regulates Platelet Activity through the Inhibition of VASP-Mediated Filopodia Formation.
Authors: Pula Giordano , Schuh Kai , Walter Ulrich , Nakayama Keiichi I. , Nakayama Keiko , Poole Alastair W. 
Published: October 2019

Abstract
Filopodia are finger-like protrusions that play a crucial role in the guidance of cells through their extracellular and cellular environment. Blood platelets provide an essential early response system in haemostasis and arterial thrombosis and display a rapid and dramatic generation of filopodia upon activation, which play an essential role regulating their adhesive events leading to platelet aggregation. Here we report a novel and unexpected control mechanism for this explosive cellular rearrangement. Using pharmacological and genetic approaches we show that the novel isoform of protein kinase C, PKCδ, is a critical negative regulator of actin polymerization, filopodia formation and platelet aggregation. We show that PKCδ interacts with vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics, and regulates VASP phosphorylation. VASP is shown to be necessary for the PKCδ-dependent regulation of actin polymerization, filopodia formation and platelet aggregation. We therefore propose a causative link between the functional interaction of PKCδ with VASP and the role of the kinase as modulator of platelet activity.
Characterization of the Mechanism of Interaction between C-Reactive Protein and GPIIb/IIIa.
Authors: Brennan Marian P. , Moriarty Roisin D. , Arasu Stephanie , Foster Timothy J. , Cox Dermot 
Published: October 2019

Abstract
C-reactive protein (CRP) is a well established marker for inflammation, and a good predictor of coronary heart disease. It is also known to interact with the platelet FcγRIIa and to enhance the inhibition of platelet aggregation by aspirin by an unknown mechanism. CRP has also recently been demonstrated to compete for PAC-1 binding in collagen stimulated platelets, suggesting that CRP interacts with GPIIb/IIIa. In order to study the mechanism of interaction with platelets directly, we carried out platelet adhesion assays. We coated plates with recombinant CRP which was in the native pentameric form as confirmed by size exclusion chromatography. Platelets adhered to immobilized CRP and to immobilized fibrinogen to a similar extent. Adhesion to CRP and fibrinogen was inhibited by GPIIb/IIIa antagonists but not by antibody to the FcγRIIa (IV.3). Platelet adhesion to CRP was increased 5-fold when platelets were treated with 1 mM MnCl2. Adhesion of MnCl2 treated platelets was also inhibited by GPIIb/IIIa antagonists. When viewed by confocal microscopy, the adherent platelets displayed pseudopodia, but did not spread fully. Treatment of platelets with MnCl2 stimulated lamellipodia formation and full platelet spreading on CRP. Analysis of the exposed residues on the surface of the CRP crystal structure identified two RGD-like sequences, RQD and DGK. The peptides KRQDN and VDGKP derived from CRP inhibited platelet adhesion to fibrinogen suggesting that these sequences could be responsible for the interaction with GPIIb/IIIa. Our data demonstrates that CRP in the native pentameric form interacts with GPIIb/IIIa and that it has an increased affinity for the activated conformation of GPIIb/IIIa. This interaction is likely to be due to an interaction with the surface exposed KRQDN and VDGKP CRP sequences. We have previously demonstrated that CRP acts as a GPIIb/IIIa antagonist when in solution. Furthermore, pentameric CRP cannot support platelet adhesion under shear conditions. Thus it is likely that elevated circulating levels of soluble CRP reduces the overall thrombotic potential.
Charaterization of Human Primitive Megakaryocytes and “Platelets”.
Authors: Chen Dong , Lewis Rachel L. , Daigh Christine A. , Thomson James A. 
Published: October 2019

