bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protease-Activated Receptor 3 Negatively Regulates Plasmin-Mediated Protease-Activated Receptor 4 Activation in Platelets.
Authors: Mao Yingying , Jin Jianguo , Kunapuli Satya P. 
Published: April 2020

Abstract
Plasmin, a major extracellular protease, causes intracellular signals to mediate platelet aggregation. Previously, we reported that plasmin-mediated platelet aggregation predominantly occurs through proteolytic cleavage of protease-activated receptor 4 (Quinton et al J. Biol. Chem 2004). We showed that plasmin caused aggregation of mouse platelets more readily than human platelets. In this study, we investigated the mechanism of such a differential sensitivity of mouse platelets to plasmin, using transfected cell lines and platelets. In the platelet system, plasmin caused both human and mouse platelets to shape change and aggregate in a concentration-dependent manner with a different efficiency. Whereas 0.1 U/ml of plasmin causes full aggregation of mouse platelets, that dose only induces shape change in human platelets. In transfected COS7 cells, 1 U/ml plasmin caused a higher intracellular calcium mobilization through mouse PAR4 (mPAR4) than human PAR4 (hPAR4) activation. These results indicate that the mPAR4 is primarily more readily activated by plasmin than hPAR4, possibly due to differences in the primary sequence. Further, mouse and human platelets also differ in terms of the expression of PAR3. It is known that PAR3 acts as a co-receptor for thrombin-induced PAR4 activation. In order to evaluate the contribution of mouse PAR3 to plasmin-mediated mPAR4 activation, we co-expressed mPAR3 and mPAR4 in COS7 cells. Plasmin caused lower mobilization of intracellular calcium when mPAR3 and mPAR4 are co-expressed, compared to the expression of mPAR4 alone. These results indicate that mPAR3, instead of acting as a cofactor to potentiate mPAR4 activation, inhibits plasmin-mediated mPAR4 activation. Consistent with these results, PAR3 null mouse platelets also show a greater plasmin-induced calcium mobilization and aggregation compared to wild-type mouse platelets, which express PAR3 and PAR4. In conclusion, mouse platelets are more readily activated by plasmin than human platelets due to differences in the primary sequence of PAR4. In addition, mPAR3 acts as an inhibitory receptor to plasmin-mediated PAR4 activation, instead of acting as a co-receptor.
P-REX1: A Novel RAC Activing Guanine-Nucleotide Exchange Factor in Human Platelets.
Authors: Morgan-Spencer Alex , Greenberg Daniel 
Published: April 2020

Abstract
A major signaling pathway that regulates platelet shape change and reorganization of the cytoskeleton involves the Rho family of GTPases whose members include Rac, CDC42 and RhoA. These GTPases are converted from their inactive or GDP-loaded state to the active or GTP-loaded state by a class of enzymes called Guanine-nucleotide Exchange Factors (GEFs). GEFs are a family of multi-domain proteins that contain a GDP-GTP exchange domain (DH-PH) as well as other protein interacting domains that are regulated by the activation of receptors present on the platelet surface. We used an affinity binding technique followed by mass spectrometry analysis to identify novel Rac binding GEFs from platelet lysates. Recombinant GST-Rac fusion proteins bound to agarose beads were prepared in the GTP, GDP and nucleotide-free states and incubated with human platelet lysates. Platelet lysate proteins associated with the different GST-Rac preparations (GTP-bound, GDP-bound or nucleotide-free) were eluted and run on SDS-PAGE. Gel slices were then cut out, trypsin digested and analyzed by mass spectrometry. Platelet GEFs were identified by the presence of a DH-PH domain. Using this technique we identified three novel Rac-associated platelet GEFs including P-REX1, a G-βγ protein and phosphatidylinositol (3,4,5)-triphosphate regulated GEF previously found in neutrophils and neurons (Welch, H.C.E et al., Cell108;809–822, 2002). PREX-1 is a 196kDa peptide composed of 1659 amino that specifically activates Rac in neutrophils. In addition to the DH-PH exchange domain, PREX-1 also contains tandem PDZ and DEP domains as well as significant similarity over its C-terminal half to Inositol Polyphosphate 4-Phosphatase. Western analysis with anti-P-REX1 antibody (gift of H.C. Welch, Barbraham Institute, U.K.) showed that PREX-1 is present in purified whole platelet lysates. We have also shown that platelet PREX-1 specifically associates with GST-Rac by affinity pull-down experiments. Functional studies to characterize the exchange activity of platelet PREX-1 are ongoing. PREX-1 is the only known GEF that is directly regulated by G-βγ protein (Hill, K. et al., J Biol Chem280(6):4166–4173, 2005) and represents a novel pathway for platelet G protein coupled receptor signaling through Rac in human platelets.
