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1
Hayman, Melissa Anne. "Genomic influences on platelet function." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/36221.
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The study of platelet messenger and micro-RNAs is of increasing interest owing to the fact that platelets contain the machinery to splice and translate mRNA into proteins in response to inhibitory or activating signals. However, the relatively small size (roughly 4000-5000 transcripts) and short half-life of the platelet transcriptome makes this a technically challenging aspect of platelet biology to investigate. The aims of these thesis investigations were therefore to optimise protocols for the isolation of platelets for downstream RNA analyses and function testing, to investigate the functional capabilities of platelet subpopulations rich in RNA, and to understand the functional and transcriptomic impact of gene mutations predicted to influence platelet function. I found that the optimal method for isolating platelets from whole blood is to use simple single step centrifugation to obtain platelet rich plasma. This method is as effective as more involved methods at reducing white blood cell contamination whilst causing minimal platelet activation. Using this method in combination with flow cytometric cell sorting techniques I was able to isolate the newly formed reticulated platelet sub-population and to confirm the link between reticulation status and increased RNA content. Furthermore, using a range of platelet function assays I demonstrated that reticulated platelets are more reactive than non-reticulated platelets. By obtaining blood samples from a patient with a PLA2G4A mutation I was able to show that loss of cPLA2α enzymatic activity alters both platelet function and the expression of certain mRNA transcripts. My investigations using samples from a range of patients with bleeding tendencies show the benefit of combining deep platelet phenotyping with next generation sequencing to understand the causation of bleeding disorders. Together these investigations highlight the utility of genomic DNA and platelet specific mRNA studies in providing novel insights in to pathways regulating platelet reactivity.
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Wong, Truman. "Dynamics of platelet shape change and aggregation size-dependent platelet subpopulations." Thesis, McGill University, 1988. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=61778.
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Gupta, Nilaksh. "UBIQUITIN-PROTEASOME SYSTEM MODULATES PLATELET FUNCTION." Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1408896695.
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Wenger, Roland Hugo. "Platelet molecular biology : cloning and characterisation of the platelet-specific genes CTAP-III and GPIba /." Bern, 1990. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Hill, Sarah Kathleen. "The tetraspanin CD9 localizes to platelet-platelet contacts and regulates thrombus stability." View the abstract Download the full-text PDF version, 2008. http://etd.utmem.edu/ABSTRACTS/2008-036-Hill-index.html.
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Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008.Title from title page screen (viewed on February 2, 2009). Research advisor: Lisa K. Jennings, Ph.D. Document formatted into pages (xv, 126 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 104-126).
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Gomez, Jorge. "Characterization and regulation of platelet activating factor receptors." Diss., The University of Arizona, 1990. http://hdl.handle.net/10150/185248.
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Platelet activating factor (PAF) is a potent mediator in a variety of inflammatory events. Determining whether PAF participates in the bronchial hyperresponsiveness characteristic of asthma is the long term obj ecti ve for which the studies described here represent an initial step. PAF is a potent agonist that causes contraction of guinea pig peripheral lung strips. To determine if specific receptor sites for PAF could be demonstrated in guinea pig lung membranes (GPLM), direct radioligand binding studies were performed with [³H]C₁₆-PAF (l-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) and the PAF antagonists [³H]WEB 2086 and [³H]RP52770. Binding parameters were compared to those from rabbit platelet membranes (RPM). These studies demonstrated specific binding sites for [³H] C₁₆-PAF of high affinity in GPLM with a Kd of 3 nM,• and in RPM with a K(d) of 1 nM. [³H]C₁₆-PAF identified receptor densities in GPLM of 200 fmol/mg protein and in RPM of 1922 fmol/mg protein. In both tissue preparations binding of inhibited to the same maximum degree by C₁₆-PAF, C₁₈-PAF, WEB 2086, and RP52770, all with pseudo-Hill coefficients of unity. The PAF antagonist [³H]WEB 2086 identified a receptor density similar to that of [³H]C₁₆-PAF. The binding of [³H]WEB 2086 was inhibited to the same degree by C₁₆-PAF, C₁₈-PAF, WEB 2086 and RP52770, indicating WEB 2086 and PAF interact at the same receptor sites in both GPLM and RPM. Although inhibition curves for antagonists yielded pseudo-Hill coefficients of unity, inhibition by agonists yielded shallow inhibition curves suggesting two types or states for the PAF receptor. The PAF antagonist [³H]RP52770 was found to be an unsuitable ligand because it labeled a much larger density of binding sites (1200 fmol/mg protein in GPLM, and 10105 fmol/mg protein in RPM) and was inhibited to little or no extent by C₁₆-PAF, C₁₈-PAF, WEB 2086 or lyso-C₁₆-PAF . studies of signal transduction suggest that the binding affinity of the agonists C₁₆-PAF and C₁₈-PAF (but not for the antagonist WEB 2086) is regulated by GTPgamroa- S and Na⁺, providing indirect evidence that the PAF receptor in both tissue preparations is coupled to a guanine nucleotide regulatory protein. However, agonist binding retained shallow inhibition curves indicating heterogeneity of sites with respect to this regulation. Binding affinity for the agonists was not affected by cholera toxin or pertussis toxin. These results indicate PAF receptors in lung tissue could not be distinguished from those in RPM, however, both tissues appear to show heterogeneity of binding indicating the existence of receptor subtypes or states.
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Bonin, Fanny. "Cytoprotective effects of intracellular platelet activating factor acetylhydrolases." Thesis, University of Ottawa (Canada), 2003. http://hdl.handle.net/10393/26529.
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Platelet activating factor (PAF) is a biologically active phospholipid implicated in the developmental brain disorder Miller-Dieker Syndrome (MDS) and purported to be a primary mediator of cell death in HIV-dementia, ischemia, and epilepsy. As part of my honour's thesis, I demonstrated that PAF can elicit cell death independently of its G-protein coupled receptor (PAFR) in PC12 cells. In my M.Sc. research, I have sought to identify how PAF-mediated cell death is regulated in PC12 cells. PAF is inactivated in brain by two intracellular PAF-acetylhydrolases (PAF-AHs): PAF-AH I and PAF-AH II. PAF-AH I is a trimeric complex composed of two catalytic subunits (alpha1 and alpha2) and one regulatory subunit (beta). Mutations in the Lis1 gene, coding for the beta subunit of PAF-AH I, are the genetic determinant of MDS. However, it is not clear whether these mutations impact on PAF-AH I enzymatic activity in MDS. Furthermore, it is not known whether cytosolic PAF-AH activity regulates the kinetics of neuronal loss following pathophysiological challenge. To begin to address these questions, I sought to identify an in vitro model system suitable for study of PAF-AH activity.* (Abstract shortened by UMI.)
*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.
8
Kabbani, Nazir. "Chemical-genetic profiling of platelet-activating factor in yeast." Thesis, University of Ottawa (Canada), 2009. http://hdl.handle.net/10393/28189.
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The basic biological processes between the yeast Saccharomyces cerevisiae and mammals are highly conserved. Yeast posses many genes that are implicated in human diseases and have been successfully used as a model for the study of neurodegeneration. Platelet-Activating Factor (C16:0 PAF) causes neuronal cell death independent of its receptor and has been implicated in Alzheimer's disease. I hypothesized that yeast could be used as a model system for deciphering PAF receptor-independent signalling and have utilized genome-wide chemical genomic screening in yeast to further characterize the molecular mechanism of PAF toxicity. Two complementary screens implicate PAF in many cellular processes, some of which parallel results obtained in mammalian studies. I have found that PAF challenge is cytotoxic, delays cell cycle progression, and affects actin stability leading to spindle misorientation and bi-nucleate mother cells.
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Buitrago, Murcia Claudia Lorena. "Cbl proteins in platelet functional responses." Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/198139.
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PhysiologyPh.D.
c-Cbl protein functions as an E3 ligase and scaffolding protein, where three residues, Y700, Y731, and Y774, upon phosphorylation, have been shown to initiate several signaling cascades. In this study, we investigated the role of these phospho-tyrosine residues in the platelet functional responses upon integrin engagement. We observed that c-Cbl Y700, Y731 and Y774 undergo phosphorylation upon platelet adhesion to immobilized fibrinogen, which was inhibited in the presence of PP2, a pan-src family kinase (SFK) inhibitor, suggesting that c-Cbl is phosphorylated downstream of SFKs. However, OXSI-2, a Syk inhibitor, significantly reduced c-Cbl phosphorylation at residues Y774 and Y700, without affecting Y731 phosphorylation. Interestingly, PP2 inhibited both platelet spreading on fibrinogen as well as clot retraction, whereas OXSI-2 blocked only platelet spreading, suggesting a differential role of these tyrosine residues. The physiological role of c-Cbl and Y731 was studied using platelets from c-Cbl KO and c-CblYF/YF knock-in mice. c-Cbl KO and c-Cbl YF/YF platelets had a significantly reduced spreading over immobilized fibrinogen. Furthermore, clot retraction with c-Cbl KO and c-Cbl YF/YF platelets was drastically delayed. These results indicate that c-Cbl and particularly its phosphorylated residue Y731 plays an important role in platelet outside-in signaling contributing to platelet spreading and clot retraction
Temple University--Theses
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Chase, Peter Burritt 1955. "The molecular pharmacology of a human platelet-activating factor receptor." Diss., The University of Arizona, 1996. http://hdl.handle.net/10150/290574.
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Platelet-activating factor (PAF) is a broadly bioactive family of phospholipids which contribute to the pathogenesis of numerous diseases as well as to many normal physiologic processes. The pleiotropic nature of PAFs actions may be due to the activation of several intracellular signaling pathways or the presence of PAF receptor subtypes. Therefore, to begin to understand the complex mechanisms by which PAF molecules induce cellular responses, a molecular approach was initiated to provide tools to investigate many of the issues surrounding PAF receptors. Using a strategy based upon homology cross hybridization, a coding sequence homologous to that of the guinea pig PAF receptor cDNA was identified in a 20 kb insert obtained from human genomic DNA. A portion of the insert was sequenced and appears to be the human homolog of the cloned guinea pig receptor. Although the sequence identity shows that the gene for the human PAF receptor does not contain introns in the coding region, the 5'-untranslated sequence deviates from previously reported cDNA sequences suggesting that at least one intron is present in the untranslated region and represents evidence for alternative mRNA splicing. The 20 kb human genomic fragment also allowed for regional mapping of the PAF receptor gene by fluorescence in situ hybridization and found to localize to chromosome 1 (1p35-> p34.3). Specific localization of the PAF receptor gene to the distal portion of chromosome 1 may assist in understanding the genetic predisposition of certain patients to inflammatory diseases. To examine the second messenger coupling of the cloned PAF receptor and adenylyl cyclase, a cAMP-responsive reporter gene has been used in transiently transfected human choriocarcinoma cells. Preliminary data suggests that PAF receptor signal transduction does result in inhibition of basal and agonist-stimulated adenylyl cyclase activity. PAF receptor specific antibodies could assist in tissue localization of the cloned PAF receptor as well as provide evidence for PAF receptor subtypes. Antibodies were produced against fusion protein consisting of glutathione-S-transferase and a peptide from the purported 2nd extracellular region of the cloned PAF receptor which recognized the native protein in transfected COS-7 cells.
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Manne, Bhanu Kanth. "CLEC-2 SIGNAL TRANSDUCTION IN PLATELET ACTIVATION." Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/340495.
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PhysiologyPh.D.
Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. CLEC-2 is a C-type lectin-like receptor that is highly expressed in platelets and lesser extent, in other cell types such as activated dendritic cells and B cells. Rhodocytin was the first ligand used to identify CLEC-2 receptor and it’s signaling on platelets. In the first chapter we identified a new agonist for CLEC-2 receptor. Fucoidan, a sulfated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylation of Syk and Phospholipase Cγ−2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. Lipid rafts are distinct areas of the plasma membrane implicated in the regulation of signaling in a variety of cells including platelets. A previous study C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling. Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an Immune Tyrosine Activation Motif (ITAM) and hemi-ITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In chapter 3, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3-Kinase, which demonstrates that PI3-Kinase regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus our data show, for the first time, that PI3-Kinase and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of CLEC-2 receptor.
Temple University--Theses
12
Cohen, Zoe. "Mechanisms of platelet activation in type 2 diabetes." Diss., The University of Arizona, 2003. http://hdl.handle.net/10150/289926.
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Diabetics suffer from a pro-thrombotic condition. It is established that platelets are activated in type 2 diabetes. However the mechanisms of platelet activation in this disease are not yet known. The purpose of these studies was to elucidate the mechanisms of platelet activation and the effect these mechanisms have on type 2 diabetic platelets. Apoptosis of cells is regulated by caspases, a group of cysteine proteases. When platelets become activated, they express phosphatidylserine (PS) on the outer leaflet of the plasma membrane as well as form platelet microparticles (PMPs). In addition, platelets aggregate when activated. We found that platelets from diabetic subjects contain activated caspases. These platelets also formed increased numbers of PMPs compared to platelets from non-diabetic subjects. We observed a 30-fold increase in thrombin activity in the plasma from diabetics. To determine if caspases were involved in platelet activation, we determined if caspase inhibition (using the pan-caspase inhibitor zVAD-fmk); (1) decreased PS expression and (2) decreased platelet aggregation following activation. We found that platelets treated with zVAD-fmk significantly decreased A23187-induced PS exposure as well as aggregation. There is limited information on the role of caspases on platelet adhesion proteins such as P-selectin or GPIIb/IIIa during platelet activation. Therefore, we tested if caspase inhibition attenuated P-selectin and GPIIb/IIIa expression. Using blood from non-diabetic volunteers, we found that treatment with zVAD-fmk caused a significant attenuation of P-selectin expression in stimulated platelets. Together these data suggest that caspases play a novel role in platelet activation. We also wanted to determine if treating type 2 diabetic rats (Zucker Diabetic Fatty rats, ZDF) with the caspase inhibitor zVAD-fmk in vivo ; (1) attenuated PS expression, (2) attenuated platelet microparticle (PMP) formation and (3) attenuated platelet aggregation. ZDF rats were treated in vivo with 40 μg zVAD-fmk for 4 days. We found attenuated PS exposure, PMP formation as well as decreased aggregation in ZDF rats treated with zVAD-fmk. Our overall results demonstrate a novel role of caspases in platelet activation. Together, these studies may lead to development of novel treatments for pathophysiologic states associated with platelet activation such as diabetes, myocardial infarction, and stroke.
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Toledano, Baruch Joseph. "Platelet activating factor's role in regulating apoptosis in immature B cells." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0006/MQ44299.pdf.
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Gavin, Rebecca Louise. "The tetraspanin Tspan18 regulates GPVI induced platelet activation and Ca²⁺ mobilisation." Thesis, University of Birmingham, 2015. http://etheses.bham.ac.uk//id/eprint/5903/.
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Platelet activation and subsequent thrombus formation are important for preventing excessive blood loss at sites of vascular injury, a process termed haemostasis. However, excessive platelet activation at sites of atherosclerotic plaque rupture can lead to thrombus formation, which may occlude the vessel and cause heart attack or stroke. The platelet collagen receptor GPVI is essential for thrombus formation, but is largely dispensable for haemostasis. Tetraspanins are transmembrane proteins which compartmentalise the membrane through formation of dynamic tetraspanin-enriched microdomains. Due to regulation of a wide range of partner proteins, tetraspanins have been implicated in many cellular processes, including platelet activation, though most platelet tetraspanins have not been characterised. The aim of this thesis was to investigate the novel platelet tetraspanin Tspan18, using the Tspan18 knockout mouse. Tspan18 was shown to have a role in platelet activation and platelet Ca\(^2\)\(^+\) signalling specifically downstream of GPVI. Tspan18 also appeared to have a role in haemostasis, as Tspan18 deficient mice displayed a severe bleeding phenotype. The bleeding was shown to be driven by non-haematopoietic cells and is therefore unlikely to be platelet-driven. Additionally, Tspan18-induced Ca\(^2\)\(^+\) mobilisation was shown to be dependant on functioning Orai1 Ca\(^2\)\(^+\) channels and a novel interaction between Tspan18 and the Orai family was identified. Together, these findings suggest a role for Tspan18 in platelet activation and regulation of Ca\(^2\)\(^+\) mobilisation, potentially via interaction with Orai proteins.
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Marrache, Anne Marilise. "Platelet activating factor receptors : nuclear localization and signaling in microvascular endothelial cells." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=82928.
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It has been postulated that intracellular binding sites for platelet activating factor (PAF) contribute to pro-inflammatory responses to PAF. Isolated nuclei from porcine cerebral microvascular endothelial cells (PCEC) produced PAF-molecular species in response to H2O2 . Using fluorescent-activated cell sorter analysis, we demonstrated the expression of PAF receptors (PAFR) on cell and nuclear surfaces of PCEC. Confocal microscopy studies performed on PCEC, Chinese hamster ovary cells stably overexpressing PAFR and on isolated nuclei from PCEC also showed a robust nuclear distribution of PAFR. Presence of PAFR at the cell nucleus was further revealed in brain endothelial cells by radioligand binding experiments, immunoblotting, and in situ in brain by immunoelectron microscopy. Stimulation of nuclei with PAF evoked a decrease in cAMP production and a pertussis toxin (PTX)-sensitive rise in nuclear calcium, unlike observations in plasma membrane, which exhibited a PTX-insensitive elevation in inositol phosphates. Moreover, on isolated nuclei C-PAF evoked the expression of pro-inflammatory genes inducible nitric oxide synthase and cyclooxygenase-2 (COX-2), and was associated with augmented ERK1/2 phosphorylation and NF-kappaB binding to DNA consensus sequence; COX-2 expression was prevented by MEK/ERK1/2 and NF-kappaB inhibitors. This study describes for the first time the nucleus as a putative organelle capable of generating PAF and expresses its receptor, which upon stimulation induces the expression of pro-inflammatory gene COX-2.
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Chen, Kan. "Prothrombotic Platelet Signaling By the Scavenger Receptor CD36." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1226608419.
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Bennett, Cavan. "Cytokine receptor-like factor 3 (CRLF3) : a novel regulator of platelet biogenesis and potential drug target for thrombocythaemia." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/277068.
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Thrombocythaemia is defined as a circulating platelet count above 450x10$^9$/L in humans. The major cause of thrombocythaemia is reactive $(secondary)$ thrombocythaemia which occurs secondary to many conditions such as infection, cancer and inflammation. However, acquired clonal mutations in mainly Janus Kinas 2 $(JAK2)$, CALR and MPL cause essential thrombocythaemia $(ET)$. ET is a rare disease that leads to an increased risk of cardiovascular thrombotic events. Current treatment of ET uses combination of low dose aspirin to decrease platelet function and cytoreductive agents to decrease thrombopoiesis. The most commonly used cytoreductive agents are hydroxyurea, anagrelide and interferon-$alpha$ and all have unwanted side effects. Cytokine receptor-like factor 3 $(CRLF3)$ is a 2.4kb gene that is ubiquitously expressed throughout the haematopoietic system. Very little is known about the function of CRLF3, with only one peer reviewed journal article in the literature which shows that CRLF3 may negatively regulate the cell cycle at the G0/G1 phase. However, nothing is known about the role of CRLF3 in platelet biology. Using a Crlf3 knockout mouse $(Crlf3-/-)$ developed by the Wellcome Trust Sanger Institute we show CRLF3’s role in platelet biogenesis and how it could be used as a novel therapeutic target to treat ET. Crlf3-/- mice have an isolated and sustained 25-40$\%$ decrease in platelet count compared to wildtype $(WT)$ controls. Platelet function is unaffected as demonstrated in a range in a range of in vitro assays. The thrombocytopenia is a consequence of abnormalities in hematopoietic cells, as shown by bone marrow transplantations. Megakaryopoiesis is upregulated in Crlf3-/- mice and proplatelet morphology is unaffected, suggesting the thrombocytopenia is due to increased platelet clearance. Indeed, splenectomised Crlf3-/- mice show normalised platelet counts within 7 days, showing rapid splenic removal of platelets is responsible for the thrombocytopenia. Abnormal large platelet structures that resemble proplatelets shafts $(preplatelets)$ are abnormally present in the circulation of elderly Crlf3/- mice. Immunohistochemistry showed increased and aberrant tubulin expression in Crlf3-/- platelets compared to WT controls, especially in the preplatelet forms. Cold induced depolymerisation of microtubules was decreased in Crlf3-/- platelets, suggestive of increased tubulin stability, however, the ratio of detyrosinated to tyrosinated tubulin was not altered. We then crossbred Crlf3-/- mice with JAK2 V617F ET mice, to determine the effect of Crlf3 ablation of thrombocythaemia. Crossbred mice showed restoration of platelet counts to WT values without grossly affecting platelet function or other blood lineages, providing the rational for CRLF3 as a novel therapeutic target for treatment of ET. Finally, we aimed to resolve the crustal structure of CRLF3 and discover its interactome. To this end, we were able to resolve the crystal structure of a C-terminal portion of the full length protein containing the predicted fibronectin type III domain. To shed light on the interactome of CRLF3, endogenous CRLF3 was tagged with a tandem affinity purification $(TAP)$ tag using CRISPR/Cas9 technology in induced pluripotent stem cells $(iPSCs)$. We have been able to produce megakaryocytes from these TAP-tagged iPSCs by forward programming. However, as yet we have not been able to generate enough MKs to have adequate material to perform immunoprecipitation assays. Therefore, the interactome of CRLF3 in MKs remains unknown. In conclusion, we identified a mechanism by which Crlf3 controls platelet biogenesis. Slowed maturation of Crlf3-/- preplatelets in the peripheral circulation potentially due to increased structural stability leads to rapid removal of these immature forms by the spleen and therefore a decrease in platelet count. The isolated effect on platelet numbers and normalisation of platelet count in ET mice deficient of Crlf3 provides the rational for further study on CRLF3 drug targeting as a novel therapeutic strategy for ET.
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Rudmann, Sally V. "The effect of twenty minutes of aerobic exercise on in vivo platelet release in moderately trained females : radioimmunoassay of platelet factor 4 beta-thromboglobulin /." The Ohio State University, 1986. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487266362337217.
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Mooney, Robert Francis. "The regulation of platelet aggregation by glycoprotein IIb-IIIa receptor and fibrinogen /." Thesis, McGill University, 1991. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=60500.
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We compared both the rate and extent of platelet aggregation with the extent of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted ten-fold) was maximally pre-activated by incubating with adenosine diphosphate (ADP) at room temperature and then stirred. Platelet aggregation was determined from the decrease in the total number of particles. The number of fibrinogen receptors or bound Fg was measured from mean fluorescence values obtained with FITC-labelled IgM monoclonal antibody PAC1, and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry. The expression of fibrinogen receptors occurs within seconds of activation. The fraction of platelets with fluorescence values above one critical threshold value increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r $>$ 0.9). Aggregation was not rate-limited by fibrinogen receptor expression nor by Fg binding. It appears that each platelet expresses $>$90% of its maximal Fg receptors at a critical ADP concentration i.e, occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and platelet aggregation.
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Mao, Yingying. "ROLE OF PROTEASE-ACTIVATED RECEPTORS IN PLATELET ACTIVATION." Diss., Temple University Libraries, 2009. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/47279.
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PhysiologyPh.D.
Platelets act as a fundamental component of the hemostatic process and their activation leads to the formation of a stable clot at the injured endothelium surface. Thrombin, as the important physiological agonist, activates platelets through protease-activated receptors (PARs). Protease-activated receptors are one of the major receptors in platelets and belong to the seven-transmembrane G-protein couple receptor family. Four protease-activated receptors are found, named as PAR1, PAR2, PAR3 and PAR4. Human platelets express PAR1 and PAR4 and murine platelets express PAR4 and PAR3 instead of PAR1. Thrombin activates PARs through a unique mechanism, involving the cleavage of N-terminus of PAR receptors and the newly exposed N-terminus acts as its own tethered ligand to bind and activate the receptor. In this study, we characterized a new PAR1 specific activating peptide (TFRRRLSRATR), generated from the c-terminus of human platelet P2Y1 receptor, and evaluated its biological function. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Its activation is completely inhibited by using BMS200261, a PAR-1 specific antagonist. Its specificity to PAR1 receptor is further confirmed by using TFRRR-peptide-pretreated washed platelets and murine platelets. The shape change induced by 10 microM peptide was totally abolished by Y-27632, an inhibitor of p160ROCK which is the downstream signal of G12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increased concentrations. Similar to SFLLRN, the TFRRR-peptide caused phosphorylation of Akt and Erk in a P2Y12 receptor-dependent manner, and p-38 MAP kinase activation in a P2Y12-independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. Beside thrombin, PARs also can be activated by other proteases. Previous studies in our lab show that plasmin, a major extracellular protease, activates both human and murine platelets through prototypical cleavage of PAR4 (Quinton et al., 2004). In this study, we continue our study and investigate the molecular basis for the differential activation of murine and human platelets by plasmin. Plasmin-induced full aggregation is achieved at lower concentrations (0.1 U/mL) in murine platelets as compared to human platelets (1 U/mL). In COS7 cells expressing the murine PAR4 (mPAR4) receptor, 1 U/mL plasmin caused a higher intracellular calcium mobilization than in cells expressing the human PAR4 (hPAR4) receptor. This difference was reversed when the tethered ligand sequences of mPAR4 and hPAR4 were interchanged through site-directed mutagenesis. This difference between human and murine PAR4 is not because of the cofactor effect of PAR3 in murine platelets by showing that in both transfected cell lines and platelet system, PAR3 inhibits plasmin-induced PAR4 stimulation. All of the data suggest that murine platelets are more sensitive to activation by plasmin than human platelets due to differences in the primary sequence of PAR4. In contrast to thrombin-dependent activation of platelets, wherein PAR3 acts as a co-receptor, mPAR3 inhibits plasmin-induced PAR4 activation. Abnormal platelet activation causes thrombus formation and induces pathological conditions including stroke and atherosclerosis. Antithrombotic therapy is a widely used therapeutic method for stroke. However, currently used agents based on the irreversible inhibition of the platelet cyclooxygenases 1 and 2 or inhibition of P2Y12 receptors can cause unexpected bleeding or resistant side effects. Antithrombotic therapy targeting thrombin signaling is one of the new treatments under investigation and PAR1 antagonists are now in clinical trials. In this study, we investigate the effect of one of thrombin receptors, protease-activated receptor 4 (PAR4) in mice transient middle cerebral artery occlusion/ reperfusion (tMCAO/R) model. Our data show that PAR4 -/- mice have more than 80% reduction in infarct volume and significant improved neurological and motor function after 1 h MCAO followed by 23 h reperfusion. Examination of cellular responses to tMCAO/R indicates that PAR4-/- mice have less cellular death. Platelet/endothelial and leukocyte/endothelial interactions have been shown to play a critical role in the inflammatory responses during cerebral ischemic/reperfusion injury. Comparing wild-type with PAR4-/- mice platelets/endothelial and leukocyte/endothelial interactions, deficiency of PAR4 causes a significant decrease in both platelet/endothelial and leukocyte/endothelial interactions. In addition, PAR4-/- mice attenuate blood-brain barrier (BBB) disruption during tMCAO/R. All the data suggest that deficiency of PAR4 will protect against brain ischemic injury though attenuation of cerebral inflammatory responses including inflammatory cells extravasation and BBB disruption. Protease-activated receptor 4 (PAR4) is the only thrombin receptor existing in both human and murine platelets. The data we get in this study also have a beneficial effect for human study and inhibition of PAR4 may provide a novel potential therapeutic strategy for ischemic injury.
Temple University--Theses
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Al, Ghaithi Rashid Hafidh Rashid. "Laboratory investigation of platelet function in patients with mild bleeding disorders." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8173/.
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Platelets play a crucial role in haemostasis by preventing bleeding at sites of vascular injury. Inherited or acquired platelet defects can impair haemostasis resulting in bleeding symptoms of varying severity ranging from mild to excessive which can be life threatening. Diagnosis of mild platelet-based bleeding disorders is challenging due to the absence of a gold standard technique and their variable bleeding symptoms and bleeding phenotypes observed in healthy individual as well as other haemostatic disorders. The work in this thesis built on the previous studies in the genotyping and platelet phenotyping project allowing further characterization of inherited platelet function defects in individuals with mild bleeding disorders. Platelet aggregation and secretion in samples from 206 patients were investigated during the course of this thesis and were categorised on the basis of the observed defects. Surprisingly, in over a half of these patients, an ex vivo platelet function defect was not found. The genetic investigation of selected cases using whole exome sequencing identified mutations in number of genes previously known to be critical in platelet biology. This thesis also focused on evaluation of three other platelet techniques by comparison with lumi-aggregometry to assess their overall potential in detecting platelet function defects. Further studies are still needed to further assess the potential of these techniques before they can be applied in routine clinical diagnosis.
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Clancy, Lauren R. "Platelet Transcriptome Heterogeneity: A Role for RNA Uptake in Vascular Health and Disease." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/922.
Full textAbstract:
As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated.
Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond.
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Clancy, Lauren R. "Platelet Transcriptome Heterogeneity: A Role for RNA Uptake in Vascular Health and Disease." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/922.
Full textAbstract:
As our understanding of the platelet’s systemic role continues to expand beyond hemostasis and thrombosis, interrogation of the platelet’s ability to affect diverse biological processes is required. Studies of the platelet’s non-traditional roles have focused on developing our understanding of the platelet’s relation to specific disease phenotypes as well as elucidation of platelet characteristics, content, and function. The generic content, traditional function and heterogeneity of platelets have long been accepted; more ambiguous and controversial has been how these factors are interrelated.
Investigation of platelet content revealed the presence of biologically functional RNA in anucleated platelets, the correlation of platelet RNA to distinct phenotypes, and the ability of platelets to transfer RNA to other vascular cells; however how these processes occur is unclear. To further interrogate platelet RNA processes, we utilized sorting and RNA sequencing to develop platelet subpopulation transcriptome profiles. We found that platelet heterogeneity extends to the platelet transcriptome: distinct RNA profiles exist dependent on platelet size. We hypothesized that this RNA heterogeneity is the result of RNA transfer between platelets and vascular cells. Using in vitro and in vivo modeling, we were able to show the novel ability of platelets to take up RNA from vascular cells, correlating to the unique functional profile associated with small platelet transcriptomes. These findings reveal a role for platelet RNA transfer in platelet RNA heterogeneity, with potential correlation to platelet functional diversity previously proposed. The ability of the platelet to bidirectionally transfer RNA within circulation has implications for vascular health and beyond.
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Douglas, Cheryl E. "Modulation of rat platelet phospholipase A2 and 12-lipoxygenase activities by dietary vitamin E." Thesis, University of Ottawa (Canada), 1986. http://hdl.handle.net/10393/4552.
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Saggu, Gurpanna. "Role of Complement Regulatory Protein Properdin in Complement Activation on Platelets and in the Formation of Platelet-Leukocyte Aggregates." University of Toledo Health Science Campus / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1392998532.
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Alshehri, Osama Mohammed D. "The role of GPVI and CLEC-2 in platelet activation by miscellaneous ligands." Thesis, University of Birmingham, 2016. http://etheses.bham.ac.uk//id/eprint/6880/.
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Platelets are an essential factor in wound repair and blood clotting, where exposed sub-endothelial extracellular matrix (ECM) proteins induce activation during vascular injury. However, platelets can also be activated by a diverse range of stimuli that share little-to-no resemblance in structure to each other, or to recognized ligands of platelet receptors. These stimuli include diesel exhaust particles (DEP), various peptides including 4N1-1 and Champs, lipoproteins such as PAM3-CSK4, and large polysaccharides for example, fucoidan, and dextran. In this thesis, I demonstrate that this seemingly miscellaneous group of stimuli cause aggregation of human and mouse platelets through Src and Syk tyrosine kinases in association with stimulus-specific tyrosine phosphorylation of the GPVI/FcRγ-chain complex and/or CLEC-2. A critical role for GPVI and/or CLEC-2 in mediating aggregation is shown using platelets from receptor-deficient mouse platelets. Additionally, in double deficient mouse platelets these stimuli activate Src tyrosine kinases independent of GPVI and CLEC-2. DEP, fucoidan and dextran were shown to activate transfected GPVI or CLEC-2 in a cell line model. However, 4N1-1, Champs and PAM3-CSK4 did not activate transfected GPVI or CLEC-2 in a cell line model, nor could they bind to recombinant forms of either receptor. In addition, I demonstrate the unexpected observation that fibrin also activates GPVI revealing a new stage of haemostasis in which the generation of fibrin from fibrinogen reinforces platelet activation.
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Narayanan, Padmini. "Interleukin 1 Receptor1 signaling in Platelet Inflammatory responses Interleukin-1ß processing and secretion." Cleveland State University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=csu1425911007.
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Griffin, Christina Michele. "Investigation of the role of Platelet-Derived Growth Factor (PDGF) in the development of breast carcinomas." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/86279.
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Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, September 2002.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 30-33).
by Christina Michele Griffin.
S.M.
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Unsworth, Amanda J. "The role of protein kinase C in platelet activation." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:114582b8-185a-41f5-958c-77038fb185df.
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The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.
30
Joshi, Smita. "CONTROLLING PLATELET SECRETION TO MODULATE HEMOSTASIS AND THROMBOSIS." UKnowledge, 2018. https://uknowledge.uky.edu/biochem_etds/37.
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Upon vascular injury, activated blood platelets fuse their granules to the plasma membrane and release cargo to regulate the vascular microenvironment, a dynamic process central to platelet function in many critical processes including hemostasis, thrombosis, immunity, wound healing, angiogenesis etc. This granule- plasma membrane fusion is mediated by a family of membrane proteins- Soluble N-ethyl maleimide Attachment Receptor Proteins(SNAREs). SNAREs that reside on vesicle (v-SNAREs) /Vesicle-Associated Membrane Proteins(VAMPs) interact with target/t-SNAREs forming a trans-bilayer complex that facilitates granule fusion. Though many components of exocytic machinery are identified, it is still not clear how it could be manipulated to prevent occlusive thrombosis without triggering bleeding. My work addresses this question by showing how the rates and extents of granule secretion could be regulated by various v-SNAREs. We also show that the granule cargo decondensation is an intermediate to secretion that also contributes to rates of cargo release.
Platelets contain four major VAMP isoforms (-2, -3, -7, and -8), however, VAMP-8 and -7 play a primary role while VAMP-2 and -3 are ancillary in secretion. To exploit this heterogeneity in VAMP usage, platelet-specific V-2/3-/- and V-2/3/8-/- mouse models were generated and characterized to understand how secretion influences hemostasis. We found that each VAMP isoform differentially contributes by altering the rates and extents of cargo release. The loss of VAMP-2 and -3 had a minimal impact while the loss of VAMP-2, -3 and -8 significantly reduced the granule secretion. Platelet activation and aggregation were not affected though the spreading was reduced in V-2/3/8-/- platelets indicating the importance of secretion in spreading. Though coagulation pathways were unaltered, PS exposure was reduced in both V-2/3-/- and V-2/3/8-/- platelets suggesting diminished procoagulant activity. In vivo experiments showed that V-2/3/8-/- animals bled profusely upon tail transaction and failed to form occlusive thrombus upon arterial injury while V-2/3-/- animals did not display any hemostatic deficiency. These data suggest that about 40-50% reduction in secretion provides protection against thrombosis without compromising hemostasis and beyond 50% secretion deficiency, the animals fail to form functional thrombi and exhibit severe bleeding. Additionally, detailed structural analysis of activated platelets suggests that the post-stimulation cargo dissolution depends on an agonist concentration and stimulation duration. This process is VAMP-dependent and represents intermediate steps leading to a full exodus of cargo. Moreover, we also show that VAMP-8 is important for compound fusion events and regulates fusion pore size.
This is a first comprehensive report that shows how manipulation of the exocytic machinery have an impact on secretion and ultimately on hemostasis. These animals will be instrumental in future investigations of platelet secretion in many other vascular processes.
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Chari, Ramya. "Molecular Mechanisms Underlying Differential Regulation of Platelet Dense Granule Secretion by Protein Kinase C delta." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/77283.
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PhysiologyPh.D.
Protein Kinase C delta (PKCδ) is expressed in platelets and activated downstream of protease-activated receptors (PAR)s and glycoprotein VI (GPVI) receptors. We evaluated the role of PKCδ in platelets using two approaches - pharmacological and molecular genetic approach. In human platelets pretreated with isoform selective antagonistic RACK peptide (δV1-1)TAT, and in the murine platelets lacking PKCδ, PAR4-mediated dense granule secretion was inhibited, whereas GPVI-mediated dense granule secretion was potentiated. These effects were statistically significant in the absence and presence of thromboxane A2 (TXA2). Furthermore, TXA2 generation was differentially regulated by PKCδ. However, PKCδ had a small effect on platelet P-selectin expression. Calcium- and PKC-dependent pathways independently activate fibrinogen receptor in platelets. When calcium pathways are blocked by dimethyl-BAPTA, AYPGKF-induced aggregation in PKCδ null mouse platelets and in human platelets pretreated with (δV1-1)TAT, was inhibited. In a FeCl3-induced injury in vivo thrombosis model, PKCδ-/- mice occluded similar to their wild-type littermates. Hence, we conclude that PKCδ differentially regulates platelet functional responses such as dense granule secretion and TXA2 generation downstream of PARs and GPVI receptors, but PKCδ deficiency does not affect the thrombus formation in vivo. We further investigated the mechanism of such differential regulation of dense granule release by PKCδ in platelets. SH2 domain-containing Inositol Phosphatase (SHIP)-1 is phosphorylated on Y1020, a marker for its activation, upon stimulation of human platelets with PAR agonists, SFLLRN and AYPGKF, or GPVI agonist, convulxin. GPVImediated SHIP-1 phosphorylation occurred rapidly at 15 sec whereas PAR-mediated phosphorylation was delayed, occurring at 1 min. Lyn and SHIP-1, but not SHIP-2 or Shc, preferentially associated with PKCδ upon stimulation of platelets with a GPVI agonists, but not with a PAR agonist. In PKCδ null murine platelets, convulxin-induced SHIP-1 phosphorylation was inhibited, suggesting that PKCδ regulates the phosphorylation of SHIP-1. Furthermore, in Lyn null murine platelets, GPVI-mediated phosphorylations on Y-1020 of SHIP-1, Y311 and Y155 of PKCδ were inhibited. In murine platelets lacking Lyn, or SHIP-1, GPVI-mediated dense granule secretions were potentiated, whereas PAR-mediated dense granule secretions were inhibited. Phosphorylated SHIP-1 associated with phosphorylated-Y155 PKCδ peptide. Therefore, we conclude that Lyn-mediated phosphorylations of PKCδ and SHIP-1 and their associations negatively regulate GPVI-mediated dense granule secretion in platelets.
Temple University--Theses
32
Greenstone, Elliot Ari. "Platelet-derived growth factor expression in a rat model of allergic bronchoconstriction." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ55062.pdf.
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Liu, Qingde 1963. "Molecular and physical determinants of fibrinogen-dependent platelet aggregation and adhesion in flow." Thesis, McGill University, 1998. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=35909.
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Fibrinogen (Fg) mediates platelet aggregation and adhesion to artificial surfaces by interacting with its receptor, glycoprotein IIb and IIIa complex (GPIIbIIIa, or integrin alphaIIbbeta 3), on the platelet membrane. Considerable evidence has demonstrated that the (H12) on the gamma chain carboxyl terminus is required for the binding of Fg from solution to activated platelet GPIIbIIIa, while the RGD sites, the universal integrin recognition domain on adhesive ligands, are not involved. In this study, using recombinant Fg, well-defined Fg plasmin digestion fragments, and specific monoclonal anit-Fg antibodies, we demonstrated that the same sequence, the H12, or more precisely, the AGDV on the extreme carboxyl terminus of the gamma chain (gamma408--411), is also required for platelet-bound Fg to support platelet aggregation (crosslinking), thus experimentally verifying the "two sticky ends" theory of Fg-mediated platelet aggregation The RGD-containing domains on the Aalpha chains are not involved in aggregation. The AGDV sequence on the gamma chain carboxyl terminus is also necessary and sufficient for activated platelets to adhere to surface-adsorbed Fg, while the RGD sequences we similarly not required. A receptor induced binding site (RIBS), the Fg RIBS-I site (gamma373--385), on Fg either bound to its GPIIbIIIa-receptor or on a surface, is not directly involved in interactions between platelet GPIIbIIIa and immobilized Fg. The inhibitory effects of the anti-Fg-RIBS-I antibody are due to steric hindrance of the accessibility of the AGDV site to platelet GPIIbIIIa. Thus, the extreme carboxyl terminus of the gamma chain is the only site in both fluid and solid phase Fg that is involved in platelet GPIIbIIIa-Fg interactions.Though resting platelets are able to adhere to surface-bound Fg, this adhesion efficiency is much lower than that of the adhesion of the activated platelets. The adhesion efficiency of both resting and activated platelets to surface-adsorbed Fg decreases with increasing shear rate from 100 s -1 to 2,000 s-1. However, the decrease of the adhesion efficiency of the resting platelets is more marked than the decrease of the adhesion efficiency of the activated ones. Thus, the higher the shear rates, the larger the difference in the adhesion efficiencies between resting and activated platelets. However, due to the higher collision frequencies at higher shear rates, the adhesion of resting platelets was maintained at a similar level from shear rates of 300--2,000 s-1, while the adhesion of activated platelets kept increasing from 100 s -1 to 2,000 s-1. These data indicate that platelet activation is an efficient regulation pathway for platelet adhesion to surfaces.
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Meikle, Claire K. "Platelet-Leukocyte Aggregation in Lung Cancer Patients." University of Toledo Health Science Campus / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=mco1555937904448281.
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Chauhan, Abhishek. "Contribution of the platelet receptor CLEC-2 and its ligand podoplanin to the pathogenesis of liver disease." Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8234/.
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Increasing lines of evidence place platelets as having a central role in liver disease. Platelets are recruited to the liver and, depending upon stage and type of liver injury play varying roles ranging from driving liver fibrosis to aiding regeneration. However the molecular basis and consequences of platelet activation in the liver are less clear. The work presented in this thesis demonstrates for the first time that platelet activation via CLEC-2 is important in the pathogenesis of liver disease. In chronic human diseases (CLD) such as Primary Biliary Cirrhosis, and Alcoholic Liver disease I have demonstrated that the ligand for CLEC-2, podoplanin is upregulated on portal venules and increases proportionately to disease activity. I also note podoplanin staining on macrophage populations in CLD. Furthermore I show that this enhanced podoplanin expression may be a useful predictor of portal venous thrombosis, and correlates with MELD score for some categories of disease. In acute liver injury, CLEC-2-depended platelet activation has a profound effect on disease development. Here podoplanin expression occurs upon Kupffer cells in both humans and mice. Using carbon tetrachloride and paracetamol to induce acute liver injury in mice, I show that macrophage-expressed podoplanin activates platelets via CLEC-2. This interaction worsens liver injury, I next show that by blocking this interaction (using either CLEC-2 or podoplanin-deficient mice, or by using a function- blocking podoplanin antibody) liver recovery from toxic liver injury was remarkably enhanced. This was dependent upon enhanced hepatic neutrophil recruitment in a TNF dependent fashion.
36
Bhavaraju, Kamala. "MOLECULAR PHYSIOLOGY OF THROMBOXANE A2 GENERATION IN PLATELETS." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/92746.
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Molecular and Cellular PhysiologyPh.D.
Cardiovascular diseases are a major cause of mortality and morbidity in the developed countries. Anti-platelet therapy is a cornerstone treatment for patients with cardiovascular diseases. Patients are routinely managed with a combination therapy consisting of aspirin and clopidogrel. Aspirin inhibits cyclooxygenase 1 (COX 1) a crucial intermediate enzyme involved in thromboxane biosynthesis. Clopidogrel on the other hand antagonizes ADP receptor P2Y12. ADP is a weak platelet agonist stored in platelet dense granules and is released upon platelet activation. ADP activates platelets through two purinergic receptors namely P2Y1 and P2Y12 these receptors couple to Gq and Gi class of G-proteins, respectively. P2Y1 causes calcium mobilization through activation of PLC-β. P2Y12 inhibits adenylyl cyclase, causes activation of Rap1B and Akt. Signaling from both the receptors is required for complete integrin activation, thromboxane generation and Erk activation. Previous studies have shown that P2Y12 potentiates fibrinogen receptor activation, secretion, thrombi stabilization, thrombin generation, platelet leukocyte aggregation formation. ThromboxaneA2 (TXA2) is a potent platelet agonist generated through arachidonic acid metabolism in platelets. TXA2 thus, generated after platelet activation acts as a positive feedback mediator along with ADP. Under physiological conditions, platelet activation leads to thrombin generation through coagulation cascades. Generated thrombin activates PAR receptors and ADP is released from dense granules, which further potentiates thromboxane generation downstream of PARs. Current anti-platelet therapy regimens often include P2Y12 antagonists and aspirin in management of patients with acute coronary syndrome (ACS) and in those undergoing percutaneous coronary intervention (PCI) with stent implantation. However, there still exists a need for improved treatment strategies as not all patients benefit from this dual combination therapy. Reasons include, poor responders either to P2Y12 antagonists or to aspirin, or if aspirin is contraindicated in these patient populations. In the current study we evaluated the role of P2Y12 in thromboxane generation under physiological conditions. We studied serum thromboxane generation in a model system wherein P2Y12 was antagonized or deficient. Using pharmacological approaches we show that dosing mice with 30mg/Kg/body weight clopidogrel or 3mg/Kg/body weight prasugrel decreased serum thromboxane levels when compared to the control mice. Pre-treatment of human blood ex vivo with active metabolites of clopidogrel (R361015) or prasugrel (R138727) also led to reduction in thromboxane levels. We also evaluated serum thromboxane levels in P2Y receptor null mice, serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, and were inhibited in P2Y12 null mice. Furthermore, serum thromboxane levels in P2Y12 deficient patients, previously described in France and Japan, were also evaluated and these patients had lower serum thromboxane levels compared to normal controls. In a pilot study, serum thromboxane levels were radically reduced in healthy human volunteers upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y12 antagonism alone can decrease physiological thromboxane levels. Thus P2Y12 regulates physiological thromboxane levels. Further it is known that ADP-induced thromboxane generation is integrin-dependent. However it is not clear if other potent platelet agonists like thrombin require outside-in signaling for thromboxane generation. Our results show that thrombin-induced thromboxane generation was independent of integrins i.e. when platelets were stimulated with PAR agonists in presence of fibrinogen receptor antagonist thromboxane generation was not affected. Since PAR agonists, unlike ADP, activate G12/13 signaling pathways. Hence, we hypothesized that these pathways might play a role in TXA2 generation. Our results show, that inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by costimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Next, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. Our results showed that c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. We observed differential activation of FAK downstream of integrins and G12/13 pathways. ADP-induced activation of FAK was integrindependent and SFK-independent. On the other hand selective activation of G12/13 pathway lead to FAK activation, in SFK and Rho dependent manner. We also evaluated specificity of new FAK inhibitor TAE-226 to understand the role of FAK in TXA2 generation. Our results showed that TAE-226 exhibited non-specific effects at higher concentrations. Furthermore, in comparison to WT mice, FAK null mice did not show any difference in TXA2 generation. Therefore, we concluded that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule downstream of integrins and G12/ 13 pathways contributing to TXA2 generation in platelets remains elusive.
Temple University--Theses
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Malboubi, Saeid. "In vitro actions of platelet rich plasma and resolvin E1 on osteoblast and osteoclast activity." Thesis, Boston University, 2009. https://hdl.handle.net/2144/35619.
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Thesis (MSD) --Boston University, Henry M. Goldman School of Dental Medicine, 2009 (Department of Periodontology and Oral Biology).Includes bibliographic references: leaves 52-59.
Platelet-rich plasma (PRP) is a concentrated gel of platelets that contains several growth factors. Growth factors have been recognized as the part of PRP that play role in regeneration of the bone. It is not clear how these growth factors in PRP affect the bone regeneration. Resolvin El (RvEl; 5S,12R,18R-trihydroxyeicosapentaenoic acid) is an pro-resolving lipid mediator derived from omega-3 fatty acid eicosapentaenoic acid and shown to have potent effects on the resolution of inflammation. The purpose of this study was to analyze the action of PRP and RVEl on the proliferation and behavior of osteoblasts and osteoclasts in vitro. PRP was prepared from 14 healthy donors. Osteoblast cultures were from a cell line (Saos2) of osteosarcoma cells. Osteoclasts were differentiated from primary human peripheral blood monocytes. Osteoclastic morphology was studied and activity was analyzed via resorption on dentin discs using SEM. PRP and RVE 1 were added at different doses and time-points. Osteoblast function was analyzed by osteocalcin expression and release. Osteoclast activity was assessed by resorption and cathepsin K expression. PRP and RvEl comparably increased the osteoblastic activity and suppressed the osteoclast differentiation and function. These results suggest that multiple tools are available to reverse the inflammation and restore the lost bone architecture as a result of periodontal disease.
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Mukkamala, Muralikrishna. "Regulation of Platelet-Activating Factor Acetylhydrolase by Oxidized Phospholipids and Proinflammatory Cytokines." VCU Scholars Compass, 2008. http://scholarscompass.vcu.edu/etd/653.
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Platelet-Activating Factor Acetylhydrolase (PAFAH) is a monocyte-derived phospholipase A2 that catalyzes the hydrolysis of platelet-activating factor (PAF) and has been implicated in atherosclerosis. Although PAF and other proinflammatory stimuli are postulated to induce the enzyme, mechanisms controlling PAFAH expression are largely unknown at present. We have shown that PAFAH induction in monocytes is increased in response to oxidized phospholipids. The PAFAH 5' flanking region has at least 10 putative Stat elements, and IL-6 has been shown to be downstream from the prostaglandin receptor, EP2, which has been shown to bind oxidized phospholipids, prompting the hypothesis that Stat proteins might regulate its expression. To test this hypothesis, we treated human monocytes with IL-6, a monocyte-derived cytokine that activates Stat3, IL-8, a monocyte-derived cytokine induced by Stat3, and oxidized 1-palmitoyl-2-arachidonoyl-sn-3-phosphocholine (oxPAPC), a major component of the oxidized LDL particle. Two monocyte-derived cell types, macrophages (MO) and dendritic cells (DC) were prepared from primary human monocytes. The cells were treated with various doses of IL-6, IL-8, or oxPAPC for various time frames in the absence of serum. Culture supernatants from the cytokine-treated cells were harvested and screened for PAFAH protein and activity and cell monolayers were assessed for PAFAH mRNA by quantitative real time PCR (qPCR). Cells treated with oxPAPC were further analyzed for secreted IL-6 using ELISA and activation of Stat3 using Western Blot. Both IL-6 and IL-8 induced PAFAH expression in a dose-dependent manner. Although both MO and DC responded to the cytokines, preliminary experiments suggested that induction of PAFAH is more robust in DC than MO. Cytokine-treated cells exhibited increased PAFAH activity in their culture supernatants that correlated with increased PAFAH protein levels. Treatment with oxPAPC induced IL-6 secretion and subsequent Stat3 activation in DC. Together, these data support the hypothesis that PAFAH expression is regulated by oxidized phospholipids and proinflammatory cytokines in developing atheromas.
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Dai, Yuheng. "The Commercilazation of a Noval Antithrombotic Drug." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1505303242046038.
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Huang, Yunjie. "ADP-RIBOSYLATION FACTOR 6 (ARF6) REGULATES INTEGRIN αIIbβ3 TRAFFICKING, PLATELET SPREADING, AND CLOT RETRACTION." UKnowledge, 2015. http://uknowledge.uky.edu/biochem_etds/20.
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Endocytic trafficking of platelet surface receptors plays a role in the accumulation of granule cargo (i.e. fibrinogen and VEGF) and thus could contribute to hemostasis, angiogenesis, or inflammation. However, the mechanisms of platelet endocytosis are poorly understood. The small GTP-binding protein, ADP-ribosylation factor 6 (Arf6), regulates integrin trafficking in nucleated cells; therefore, we posited that Arf6 functions similarly in platelets. To address this, we generated platelet-specific, Arf6 knockout mice. Arf6-/- platelets had a storage defect for fibrinogen but not other cargo, implying Arf6’s role in integrin αIIbβ3 trafficking. Additionally, platelets from Arf6-/- mice injected with biotinylated-fibrinogen, showed lower accumulation of the modified protein than did WT mice. Resting and activated αIIbβ3 levels, measured by FACS, were unchanged in Arf6-/- platelets. Arf6-/- platelets had normal agonist-induced aggregation and ATP release; however, they showed faster clot retraction and enhanced spreading, which appears due to altered αIIbβ3 trafficking since myosin light chain phosphorylation and Rac1 activation, in response to thrombin, were unaffected. Arf6-/- mice showed no hemostasis defect in tail-bleeding or FeCl3–induced carotid injury assays. These data suggest a role for Arf6 in integrin αIIbβ3 trafficking in platelets.
Additionally, the regulation of Arf6 in platelets was also investigated, focusing on integrin αIIbβ3 outside-in signaling which was suggested to be responsible for the second wave of Arf6-GTP loss. G protein-coupled receptor kinase-interacting protein 1 (GIT1), a GTPase-activating protein (GAP) toward Arf6, is suggested to be involved in αIIbβ3 downstream signaling. I found that GIT1, complex with β-PIX, was translocated to the detergent-insoluble pellet upon human platelet activation, a process that is blocked by RGDS and myrArf6 peptide treatment. Moreover, tyrosine-phosphorylation of GIT1 was impaired by treatment with both peptides or with actin polymerization inhibitors. GIT1’s role in platelets was further studied using platelet-specific, GIT1 knockout mice. GIT1-/- platelets failed to show any defect, including clot retraction or fibrinogen storage. Unlike human platelets, GIT1 expression levels were much lower in mouse platelets, suggesting that GIT2 may be the functionally relevant Arf6-GAP in mouse platelets. The data in this dissertation identify that Arf6 mediates fibrinogen storage, implying its role in integrin αIIbβ3 trafficking in platelets.
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Fierros, Juancarlos. "PLATELET DERIVED GROWTH FACTOR RECEPTOR B (PDGFRB) EXPRESSING CELLS DURING ZEBRAFISH CORONARY VESSEL DEVELOPMENT." CSUSB ScholarWorks, 2017. https://scholarworks.lib.csusb.edu/etd/567.
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Coronary heart disease is a prevalent issue in developed countries throughout the world. It can have crippling effects on the quality of life and even lead to mortality, in the case of myocardial infarction. Part of the problem is the lack of a robust regenerative response in mammals after injury. Zebrafish have an amazing ability to regenerate after injury, and studies have demonstrated that the regenerative response recapitulates embryonic development. Our lab previously reported the first analysis of coronary vessel development in zebrafish and demonstrated that coronary endothelial cells undergo angiogenesis to form a vascular network. The roles of perivascular cells in this process have not been examined in zebrafish. Using a transgenic reporter line marking pdgfrb expression, I found that pdgfrb is first observed in epicardium at the AV canal. At later stages of coronary vessel development, pdgfrb positive cells become localized to the perivascular region of mature vessels. I also observe that early in development, Tcf21 and pdgfrb co-express, which suggests a close relationship between the epicardium and pdgfrb+ cells. Previous findings from our lab revealed that cxcl12b+ cells localize to large coronary vessels during development. My findings reveal that pdgfrb+ marks perivascular cells of both capillaries and large coronary vessels. Lineage tracing analysis revealed that a subset of pdgfrb+ perivascular cells derive from tcf21 labeled epicardial cells. To see if disruption of Pdgfrb signaling impacts coronary development, I examined pdgfrb mutant hearts. In the Pdgfrb mutant, a mature coronary vessel network fails to form, and instead we observe isolated endothelial cell islands. Lastly, I characterized a transgenic line that expresses a dominant negative form of Pdgfrb (dnpdgfrb) and can be potentially used for later developmental and/or regenerative studies. My findings indicate strong dnpdgfrb induction can be achieved at adult stages. My studies will greatly enhance our current understanding of coronary vessel development, and can be used as the basis for studying perivascular cells and their interactions with endothelial cells after cardiac injury in regeneration.
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Grundström, Gunilla. "Functional Studies of Collagen-Binding Integrins α2β1 and α11β1 : Interplay between Integrins and Platelet-Derived Growth Factor Receptors." Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-3686.
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Integrins are heterodimeric cell surface receptors, composed of an α- and a β-subunit, which mediate cell-extracellular matrix (ECM) interactions. Integrins mediate intracellular signals in response to extracellular stimuli, and cooperate with growth factor and other cytokine receptors. Cells execute their differentiated functions anchored to an ECM. In this thesis functional properties of the two collagen-binding integrins α2β1 and α11β1 were studied. In addition, the impact of β1 cytoplasmic tyrosines in collagen-induced signalling was analyzed.
The integrin α11β1 is the latest identified collagen-binding integrin. In this study, tissue distribution of α11 mRNA and protein during embryonal development was explored, and the first α11β1-mediated cellular functions were established. Both α11 protein and mRNA were present in mesenchymal cells in intervertebral discs and around the cartilage of the developing skeleton. α11 protein was also detected in cornea keratinocytes. α11β1 mediated cation-dependent adhesion to collagen types I and IV and localized to focal adhesions. In addition, α11β1 mediated contraction of a collagen lattice and supported cell migration through a collagen substrate. PDGF-BB and FBS both stimulated α11β1-mediated contraction and directed migration.
Expression of β1Y783,795F in β1-null cells, prevents activation of FAK in response to fibronectin, and decreases cell migration. In this study, we investigated how this mutation affected α2β1-mediated functions in response to collagen. The β1 mutation impaired collagen gel contraction and prevented activation of FAK, Cas and Src on planar collagen, but not in collagen gels. PDGF-BB stimulated contraction via αvβ3, which also induced activation of Cas in collagen gels. The YY-FF mutation also abolished β1A-dependent downregulation of β3.
In the final study integrin-crosstalk during collagen gel contraction was investigated. In cells lacking collagen-binding integrins αvβ3 mediated contraction. Clustering of β1-integrins by antibodies and PDGF-BB stimulated αvβ3-mediated contraction in an ERK-dependent way. Expression of α2β1, but not α11β1, prevented αvβ3-mediated contraction. Contraction by α2β1 and α11β1 was ERK-independent.
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Mandavyapuram, Hima Bindu. "ANTIBIOTIC DELIVERY SYSTEM FOR SURGICAL SITE INFECTION PREVENTION IN SPINAL IMPLANT SURGERY." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1275624787.
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Griffiths, Rachael. "The Regulation of Platelet Activating Factor Acetylhydrolase by Oxidized Phospholipids." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1913.
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Platelet-activating factor acetylhydrolase (PAFAH) is elevated in atherosclerosis and may play a role in pathogenesis of this disease. Molecular mechanisms regulating the expression of this lipoprotein-associated PLA2 are indistinct. Mildy oxidized low density lipoprotein (oxLDL) and monocytes (the primary source of PAFAH) are co-localized in early atheromas. Monocytes are activated by oxidized phospholipids (oxPL) in the oxLDL particle. We hypothesized that oxPL-activated monocytes are the source of increased levels of PAFAH in atherosclerosis. We found that PAFAH expression is significantly induced by OxPAPC and in particular long-chain fractions of oxPAPC in monocytes and cytokine-differentiated DC, but not cytokine-differentiated MO. Furthermore, spontaneously differentiated MO and DC from monocytes of non-periodontitis and aggressive periodontitis subjects, oxPAPC induced PAFAH in DC alone. 1-palmitoyl-2-epoxyisoprostane-sn-glycero-3-phosphocholine (PEIPC) is a particularly bioactive component of long-chain oxPAPC fractions that binds the prostaglandin receptor subtypes DP1 and EP2. We revealed using selective agonists and antagonists of these receptors that DP1 and EP2 are required for the induction of PAFAH expression. OxPAPC stimulates IL-6 release from monocytes and this cytokine is required for oxPAPC-induced PAFAH expression. We next tested the hypothesis that oxPAPC did not induce PAFAH in MO because a key component of the signaling machinery was lacking. Flow cytometric and immunoblot analyses demonstrated that MO express very low levels of IL-6 receptor in comparison to DC and monocytes. Based on these observations, we propose that long-chain oxPL induce PAFAH expression by binding DP1 and/or EP2 and stimulating IL-6 production. These data strongly support the hypothesis that oxLDL-activated DC are the source of high PAFAH levels in atherosclerosis. Platelet activating factor (PAF) is the inflammatory phospholipids for which PAFAH is named. PAF has been shown by other investigators to induce the expression of PAFAH. In our physiologically relevant monocytes, PAF suppresses PAFAH transcription and expression. 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphatidylcholine (POVPC) is a short-chain oxPL that signals through the PAF receptor. Our preliminary data suggest that like PAF, POVPC suppresses PAFAH expression in monocytes. Further investigation into the effects of the short-chain oxPL are warranted. Our data support the hypothesies that oxPL-activated DC are the source of high PAFAH levels in atherosclerosis.
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Hedlund, Eva-Maria. "Molecular mechanisms of angiogenic synergism between Fibroblast Growth Factor-2 and Platelet Derived Growth Factor-BB." Thesis, Södertörn University College, School of Life Sciences, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-932.
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Bynagari, Yamini Saraswathy. "Molecular Physiology of Novel Class of Protein Kinase C isoforms in Platelets." Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/103230.
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Molecular and Cellular PhysiologyPh.D.
Platelets are primary components of hemostasis. However, incongruous activation of platelets lead to thrombosis, which result in multiple cardio-vascular and cerebrovascular complications. Thus, platelet activation is tightly regulated. Molecular components that aid in activation of platelets have been extensively studied. However, molecular pathways that negatively regulate platelet activation and prevent accidental activation of platelets are poorly understood. In this study we investigated the molecular mechanisms that negatively regulate platelet activation. Protein Kinase C isforms (PKCs) are serine threonine kinases that regulate various platelet functional responses leading to hemostasis. Positive regulatory role of PKCs towards platelet aggregation and secretion has been extensively studied. However, we have recently demonstrated that PKCs negatively regulate ADP- induced thromboxane generation. The PKC isoforms and mechanism involved in this process have not been known. Thus, in this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms that regulate thromboxane generation. Thromboxane generation in platelets is a multi-step process beginning with cPLA2 activation. cPLA2 activation is the rate limiting step in the process of thromboxane generation. Furthermore, cPLA2 activation is regulated by ERK and calcium in various cell systems including platelets. PKC inhibition potentiated both cPLA2 and ERK activation, suggesting that PKCs negatively regulate thromboxane generation by regulating ERK activation, which in turn regulates cPLA2 activation. Furthermore, we have also shown that PKCs negatively regulate ADP-induced calcium mobilization. ADP activates platelets via P2Y1 and P2Y12 receptors. P2Y12 receptor-mediated signaling is shown to positively regulate P2Y1-mediated calcium mobilization in platelets. Furthermore, PKCs are shown to negatively regulate P2Y12 receptor desensitization in platelets. Thus, we investigated if PKCs regulate calcium mobilization indirectly by regulating P2Y12 receptor function. However, PKCs regulate calcium mobilization independent of P2Y12 receptor signaling. In summary we have shown that PKC isoforms negatively regulate ADP-induced thromboxane generation by regulating calcium mobilization and ERK activation that in turn regulates cPLA2 activity. We further investigated the PKC isoforms involved in this process. Based on our results with Go-6976, a classical PKC inhibitor and GF109203X, a pan PKC inhibitor, we identified that that novel or atypical PKC isoforms are involved in negative regulation of ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We first investigated the nPKCs activated by ADP. In aspirin-treated platelets, ADP failed to activate nPKC θ and δ non-stirring conditions. Thus, we conclude that these isoforms are not involved in negative regulation of thromboxane generation. We further investigated if other non-classical PKC isoforms i. e nPKC η and ε or atypical PKC isoforms could be involved in this process. We began our investigation with the mechanism of activation and functional role of nPKC η in platelets. The mechanism of activation of PKCs has been extensively studied in various cell systems including platelets. However, the mechanism by which they are inactivated is not completely understood. In this study, we demonstrate a novel mechanism of inactivation of nPKC η isoform by integrin associated serine/threonine phosphatase. we demonstrated that ADP activates nPKC η via P2Y1 receptor coupled to Gq. As expected, Gi pathway, which does not generate DAG or mobilize calcium, has no role in regulation of nPKC η. Interestingly, we show that upon activation of platelets, αIIbβ3 mediated outside-in signaling dephosphorylates nPKCη through PP1γ phosphatase. We have also evaluated the role of nPKC η using η-RACK antagonistic peptides that interfere with enzyme-substrate interaction. Similar antagonistic peptides have been successfully used in various cell systems such as cardiomyocytes and neuronal cell. Using η-RACK antagonists we have demonstrated that nPKC η positively regulates agonist- induced thromboxane generation with no effect on agonist- induced platelet aggregation. The peptides were targeted in to the cell using TAT carrier protein, which is also used as a negative control for these experiments. The specificity of η-RACK antagonistic peptides is further elucidated by the fact that they do not affect the platelet aggregation. In summary, nPKC η is activated by ADP via P2Y1 receptor and is dephosphorylated by integrin αIIbβ3 via PP1γ phosphatase. Furthermore, activated nPKC η positively regulates ADP- induced thromboxane generation with no effect on aggregation. Since, our aim was to investigate the nPKC isoforms that negatively regulate ADPinduced thromboxane generation we investigated if nPKC ε is involved in this process. We made use of PKC ε knockout mice (PKC ε KO) for this process. We observed potentiated thromboxane generation in ADP-induced PKC ε murine platelets compared to witd type murine platelets. Thus, PKC ε might be one of the PKC isoforms involved in negative regulation of ADP-induced thromboxane generation. However, we failed to detect PKC ε in human platelets using western blot analysis. Since, PKC ε has been reported to be a part of platelet kinase repertoire, it could be limitation of our technique that we failed to detect it in western blot analysis. Since, PKCs negatively regulate ADP-induced thromboxane generation, we also investigated if PKCs also regulate PAR-mediated thromboxane generation. Similar to ADP, PAR-mediated thromboxane generation is not affected by Classical PKC isoforms. Furthermore, although non-classical PKC isoforms negatively regulate thromboxane generation, the extent of negative regulation is smaller and non-significant compared to ADP. Thus, we investigated if activation of nPKC isoforms were different between ADP and AYPGKF (PAR4 agonist). While, ADP fails to activate nPKC δ and θ, PARs activate Them. Furthermore, we have recently demonstrated that nPKC δ and θ are positive regulators of PAR-mediated platelet functional responses. Therefore, PKCinduced potentiation of thromboxane generation by ADP and PAR agonist are different due to differential activation of PKCs. This data lead to our final project, where we investigated the reason for differential activation of nPKC isoforms by various platelet agonists. Using strong and weak platelet agonists and DAG analogue, DiC8, we demonstrated that different platelet agonists differentially regulate nPKC activation due to variable amounts of DAG generated by them. Furthermore, we also have demonstrated that nPKC η and ε have higher affinities to DAG compared to nPKC δ and θ.
Temple University--Theses
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Ning, Jing 1953. "Effects of heterologous and homologous stroma-free hemoglobin and polyhemoglobin on complement activation, blood coagulation, platelet aggregation, and blood cells in rats." Thesis, McGill University, 1990. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=74319.
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This research serves to answer the following basic question: Are potential toxic effects of hemoglobin solutions due to hemoglobin itself? Homologous and heterologous stroma-free hemoglobin and polyhemoglobin were investigated for their effects on complement activation, blood coagulation, platelet aggregation, blood cell counts, plasma electrolytes, proteins and enzymes in rats. A modified Mayer's method for testing whole complement hemolytic activity (CH$ sb{50}$) was developed. C$ sb3$ and C$ sb{ rm 3a}$ were also measured. Purified homologous or heterologous stroma-free hemoglobin or polyhemoglobin did not cause complement activation when incubated with rat plasma or when infused into rats. In addition, blood coagulation parameters did not change significantly in rats who received purified homologous or heterologous stroma-free hemoglobin or polyhemoglobin. Polyhemoglobin prepared from homologous or heterologous hemoglobin did not induce any changes in total leucocyte, differential or platelet counts. Effects of PolyHb and stroma-free hemoglobin on platelet aggregation were also studied. Plasma electrolytes, proteins, and enzymes tested were all within the normal ranges in rats who received purified homologous or heterologous polyhemoglobin infusions. Infusion of membrane stroma and endotoxins caused complement activation. This suggests that it is not purified hemoglobin or polyhemoglobin, but contaminants in hemoglobin preparations, which are responsible for some of the potential toxic effects observed. This study also has practical implications. It shows that only highly purified hemoglobin preparations should be used as blood substitutes for clinical applications.
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Qureshi, Shahryar Jamshed. "Nanomaterial Charge-Dependent Platelet Activating Factor Receptor Agonism in Human Epidermal Cells." Wright State University / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=wright1535391805311408.
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Disney, Benjamin Robert. "Assessment of platelet activation and prothrombotic risk following acute upper gastrointestinal bleeding and bleeding in the context of acute coronary syndromes." Thesis, University of Birmingham, 2017. http://etheses.bham.ac.uk//id/eprint/7505/.
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Patients presenting with acute upper gastrointestinal bleeding have an increased incidence of cardiovascular events, with mortality invariably related to factors other than the bleeding itself; the majority of deaths related to cardiovascular disease. Patients presenting with bleeding complicating acute coronary syndromes are at an increased risk of recurrent cardiovascular events and have higher short-term and long-term mortality when compared to patients with uncomplicated acute coronary syndromes. This study has shown the preferential use of the Blatchford score in identifying patients suitable for early discharge; using a cut-off score of 2 could avoid up to 15% of admissions. The results show significant diurnal and seasonal variation in the presentation of acute upper gastrointestinal bleeding with fewer admissions in the winter months. This study has shown an increase in levels of platelet activation and prothrombotic markers (d-dimer and vWF) in the acute upper gastrointestinal bleeding population. No such findings were seen in the acute coronary syndromes complicated by bleeding group. These novel findings may help explain the excess of cardiovascular mortality in patients with presenting upper gastrointestinal bleeding and give a biological rationale to restarting antiplatelet agents early in these patients. Further studies are required to confirm and further investigate these findings.
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Patcharapinyopong, Thanasan. "Antimicrobial and Anti-Platelet Activity in Botanical Extracts of Plants Collected in Northern Thailand." Thesis, University of North Texas, 2019. https://digital.library.unt.edu/ark:/67531/metadc1505273/.
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The purpose of my research work was to assess a variety of Northern Thailand plants tissue extracts for antibacterial and anti-platelet aggregation activity. The Minimum Inhibitory Concentration assay method was used to assess antimicrobial activity of plant extracts, while the Zebrafish Platelet Aggregation Assay and the in vitro Whole Human Blood Impedance Aggregation Assay were used to study anti-platelet activity. Forty one plant extracts harvested from the tissues of 26 plants collected from Northern Thailand were assessed. Thirty-four plant extracts were found to have antibacterial activity against the Gram positive bacteria Staphylococcus aureus and/or Bacillus subtilis, while six plant extracts demonstrated activity against the Gram negative bacterium, Escherichia coli. Thirteen plant extracts exhibited anti-platelet aggregation activity better than the positive control. Two crude plant extracts, twigs from Garcinia sp. and twigs from Goniothalamus chilensis were selected for fractionation. Five of the 12 fractions showed anti-platelet activity. Four fractions (two from each plant extract) were selected for further sub-fractionation. Fourteen of 35 sub-fractions were selected for further testing of anti-platelet aggregation activity with 12 sub-fractions demonstrating positive antiplatelet activity. Positive sub-fractions were analyzed using liquid chromatography-mass spectrometry to determine their chemical properties. Three compounds that possessed anti-platelet activity were characterized by nuclear magnetic resonance. The compounds, all isolated from Garcinia sp., were identified as cambogin, isoxanthochymol and guttiferone F.