Abstract
Primitive megakaryocytes (PMs) have been first observed in murine embroynal yolk sac [Xu et al. Blood. 2001 Apr 1;97(7):2016–22.]. The biological characteristics and functional aspects of human primitive megakaryocytes are not well established even though they can be transiently observed by bone marrow stromal cell (OP9) co-culture methodology [Gaur, M et al J Thromb Haemost.2006 Feb;4(2):436–42.; Eto K et al. Proc Natl Acad Sci U S A.2002 Oct 1;99(20):12819–24.]. The goal of this study is to charaterize PMs’ unique biological and functional features. To obtain a pure population of human primitive megakaryocytes, human embryonic stem (hES) cells were cultured to form embryoid bodies.
CD34+/CD31+/VE-Cadherin+/Flk-1+ hemangioblasts on day 12 were isolated and further cultured for 10–12 days in media containing thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor (SCF). 30% of the non-adherent cells were strongly positive for CD31/CD41/CD61/CD42b, and weakly positive for CD42a/CD45/beta-2-microglobulin (B2M) but negative for CD117 or HLA-DR. The DNA ploidies are exclusively 2N–8N confirming the primitive status of PMs. Morphologically when compared to bone marrow derived megakaryocytes, PMs readily produce long processes with chains of “proplatelet” like structures. By live videomicroscopy, these “proplatelet” structures can actively recruit mitochondria. Interestingly, “platelet-like” fragment shedding from megakaryocytes is significantly stimulated in the presence of nitric oxide and human plasma. The “platelet-like” fragments and megakaryocytes respond to strong agonists, including thrombin and TRAP, with aggregation, alpha granule release and increased affinity for fibrinogen. In contrast, weaker agonists such as ADP only generate minimal response. This study provides preliminary evidence that primitive megakaryocytes have acquired some basic machinery of hemostasis even when the blood flow, liver function and coagulation factors are not present at the early phase of embryonal development. [We thank Dr.William L. Nichols at Mayo Clinic for his critical reviews of this abstract.]
Arrestin-2 Regulates Akt Activation by G Protein-Coupled Receptors in Platelets.
Authors: Li Dongjun , Woulfe Donna S. 
Published: October 2019

Abstract
Arrestins have been shown to play important roles in G Protein-Coupled Receptor (GPCR) function in many cells, but their roles in platelets remain uncharacterized. While the classical role of arrestins is considered to be the internalization and desensitization of GPCRs, more recent studies suggest that arrestins can serve as scaffolds to recruit phosphatidyl inositol-3 kinases (PI3K)s to Gq-coupled receptors and promote PI3K-dependent signaling. Thrombin stimulates the PI3K-dependent activation of Akt in platelets in a Gq-dependent manner. Therefore, we sought to determine whether arrestins are involved in the PI3K-dependent activation of Akt in platelets. Comparative immunoblots show that of the two non-visual mammalian arrestins, only one, arrestin-2 (β-arrestin-1), is expressed in human and mouse platelets. Immunoprecipitation of arrestin-2 or p85-PI3K from platelet lysates demonstrated that arrestin-2 associates with the p85 subunit of PI3Ka/b in thrombin or ADP-stimulated platelets, but not resting cells. The association can be inhibited by inhibitors of the P2Y12 receptor for ADP, but not by P2Y1 inhibitors. p85-arrestin association is also blocked by inhibitors of src family kinases, as is Akt phosphorylation. To determine whether src family members were part of the p85-arrestin complexes, immunoblots were re-probed with antibodies to src, lyn and fyn. The results show that Lyn is incorporated into thrombin-stimulated arrestin complexes in a P2Y12-dependent manner. To determine whether arrestin-2 is important for Akt phosphorylation in platelets, megakaryocytes differentiated in culture from mouse embryonic stem cells were used as models of platelet signaling, since these cells are amenable to genetic manipulation. Arrestin-2 was inhibited in the cultured megakaryocytes using a siRNA approach, then cells were stimulated with thrombin and Akt phosphorylation was assessed by immunoblotting. Arrestin-2 expression in the cultured megakaryocytes treated with arrestin-2 specific siRNA was suppressed by an average of 53% compared to cells treated with scrambled siRNA, while thrombin-stimulated Akt phosphorylation was suppressed by 98% compared to scrambled siRNA-treated control cells (n=3 experiments, difference is significant, p=.01, unpaired student’s t-test). In conclusion, the results show that arrestin-2, lyn and PI3Kform a tri-molecular complex following stimulation of platelets with ADP or thrombin. Formation of arrestin complexes at activated receptor sites is important for the localized recruitment and src-dependent activation of p85-PI3K, thus promoting activation of Akt by G protein-coupled receptors.
Computational Design of a Model for the Platelet Integrin αIIbβ3.
Authors: Metcalf Douglas G. , DeGrado William F. , Bennett Joel S. 
Published: October 2019