Removing the Covalent Links between GP Ibα and GP Ibβ Hampers Binding of von Willebrand Factor to GP Ib-IX Complex in CHO Cells.
Authors: Mo Xi , Luo Shi-Zhong , Peng Yuandong , Munday Adam D. , Sun Carol , Berndt Michael C. , Lopez Jose A. , Dong Jing-fei , Li Renhao 
Published: April 2020

Abstract
In the glycoprotein (GP) Ib-IX complex, GP Ibα contains the binding region for its ligand von Willebrand factor (vWF). How the other subunits in the complex, GP Ibβ and GP IX, regulate the GP Ibα-vWF interaction is not clear. Since GP Ibα connects with two GP Ibβ subunits via disulfide bonds, these inter-subunit covalent links may be important to the proper vWF-binding activity of the GP Ib-IX complex. To test this idea, we have obtained Chinese hamster ovary cells stably expressing the mutant GP Ib-IX complex (CHOαSSβIX) in which both residues C484 and C485 of GP Ibα were changed to Ser. As expected, the mutant GP Ibα did not form any disulfide bonds with GP Ibβ in these cells. Nonetheless, co-immunoprecipitation experiments in the digitonin-containing buffer demonstrated that association of GP Ibα with GP Ibβ and GP IX was retained in CHOαSSβIX cells. The expression level of GP Ibα in CHOαSSβIX cells, detected by its binding to WM23, a conformation-insensitive antibody, was comparable to that in the transfected cells stably expressing the wild type GP Ib-IX complex (CHOαβIX). In contrast, binding of CHOαSSβIX cells to AK2, SZ2 or AN51, all of which are GP Ibα-specific conformation-sensitive antibodies, was significantly different from that of CHOαβIX cells. Compared to CHOαβIX cells, ristocetin-induced binding of CHOαSSβIX cells to vWF under static conditions was reduced by about 50% as determined by flow cytometry. Consistently, rolling of CHOαSSβIX cells on the vWF-coated glass slide was significantly faster than CHOαβIX cells under various flow conditions. Thus, in a transfected cell model, removing the disulfide links between GP Ibα and GP Ibβ in the GP Ib-IX complex leads to an alteration in the conformation of the ligand-binding domain in GP Ibα and as a consequence hampers the complex binding to vWF. Our finding suggests that the relative position of GP Ibα and GP Ibβ in the receptor complex is crucial to its vWF-binding activity, which may be exploited to modulate the GP Ibα-vWF interaction.
CPI-17 Contributes to Calcium-Independent ADP-Induced Platelet Shape Change through P2Y1-Gq-PKC Pathways.
Authors: Getz Todd M. , Bhavaraju Kamala , Kunapuli Satya P. 
Published: April 2020

Abstract
The initial event in platelet activation is the reorganization of the cytoskeleton causing the platelets to change from a discoid to a spiculated spheroid shape. Platelet shape change is primarily regulated by the phosphorylation of myosin light chain kinase. We have shown that this process is mediated through both calcium-dependent and calcium-independent Rho kinase pathways. CPI-17, a Protein kinase C (PKC) phosphorylated inhibitory protein of myosin light chain phosphatase, has been shown to have a role in platelet shape change downstream of thrombin-induced platelet activation. CPI-17 is a 17 kDa protein expressed in human platelets shown to inhibit myosin light chain phosphotase activity via PKCs. In this study we examined the role of CPI-17 in ADP-induced shape change and phosphorylation of CPI-17, downstream of the Gq coupled, P2Y1, and the Gi coupled, P2Y12 receptors. CPI-17 phosphorylation occurred upon activation of platelets with 2MeSADP. This phosphorylation was abolished in the presence of the P2Y1 receptor antagonist, MRS-2179. These results indicated that Gq signaling is important for platelet shape change and phosphorylation of CPI-17. In the presence of the calcium chelator, BAPTA, platelets changed shape in response to 2MeSADP; CPI-17 phosphorylation, however, was unaffected by BAPTA treatment under these conditions. However, CPI-17 phosphorylation was inhibited in the presence of the pan PKC inhibitors. These results indicate that CPI-17 phosphorylation occurs downstream of PKC activation. In the presence of BAPTA, treatment with PKC inhibitors decreased platelet shape change possibly due to reduced CPI-17 phosphorylation. The shape change caused by p160ROCK downstream of G12/13 pathways was unaffected by pan PKC inhibitors, but abolished by p160ROCK inhibitors H1152 or Y27632. Platelets incubated with BAPTA, pan PKC inhibitors, and p160ROCK inhibitor H1152, abolished ADP-induced platelet shape change and CPI-17 phosphorylation. In conclusion, ADP-induced platelet shape change occurs through a Gq-mediated, calcium-independent signaling pathway regulated by CPI-17 phosphorylation via PKC activation.
Erythrocytosis Caused by Mutations in the PHD2 and VHL Genes.
Authors: Percy Melanie J. , Furlow Paul W. , Jones Frank G.C. , Lappin Terry R.J. , Lee Frank S. , McMullin Mary Frances 
Published: April 2020

Abstract
Human diseases have provided important insights into the function of fundamental physiological processes, and studying erythrocytosis has increased our understanding of the oxygen sensing pathway. In some cases this rare disorder arises specifically from dysregulation of the erythropoietin (Epo) axis. Epo gene transcription is under the control of a negative feedback mechanism involving the kidneys, which sense tissue hypoxia at the molecular level via the hypoxia inducible factor (HIF) transcription complex. Both subunits of the HIF complex, alpha and beta, are constitutively expressed but only the beta subunit is detectable in the presence of oxygen. Key prolines in the oxygen degradation domain of HIFalpha are hydroxylated by a family of prolyl hydroxylase enzymes, PHD1-3. Of these PHD2 has the most widespread tissue distribution. Upon hydroxylation of HIF, the von Hippel Lindau (VHL) protein, a component of an E3 ligase complex, promotes the ubiquitination of the alpha subunit. HIFalpha is then targeted to the proteasome and the transcription of HIF target genes is prevented. In hypoxia the activity of the PHD enzymes is low and the HIF complex assembles causing upregulation of genes such as Epo. Over the last ten years we have maintained a registry of individuals with erythrocytosis. To date 181 patients have been included with clinical details and consented DNA samples have been collected. There is a preponderance of males with ratio of 1.7 males to each female. The mean age of erythrocytosis individuals on the registry is 37 years. The majority of erythrocytosis patients have inappropriately normal (46%) or raised (26%) serum Epo levels as compared to PV, where the Epo level is often undetectable. Thus it can be inferred that dysregulation of the Epo axis via the oxygen sensing pathway would be a significant cause of erythrocytosis. Consequently, PHD2 and VHL have been investigated. We have now identified 2 different mutations, Pro317Arg and Arg371His, in PHD2. In the tertiary structure of PHD2, Pro317and Arg371 are close to essential iron binding residues and furthermore suggest the location of a HIF binding groove. The Arg200Trp VHL mutation, commonly described as the Chuvash mutation, has been detected in 8 Asian families and their large kindred. Two Western European individuals were found to be compound heterozygous for the Arg200Trp and either the novel Pro192Thr or Gly144Arg variants. Intriguingly 2 further individuals possess one wild type and one mutated allele, Arg200Trp or Leu188Val. Screening other genes of the oxygen sensing pathway has failed to reveal further defects in these individuals. In summary, defects in the oxygen sensing pathway are associated with the development of erythrocytosis. Mutations in VHL are more common than PHD2 and highlight the importance of these proteins in the maintenance of red cell homeostasis.
Autologous Bone Marrow Mononuclear Cell (ABMMC) Transplantation in Type 1 and Type 2 Insulin Dependent Diabetes Mellitus (IDM).
Authors: Carrasco-Yalán Antonio A. , Fernandez-Vina Roberto , Castillo-Aguirre Jorge , Rios Hugo , More L. , Gomez N. , Chirino Samuel , Alvarez Marcos , Aranda M. , Arroyo M. , Rafael J. , Patel A.N. , Tuma-Mubarack Jorge 
Published: April 2020

Abstract
Background: Recent reports have shown that bone marrow-derived stem cell may contribute to islet regeneration. The goal of our study was to evaluate the safety and efficacy of ABMMC transplantation for patients with IDM.
Methods: From June 2005 to January 2007, 28 consecutive patients: 8 Type 1 IDM (T1DM) and 20 Type 2 IDM (T2IDM); who were receiving maximal medical therapy including insulin treatment for 5 years before enrollment. Median time of disease for T2IDM patients was 13 years, without pancreatic islet auto-antibodies. After IRB approval and signed informed consent, bone marrow was harvested and ABMMC were isolated and infused directly into the pancreas via splenic artery using endovascular catheters. Glucose, glycosylated HbA1c and C peptide were measure before and after transplantation. HOMA2 Calculator v2.2 was used to calculated IR and % B (*if Glucose: 3.0 to 25.0 mmol/L and C peptide: 0.2 to 3.5 nmol/L).
Results: There were no study related complications. At 1 year follow-up, mean daily insulin requirement was the same in group T1DM and significantly reduced in group T2IDM, from 42.5 to 4.5 U/d (t=7.94, p&lt;0.001). Ten of the twenty (50%) T2IDM established complete insulin independence. Data in table 1.
Table 1: Median values PRE POST t p T2IDM (n=20) Fasting Glucose (mmol/L) 10.8 6.6 3.98 0.01 Glycosylated HbA1c (%) 9.6 8.1 3.98 0.01 C peptide (nmol/L) 0.5 0.84 5.11 &lt; 0.01 HOMA 2 IR (n=17)* 2.2 2.26 0.94 0.92 HOMA 2 %B (n=17)* 42.4 130.2 4.9 &lt; 0.01 T1DM (n=8) Fasting Glucose (mmol/L) 10.1 11.1 1.382 0.21 Glycosylated HbA1c (%) 8.7 8.7 0.45 0.66 C peptide (nmol/L) 0.17 0.16 1.00 0.35
Conclusions: The use of ABMMC transplantation for T1DM and T2IDM is safe. In this pilot study, only T2IDM patients have significant improvement in pancreatic function demostrated by better glycemic and HbA1c control, and are associated with a significant independence of the insulin. This has formed for a randomized multi-center study which is currently in progress.
Differentiation Hotspots on a Cell Cycle Related Continuum.
Authors: Colvin Gerald A. , Berz David , Dooner Mark S. , Dooner Gerri J. , Johnson Kevin , Aliotta Jason M. , Quesenberry Peter J. 
Published: April 2020

Abstract
Directed differentiation is defined as the ability to program a stem cell at the most primitive level while it still has its reproductive and full proliferative potential. This is in contrast to ex-vivo expansion where the stem cells are forced into specific lineage commitments, limiting the overall therapeutic utility. We have demonstrated differentiation “hotspots” on a cell cycle continuum (Exp Heme35:96, 2007). In this work we showed marked but reversible increases in differentiation potential to megakryocyte and granulocytes at different phases of a single cytokine induced cell cycle passage of highly purified quiescent murine lineagenegative rhodaminelowHoeschtlow (LRH) marrow stem cells. We have reproducibly induced directed stem cell differentiation by capitalizing on inherent changes in sensitivities to inductive cytokine signals in the context of cell cycle position. These cells, when exposed to thrombopoietin, FLT3-ligand and steel factor, synchronously pass through cell cycle. We have found that using a differentiation cytokine cocktail of G-CSF at 0.075ng/ml, GM-CSF at 0.0375ng/ml and steel factor at 50ng/ml, we were able to see enhanced megakaryopoiesis occurring 14-days after culture in those LRH stem cells that were in early to mid S-phase at time of inductive signaling. We have now shown that a megakaryocyte hotspot clusters around 32 hours; the G1/S interface, and that dramatic reversible changes in differentiation potential occur over one hour time intervals. We have confirmed this data by looking at LRH cells through cell cycle transit after initial cell division showing that a megakaryocyte hotspot occurs in two sequential cell cycles and still tied to S-phase at time of inductive signaling of the daughter cells. This hotspot has been demonstrated on a clonal basis, although the kinetics of the hotspot shifts when clonal as opposed to population studies are carried out. An important issue is whether in vitro cytokine exposure, separate from cell cycle status, determines the existence of the hotspot. To address this, we used Hoechst 33342 dye content to assist in separation of different cell cycle fractions (G0–1, early, mid and late components of S, G2/M) of lineage negative Sca-1+ stem cells, a cycling stem/progenitor cell population in which approximately 20% of the cells are in S-phase at isolation. These cells were only exposed to the differentiation cytokines and showed a megakaryocyte hotspot present in only early S-phase cells after 14-days of culture, showing that in vitro cell cycle phase determined the presence of the hotspot, separate from cytokine exposure. These data indicate that differentiation potential of marrow stem cells exists on a cell cycle related continuum and that this potential can be demonstrated on a single cell basis. This suggests a continuum model of stem cell regulation at the stem cell level as opposed to a pure hierarchical model.
Hepatocyte Differentiation by Highly Purified CD34+/CD34− Blast Cells from G-CSF and SCF Primed Bone Marrow from Rhesus Monkey.
Authors: Xia Xiujin , La Russa Vincent F. 
Published: April 2020

Abstract
Hematopoietic stem cells and hepatic stem cells share characteristic markers such as CD34, CD117(c-kit) and CD90 (Thy-1). In this study, we investigated the potential of primitive CD34+/CD34− blast cells to differentiate into hepatocytes in vitro. Rhesus monkeys were administered granulocyte colony stimulating factor, G-CSF, combined with stem cell factor, SCF, the ligand for c-kit receptor, for 3 days. This novel G-CSF and SCF combined cytokine treatment is believed to stimulate the self renewal of primitive blast cells in the bone marrow. Blast cells were purified by a novel technique that combines purging of T-cells and other immune-competent cells that express the Fc receptor. The immuno-depleted mononuclear fraction was labeled with biotin-conjugated SKH6.1 anti-CD34 monoclonal antibody belonging to the class-I epitope. The SKH 6.1 recognizes a heterogeneous blast cell population in rhesus bone marrow that consists mainly of approximately 80% CD34+ and a CD34− small blast cell minor population that also shares low forward and side scatter properties. Blast cells were cultured on Matrigel in a defined medium containing hepatocyte growth factor (HGF), epidermal growth factor (EGF) and fibroblast growth factor-4(FGF-4), and fed every 3–4 days. Conditioned medium was analyzed at weekly intervals for four weeks. ELISA assays were used to detect de-novo synthesis of rhesus monkey specific albumin, and urea production was measured using a quantitative colorimetric assay. Peak production of albumin occurred at 7-days and then began to continuously decrease through week 4, while urea was detected at 7-days but continued to increase through the end of the 4th week. The hepatocyte-like cells were also shown to produce significant intra-cellular glycogen as detected by periodic acid-schiff staining by their 4th week in culture. Hepatocyte function was also characterized by their ability to uptake Dil-Ac-LDL as detected by their strong fluorescence on the 4th week of culture. Our results demonstrated that G-CSF and SCF stimulate the bone marrow by increasing the frequency CD34+ / CD34− blast cells that are able to differentiate into hepatocyte-like cells.
Thrombospondin-1 as a Modulator of ADAMTS13 Activity.
Authors: Palla Roberta , Rossana Lombardi , Zwicker Jeffrey I. , Lawler Jack , De Cristofaro Raimondo , Peyvandi Flora 
Published: April 2020

Abstract
Thrombospondin-1 (TSP-1) is a large homotrimeric glycoprotein of approximatively 450 kDa, synthetised by several cell types, including platelets where it is stored in α-granules. Its multidomain structure enables TSP-1 to interact with many cell-adhesive receptors, integrins, heparan sulfate, as well as with other adhesive glycoproteins, including VWF. In a mouse model, soluble or local platelet released TSP-1 can protect unfolded endothelium-bound and subendothelial VWF from degradation by its plasma protease ADAMTS13. TSP-1 has also been shown to bind to the A3 domain of VWF suggesting a role for competitive inhibition of ADAMTS13-mediated VWF proteolysis. With this background we chose to investigate the inhibition effect of TSP-1 on ADAMTS13 activity by measuring the cleavage of recombinant (r)VWF multimers using rADAMTS13 in absence or presence of TSP-1 at different concentrations. We also assessed whether TSP-1 competes with ADAMTS13 for interaction with the A3 domain of VWF, by comparing cleavage of VWF-A1A2A3 or A1A2 or A2A3 peptides by ADAMTS13 in the presence or absence of TSP-1 by using HPLC analysis.Significant inhibition by TSP1 on ADAMTS13 activity was observed only with TSP-1 plasma concentrations reached after platelet activation (24–160 nM). However, this result was not confirmed at the low concentrations physiologically present in plasma (0.3–0.7 nM). The relative percentage of high molecular weight multimers was proportionally correlated to the TSP-1 concentration. These findings were confirmed using the collagen binding assay to assess VWF activity relative to antigen concentrations. The mean ratio of CBA/Ag was 0.22 in absence of TSP-1 compared to 0.5 and 0.78 in presence of 80 and 160nM of TSP-1. The HPLC analysis of VWF peptides cleavage by ADAMTS13 in presence of increasing concentrations of TSP-1 (0–225 nM) showed that TSP-1 acts as a competitive inhibitor of ADAMTS13 in binding both A1A2A3 and A1A2 peptides. The A1A2A3 peptide demonstrated an apparent affinity for ADAMTS-13 about three-fold higher than that of A1A2 construct (IC50=36.7±4nM and IC50=100.4±25nM, respectively). We also observed a TSP-1 inhibitory effect of ADAMTS13 activity with the A2A3 peptide (IC50=120 nM). These results indicate that the entire A1A2A3 region is necessary to achieve an optimal inhibition of the ADAMTS-13/VWF interaction by TSP-1. The A3 domain plays a central role in this inhibition, although its effect is dependent on the allosteric role played by the A1 domain. Thus, the lower inhibition observed with A2A3 peptide than with the A1A2A3 peptide, could be explained by increased accessibility of ADAMTS13 to A2 domain when the inhibition effect of A1 domain is lacking, as previously described by Nishio et al (PNAS2004;101:10578–83).