Abstract
Integrins, the principal receptors linking the cytoskeleton to extracellular ligands, are responsible, in large part, for platelet adhesion and aggregation. Like many membrane spanning proteins, the molecular structure of integrins has been difficult to determine, although crystal and NMR structures are available for water soluble portions of αVβ3 and αIIbβ3. In addition, models for the αIIb and β3 transmembrane (TM) heterodimer have been obtained using computational methods, but with conflicting results. Accordingly, we employed two different modeling strategies to distinguish between existing models. First, we used a Monte Carlo-simulated annealing (MCSA) protocol that features a selective advantage for structures that are consistent with previously characterized αIIb and β3 TM domain point mutations. During each step of the protocol, we calculated the effects of selected mutations and assigned a penalty to the step if it produced a structure that was inconsistent with experimental results. Second, we threaded the αIIb and β3 TM sequences onto a database of membrane helix dimers and rank-ordered the structures based on their calculated energies and their agreement with the cysteine crosslinking experiments reported by Luo et al (PLoS Biol 2:776–86, 2004). The MCSA and threaded TM models were similar with a root mean squared deviation (RMSD) of 1.5 Å. Moreover, they differed from a model reported by Kay Gottschalk (Structure 13:703–12, 2005) most notably by an ~50° rotation of the αIIb TM helix about its helical axis. This positions αIIb residue Leu980 in the TM interface and in van der Waals contact with β3 residue Gly708, a result consistent with mutagenesis experiments. Further, the αIIb and β3 TM helices pack along conserved glycine residues (Gly972 and Gly976) in the GXXXG motif of αIIb with a right-handed crossing angle of −18°. To create a model for the full-length integrin, the N-termini of the TM model were connected to the crystal structure of the αVβ3 extracellular domain and its C-termini were connected to the corresponding NMR structures of the αIIb and β3 cytosolic domains. The model predicts an αIIb Arg995-β3 Asp723 salt bridge in the membrane proximal cytosolic domains and a change in the secondary structure of the extracellular domains from an extended to a helical conformation that occurs at Pro965 in αIIb and Pro693 in β3, just proximal to the TM domains. Finally, the full length model provides insight into distal interactions since its extracellular portion is held close to the membrane and is therefore sterically inaccessible to large ligands such as fibrinogen, whereas cytosolic regulatory sites such as the talin binding site in β3 are accessible and not sequestered by α/β interactions. In conclusion, we have modeled the αIIbβ3 TM heterodimer using two fundamentally different computational strategies, both of which converge upon similar structures that were used to construct a model for the full-length integrin.
The Cytoplasmic Domain of Glycoprotein Ibβ Is Critical to Surface Trafficking of Glycoprotein Ib-IX Complex.
Authors: Mo Xi , Lopez Jose A. , Li Renhao 
Published: October 2019

Abstract
Platelet glycoprotein (GP) Ib-IX-V complex mediates platelet adhesion to the subendothelium of damaged vessel walls under high shear conditions. Delineating the assembly process of the GP Ib-IX complex will aid our understanding of the complex structure and shed light on the signaling mechanism underlying platelet activation. The GP Ib-IX complex comprises three polypeptides, GP Ibα, GP Ibβ and GP IX, and efficient surface expression of the complex in transfected Chinese hamster ovary (CHO) cells requires all of its three subunits, indicating that the assembly of the complex in the endoplasmic reticulum is required for its subsequent trafficking to the plasma membrane. We recently showed that the interaction between the transmembrane domain of GP Ibβ and the other subunits is critical to efficient surface expression of the GP Ib-IX complex. Here, we have explored the role of the Ibβ cytoplasmic domain in the complex assembly and surface expression. In CHO cells transiently expressing the mutant Ib-IX complex in which the Ibβ cytoplasmic domain was deleted or replaced entirely with the IX counterpart, neither cellular expression of GP Ibα nor surface expression of GP Ibα and IX was detected. In contrast, deletion and/or replacement of the Ibα or IX cytoplasmic domains did not affect significantly the assembly and surface expression of the receptor complex. Furthermore, deletion of the last six residues in the Ibβ cytoplasmic domain did not affect the cellular expression level of GP Ibα but significantly decreased its surface expression level in CHO cells. Intriguingly, further deletion of the Ibβ cytoplasmic domain that removed the binding site for 14-3-3ζ restored surface expression of the GP Ib-IX complex to a level comparable to the wild type construct. Overall, our results demonstrated an important role of the Ibβ cytoplasmic domain in both assembly and trafficking of the GP Ib-IX complex. Critical residues in the Ibβ cytoplasmic domain have been identified and further characterization is currently underway.
Hepcidin Suppression Following Phlebotomy Is Regulated by an Erythropoietic Regulator Found in Serum.
Authors: Rivera Seth , Pak Mihwa , Lopez Miguel A. , Gabayan Victoria , Ganz Tomas 
Published: October 2019

Abstract
Increasing erythropoiesis requires a dramatic increase in iron absorption or the release of iron from tissue stores. Since the iron regulatory hormone hepcidin blocks both absorption and release from stores, it is suppressed during erythropoiesis. We have recently shown that hepcidin is primarily suppressed by an erythropoietic regulator rather then anemia, hypoxia, or erythropoietin (Pak et al, Blood, epub/in press). To further understand the regulation of hepcidin during erythropoiesis, we subjected groups of eight mice to phlebotomy and sacrificed them at various time points over 3 weeks. Hepcidin mRNA fell 25-fold by day 2 (p<0.001) and did not begin to rise until day 9. Hemoglobin rose well before hepcidin mRNA began to increase confirming that anemia was not the erythropoietic regulator. Serum iron never fell, excluding this as the erythropoietic regulator. Erythropoietin levels spiked early but returned to normal well before hepcidin. Reticulocyte counts increased shortly after the fall in hepcidin and remained elevated after hepcidin mRNA returned to normal. We therefore postulate that the erythropoietic iron regulator is either made by or in concert with early erythroid progenitor cells. Using primary hepatocytes treated with serum from erythropoietin stimulated or control mice, we also demonstrate that the erythropoietic regulator of hepcidin expression is present in the circulation. Experiments are underway to isolate the erythropoietic regulator of hepcidin from serum.
Rapamycin Analogs Reduce mTORC2 Signaling and Inhibit AKT Activation in AML.
Authors: Zeng Zhihong , Sarbassov Dos D. , Giles Francis J. , Samudio Ismael , Yee Karen W.L. , Jackson C. Ellen , Sabatini David M. , Andreeff Michael , Konopleva Marina 
Published: October 2019

Abstract
The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin-insensitive, and was recently shown to regulate the pro-survival kinase AKT by phosphorylation on Ser473 (Sarbassov Science 2005;307 and Mol Cell 2006;22). We investigated the molecular effects of mTOR inhibition by rapamycin analog CCI-779 in AML cells. Unexpectedly, CCI-779 not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased phosphorylation of p70S6K, 4EPB1 and reduction in Glut-1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-Cyclins in AML cell line and 5 of 8 primary AML samples in vitro. Similar observations were made in samples from patients with hematological malignancies who were treated with the rapamycin analogs temsirolimus or everolimus: the levels of Ser473 phosphorylated AKT decreased in 3/5 patient samples at 1 or 24 hour(s) of temsirolimus treatment, and in 6/8 patient samples treated with everolimus. In the 9 samples in which AKT was inhibited, ≥2-fold decrease in Cyclin D1 mRNA was observed in 5, Cyclin D2 in 3, both, Cyclin D1 and D2 in 1 sample, and Glut-1 in 4 patient samples. In 7 of the 9 patients in whom AKT was inhibited, a >50% decrease in peripheral blood absolute blast count (3 AML, 1 ALL) or absolute lymphocyte count (1 CLL) for >1 week duration was documented, and two patients with RAEB-1 had improvements in platelet counts, one fulfilling the criteria for clinical response (hematological improvement). No change in peripheral blood counts or progression of leukemia was seen in 6 patients. Of these, decrease in pAKT was observed in 2, no change in 3 and increase in 1 (Fisher exact two-tailed p= 0.021). Altogether, our study provides first evidence that rapamycin analogs inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias.