Proteolysis of platelet receptors in humans and other species

2010 ◽  
Vol 391 (8) ◽  
Author(s):  
Jian L. Qiao ◽  
Yang Shen ◽  
Elizabeth E. Gardiner ◽  
Robert K. Andrews
Keyword(s):  
Molecular Mechanisms ◽  
Surface Expression ◽  
Altered Expression ◽  
Platelet Receptors ◽  
Published Evidence ◽  
Functional Consequences ◽  
Specific Receptors ◽  
Thrombotic Diseases ◽  
Von Willebrand

AbstractIn the past 5 years, metalloproteinase-mediated ectodomain shedding of platelet receptors has emerged as a new mechanism for modulating platelet function. By regulating surface expression of the platelet-specific receptors, glycoprotein (GP)VI that binds collagen, and GPIbα (the major ligand-binding subunit of the GPIb-IX-V complex) that binds von Willebrand factor (VWF) and other procoagulant and proinflammatory ligands, shedding not only irreversibly downregulates GPVI/GPIbα function, but generates proteolytic fragments that might be unique biomarkers or modulators in plasma. This is potentially significant because GPVI and GPIbα are involved in initiating thrombotic diseases such as heart attack and stroke, as well as autoimmune diseases where anti-platelet antibodies result in thrombocytopenia. Altered expression levels of GPIbα/GPVI are associated with both thrombotic propensity and platelet aging, suggesting an additional role in platelet clearance. Although emerging data are elucidating molecular mechanisms underlying GPIbα/GPVI shedding, evidence for the functional consequences of sheddingin vivo, either clinically or in animal models, is far more limited. Here we consider recent published evidence for GPVI or GPIbα shedding in humans, nonhuman primates and mice, and whether conservation of sheddase cleavage sites across species points to a functional role for metalloproteolytic sheddingin vivo.

Effects of microgravity and hypergravity on platelet functions

2009 ◽  
Vol 101 (05) ◽  
pp. 902-910 ◽  
Author(s):  
Yuedan Wang ◽  
Rong Yan ◽  
Quanwei Shi ◽  
Zhicheng Wang ◽  
Yanhong Yuan ◽  
...  
Keyword(s):  
Simulated Microgravity ◽  
Surface Expression ◽  
Gravity Change ◽  
Thrombotic Diseases ◽  
Von Willebrand ◽  
Willebrand Factor ◽  
The Brain ◽  
Platelet Functions

SummaryMany serious thrombotic and haemorrhagic diseases or fatalities have been documented in human being exposed to micro-gravity or hypergravity environments, such as crewmen in space, roller coaster riders, and aircrew subjected to high-G training. Some possible related organs have been examined to explore the mechanisms underlying these gravity change-related diseases. However, the role of platelets which are the primary players in both thrombosis and haemostasis is unknown. Here we show that platelet aggregation induced by ristocetin or collagen and platelet adhesion to von Willebrand factor (VWF) were significantly decreased after platelets were exposed to simulated microgravity. Conversely, these platelet functions were increased after platelets were exposed to hypergravity. The tail bleeding time in vivo was significantly shortened in mice exposed to high-G force, whereas, was prolonged in hindlimb unloaded mice. Furthermore, three of 23 mice died after 15 minutes of –8 Gx stress. Platelet thrombi disseminated in the heart ventricle and blood vessels in the brain, lung, and heart from the dead mice. Finally, glycoprotein (GP) Ibα surface expression and its association with the cytoskeleton were significantly decreased in platelets exposed to simulated microgravity, and obviously increased in hypergravity-exposed platelets. These data indicate that the platelet functions are inhibited in microgravity environments, and activated under high-G conditions, suggesting a novel mechanism for gravity change-related haemorrhagic and thrombotic diseases. This mechanism has important implications for preventing and treating gravity change-related diseases, and also suggests that special attentions should be paid to human actions under different gravity conditions.

scholarly journals Molecular Characterization of the Von Willebrand Factor Type D Domain of Vitellogenin from Takifugu flavidus

Marine Drugs ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. 181
Author(s):  
Kun Qiao ◽  
Caiyun Jiang ◽  
Min Xu ◽  
Bei Chen ◽  
Wenhui Qiu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Protein Domain ◽  
Type D ◽  
Protein Toxin ◽  
Factor Type ◽  
Von Willebrand ◽  
Willebrand Factor

The von Willebrand factor type D (VWD) domain in vitellogenin has recently been found to bind tetrodotoxin. The way in which this protein domain associates with tetrodotoxin and participates in transporting tetrodotoxin in vivo remains unclear. A cDNA fragment of the vitellogenin gene containing the VWD domain from pufferfish (Takifugu flavidus) (TfVWD) was cloned. Using in silico structural and docking analyses of the predicted protein, we determined that key amino acids (namely, Val115, ASP116, Val117, and Lys122) in TfVWD mediate its binding to tetrodotoxin, which was supported by in vitro surface plasmon resonance analysis. Moreover, incubating recombinant rTfVWD together with tetrodotoxin attenuated its toxicity in vivo, further supporting protein–toxin binding and indicating associated toxicity-neutralizing effects. Finally, the expression profiling of TfVWD across different tissues and developmental stages indicated that its distribution patterns mirrored those of tetrodotoxin, suggesting that TfVWD may be involved in tetrodotoxin transport in pufferfish. For the first time, this study reveals the amino acids that mediate the binding of TfVWD to tetrodotoxin and provides a basis for further exploration of the molecular mechanisms underlying the enrichment and transfer of tetrodotoxin in pufferfish.

Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia

Blood ◽  
2002 ◽  
Vol 99 (12) ◽  
pp. 4486-4493 ◽  
Author(s):  
Gregor Theilmeier ◽  
Carine Michiels ◽  
Erik Spaepen ◽  
Ingrid Vreys ◽  
Désiré Collen ◽  
...  
Keyword(s):  
Von Willebrand Factor ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Perfusion Studies ◽  
Von Willebrand ◽  
Willebrand Factor

Platelets are thought to play a causal role during atherogenesis. Platelet-endothelial interactions in vivo and their molecular mechanisms under shear are, however, incompletely characterized. Here, an in vivo platelet homing assay was used in hypercholesterolemic rabbits to track platelet adhesion to plaque predilection sites. The role of platelet versus aortic endothelial cell (EC) activation was studied in an ex vivo flow chamber. Pathways of human platelet immobilization were detailed during in vitro perfusion studies. In rabbits, a 0.125% cholesterol diet induced no lesions within 3 months, but fatty streaks were found after 12 months. ECs at segmental arteries of 3- month rabbits expressed more von Willebrand factor (VWF) and recruited 5-fold more platelets than controls (P < .05, n = 5 and 4, respectively). The 3-month ostia had an increased likelihood to recruit platelets compared to control ostia (56% versus 18%, P < .0001, n = 89 and 63, respectively). Ex vivo, the adhesion of 3-month platelets to 3-month aortas was 8.4-fold increased compared to control studies (P < .01, n = 7 and 5, respectively). In vitro, endothelial VWF–platelet glycoprotein (GP) Ib and platelet P-selectin– endothelial P-selectin glycoprotein ligand 1 interactions accounted in combination for 83% of translocation and 90% of adhesion (P < .01, n = 4) of activated human platelets to activated human ECs. Platelet tethering was mainly mediated by platelet GPIbα, whereas platelet GPIIb/IIIa contributed 20% to arrest (P < .05). In conclusion, hypercholesterolemia primes platelets for recruitment via VWF, GPIbα, and P-selectin to lesion-prone sites, before lesions are detectable.

scholarly journals Demonstration of reduced in vivo surface expression of activating mutant thyrotrophin receptors in thyroid sections

2002 ◽  
pp. 163-171 ◽  
Author(s):  
M Sequeira ◽  
B Jasani ◽  
D Fuhrer ◽  
M Wheeler ◽  
M Ludgate
Keyword(s):  
Molecular Mechanisms ◽  
Thyroid Tissue ◽  
Surface Expression ◽  
Germline Mutations ◽  
Antigen Retrieval ◽  
Graves Disease ◽  
Paraffin Sections ◽  
Gain Of Function

OBJECTIVE: Thyroid function and growth are controlled by TSH. Hyperthyroidism can be due to Graves' Disease (GD), in which thyroid-stimulating antibodies mimic TSH, or gain-of-function mutations in the TSH receptor (TSHR). These activating mutations have poor surface expression when assessed in non-thyroidal cells in vitro but nothing is known of their in vivo behaviour. Several TSHR antibodies have been produced but none has been applied to thyroid paraffin sections. This study aimed to develop a technique suitable for use on paraffin sections and apply it to investigate TSHR expression in thyroids harbouring activating TSHR germline mutations compared with normal and GD thyroids. DESIGN AND METHODS: Immunocytochemistry coupled with antigen retrieval, using a spectrum of antibodies to the TSHR, was applied to paraffin sections of GD thyroid tissue. Subsequently, TSHR immunoreactivity was examined in three normal thyroids, three patients with GD and three patients with familial hyperthyroidism, due to different gain-of-function TSHR germline mutations, using the optimised protocol. RESULTS: Two antibodies, A10 and T3-495, to the extracellular domain (ECD) and membrane spanning region (MSR) of the TSHR respectively, produced specific basolateral staining of thyroid follicular cells. In normal and GD thyroids, basolateral staining with T3-495 was generally more intense than with A10, suggesting a possible surfeit of MSR over ECD. Graves' Disease thyroids have more abundant TSHR than normal glands. In contrast, thyroids harbouring gain-of-function mutations have the lowest expression in vivo, mirroring in vitro findings. CONCLUSIONS: The development of an immunocytochemical method applicable to paraffin sections has demonstrated that different molecular mechanisms causing hyperthyroidism result in the lowest (mutation) and highest (autoimmunity) levels of receptor at the thyrocyte surface.

scholarly journals Bipolar-associated miR-499-5p controls neuroplasticity by downregulating the Cav1.2 L-type voltage gated calcium channel subunit CACNB2

2021 ◽  
Author(s):  
Helena Caria Martins ◽  
Oezge A Sungur ◽  
Carlotta Gilardi ◽  
Michael Pelzl ◽  
Silvia Bicker ◽  
...  
Keyword(s):  
Calcium Channel ◽  
Calcium Homeostasis ◽  
Childhood Maltreatment ◽  
Molecular Mechanisms ◽  
Short Term Memory ◽  
Surface Expression ◽  
Neurocognitive Dysfunction ◽  
Voltage Gated ◽  
Depressive Episodes

Bipolar disorder (BD) is a chronic mood disorder characterized by alternating manic and depressive episodes, often in conjunction with cognitive deficits. Dysregulation of neuroplasticity and calcium homeostasis as a result of complex genetic environment interactions are frequently observed in BD patients, but the underlying molecular mechanisms are largely unknown. Here, we show that a BD-associated microRNA, miR-499-5p, regulates neuronal dendrite development and cognitive function by downregulating the BD risk gene CACNB2. miR-499-5p expression is increased in peripheral blood of BD patients and healthy subjects at risk of developing the disorder due to a history of childhood maltreatment. This up-regulation is paralleled in the hippocampus of rats which underwent juvenile social isolation. Elevating miR-499-5p levels in rat hippocampal pyramidal neurons impairs dendritogenesis and reduces surface expression and activity of the voltage-gated L-type calcium channel Cav1.2. We further identified CACNB2, which encodes a regulatory β-subunit of Cav1.2, as a direct target of miR-499-5p in neurons. CACNB2 downregulation is required for the miR-499-5p dependent impairment of dendritogenesis, suggesting that CACNB2 is an important downstream target of miR-499-5p in the regulation of neuroplasticity. Finally, elevating miR-499-5p in the hippocampus in vivo is sufficient to induce short-term memory impairments in rats haploinsufficient for the Cav1.2 pore forming subunit Cacna1c. Taken together, we propose that stress-induced upregulation of miR-499-5p contributes to dendritic impairments and deregulated calcium homeostasis in BD, with specific implications for the neurocognitive dysfunction frequently observed in BD patients.

Inhibition of Calmodulin Triggers Apoptosis Events in Human Platelets.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3003-3003
Author(s):  
Zhicheng Wang ◽  
Suping Li ◽  
Guanglei Liu ◽  
Quanwei Shi ◽  
Rong Yan ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Conflicts Of Interest ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Human Platelets ◽  
Platelet Apoptosis ◽  
Potent Antagonist ◽  
Von Willebrand

Abstract Abstract 3003 Poster Board II-980 Calmodulin (CaM) is a calcium-sensing protein ubiquitously expressed in every eukaryotic cell type regulating biological processes such as cell proliferation, vesicular fusion, fertilization and apoptosis. CaM antagonists induce apoptosis in various tumor models and inhibit tumor cell invasion and metastasis, thus some of which have been extensively used as anti-cancer agents. Tamoxifen (TMX), a potent antagonist of CaM, has been in the center of management of hormone-sensitive breast cancer, and also represents the best example of chemo-prevention to reduce the incidence of invasive breast cancer. Furthermore, TMX is potentially useful in treatment of other kinds of cancer. However, TMX has some severe side effects, one of which is thrombocytopenia. Up to now, the pathogenesis of thrombocytopenia still remains unclear. In platelets, CaM has been found to bind directly to cytoplasmic domains of several platelet receptors. Incubation of platelets with CaM antagonists impairs the receptors-related platelet function. However, it is still unclear whether CaM antagonists, especially TMX, induce platelet apoptosis. Here, we show that CaM antagonists TMX and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W7) dose-dependently induce apoptotic events in human platelets, including depolarization of mitochondrial inner transmembrane potential, caspase-3 activation, gelsolin cleavage and phosphatidylserine (PS) exposure. CaM antagonist did not incur platelet activation as detected by P-selectin surface expression and PAC-1 binding. However, ADP- and botrocetin-induced platelet aggregation and platelet adhesion and spreading on von Willebrand factor surface were significantly reduced in platelets pre-treated with CaM antagonist. Therefore, these findings indicate that CaM antagonists induce platelet apoptosis, which suggests a possible pathogenesis of thrombocytopenia in some patients treated with CaM antagonist drugs, and also may present as a novel mechanism for platelet clearance and dysfunction in vivo or in vitro. The elevation of the cytosolic Ca2+ level may involve in the regulation of CaM antagonist-induced platelet apoptosis. Disclosures: No relevant conflicts of interest to declare.

Lenalidomide Reduces Survival of Chronic Lymphocytic Leukemia Cells in Primary Co-Cultures by Altering the Myeloid Microenvironment

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3894-3894
Author(s):  
Angela Schulz ◽  
Claudia Dürr ◽  
Thorsten Zenz ◽  
Stephan Stilgenbauer ◽  
Peter Lichter ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Drug Effects ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Cell Surface Expression ◽  
Clinical Activity ◽  
Altered Expression

Abstract Abstract 3894 Chronic lymphocytic leukemia (CLL) cells are highly dependent on their microenvironment. External stimuli provided by bone marrow stromal cells or non-malignant leukocytes are required for their survival and proliferation. Interestingly, peripheral blood-derived monocytes differentiate in the presence of CLL cells to so-called Nurse-like cells (NLCs), which are round or fibroblast-shaped adherent cells that were shown to promote survival of CLL cells in vitro and to exist in lymph nodes of CLL patients. In search of new therapeutic options for patients with CLL, the immunomodulatory drug lenalidomide turned out to have significant clinical activity in CLL. Lenalidomide does not induce apoptosis in CLL cells directly, but is rather believed to act via the microenvironment. Several studies described that it alters cytokine levels and the activation status of the cells. Further, a CLL-specific T-cell defect was shown to be repaired by lenalidomide, which might represent a major activity of this drug in CLL. However, its mechanism of action seems to be complex and is not well understood. As monocytes as well as NLCs are very effective in maintaining survival of CLL cells, we aimed to investigate whether lenalidomide interferes with these supportive cell-cell interactions. To do this, we established primary co-cultures of monocytes and CLL cells in the presence or absence of lenalidomide and observed a significantly decreased viability of CLL cells after 14 days of treatment, suggesting an impact of this drug on the survival support of NLCs. Therefore, we analyzed the immunophenotype of NLCs by flow cytometry, as well as the secretion of cytokines in the co-cultures by ELISA and antibody-coupled bead arrays. Among the effects induced by lenalidomide, we observed reduced cell surface expression of the MHC II protein HLA-DR on NLCs as well as lower levels of the chemokine CCL2, but higher levels of IL-10 in the culture supernatant, indicating an altered inflammatory milieu in the co-cultures. The enhanced IL-10 levels resulted in an increase in STAT1 phosphorylation in CLL cells as measured by Western blot analysis. As a consequence, enhanced expression of the adhesion molecule ICAM-1 (CD54) and an altered expression of cytoskeletal genes (e.g. RHOC and CORO1B) were observed in CLL cells after lenalidomide treatment. Chemotaxis assays using transwell culture dishes and SDF1-α as chemoattractant revealed an impaired migratory potential of lenalidomide-treated CLL cells, which was not due to reduced expression of the SDF1-α receptor CXCR4. In summary, our data show that lenalidomide reduces the survival support of NLCs for CLL cells in vitro, suggesting that this drug effects the myeloid microenvironment in CLL in vivo. Furthermore, lenalidomide impairs the migratory potential of CLL cells which may affect circulation and homing of CLL cells in vivo. Disclosures: No relevant conflicts of interest to declare.

Mechanism of platelet functional changes and effects of anti-platelet agents on in vivo hemostasis under different gravity conditions

2010 ◽  
Vol 108 (5) ◽  
pp. 1241-1249 ◽  
Author(s):  
Suping Li ◽  
Quanwei Shi ◽  
Guanglei Liu ◽  
Weilin Zhang ◽  
Zhicheng Wang ◽  
...  
Keyword(s):  
Therapeutic Potential ◽  
Surface Expression ◽  
Filamin A ◽  
Modeled Microgravity ◽  
Functional Changes ◽  
Cytoskeleton Reorganization ◽  
Intracellular Ca ◽  
Thrombotic Diseases ◽  
Platelet Functions

Serious thrombotic and hemorrhagic problems or even fatalities evoked by either microgravity or hypergravity occur commonly in the world. We recently reported that platelet functions are inhibited in microgravity environments and activated under high-G conditions, which reveals the pathogenesis for gravity change-related hemorrhagic and thrombotic diseases. However, the mechanisms of platelet functional variations under different gravity conditions remain unclear. In this study we show that the amount of filamin A coimmunoprecipitated with GPIbα was enhanced in platelets exposed to modeled microgravity and, in contrast, was reduced in 8 G-exposed platelets. Hypergravity induced actin filament formation and redistribution, whereas actin filaments were reduced in platelets treated with modeled microgravity. Furthermore, intracellular Ca2+ levels were elevated by hypergravity. Pretreatment of platelets with the cell-permeable Ca2+ chelator BAPTA-AM had no effect on cytoskeleton reorganization induced by hypergravity but significantly reduced platelet aggregation induced by ristocetin/hypergravity. Two anti-platelet agents, aspirin and tirofiban, effectively reversed the shortened tail bleeding time and reduced the death rate of mice exposed to hypergravity. Furthermore, the increased P-selectin surface expression was obviously reduced in platelets from mice treated with aspirin/hypergravity compared with those from mice treated with hypergravity alone. These data suggest that the actin cytoskeleton reorganization and intracellular Ca2+ level play key roles in the regulation of platelet functions in different gravitational environments. The results with anti-platelet agents not only further confirm the activation of platelets in vivo but also suggest a therapeutic potential for hypergravity-induced thrombotic diseases.

scholarly journals MARP Protein Family: A Possible Role in Molecular Mechanisms of Tumorigenesis

2010 ◽  
Vol 29 (3) ◽  
pp. 157-164 ◽  
Author(s):  
Snežana Kojić
Keyword(s):  
Molecular Mechanisms ◽  
Striated Muscle ◽  
Muscular Atrophy ◽  
Protein Family ◽  
Renal Oncocytoma ◽  
Cardiac Muscle Cells ◽  
Altered Expression ◽  
Duchene Muscular Dystrophy

MARP Protein Family: A Possible Role in Molecular Mechanisms of TumorigenesisThe MARP (muscle ankyrin repeat protein) family comprises three structurally similar proteins: CARP/Ankrd1, Ankrd2/Arpp and DARP/Ankrd23. They share four conserved copies of 33-residue ankyrin repeats and contain a nuclear localization signal, allowing the sorting of MARPs to the nucleus. They are found both in the nucleus and in the cytoplasm of skeletal and cardiac muscle cells, suggesting that MARPs shuttle within the cell enabling them to play a role in signal transduction in striated muscle. Expression of MARPs is altered under different pathological conditions. In skeletal muscle, CARP/Ankrd1 and Ankrd2/Arpp are up-regulated in muscle in patients suffering from Duchene muscular dystrophy, congenital myopathy and spinal muscular atrophy. Mutations inAnkrd1gene (coding CARP/Ankrd1) were identified in dilated and hypertrophic cardiomyopathies. Altered expression of MARPs is also observed in rhabdomyosarcoma, renal oncocytoma and ovarian cancer. In order to functionally characterize MARP family members CARP/Ankrd1 and Ankrd2/Arpp, we have found that both proteins interact with the tumor suppressor p53 bothin vivoandin vitroand that p53 up-regulates their expression. Our results implicate the potential role of MARPs in molecular mechanisms relevant to tumor response and progression.

Transgenic models for the study of lung biology and disease

1994 ◽  
Vol 266 (4) ◽  
pp. L319-L353 ◽  
Author(s):  
Y. S. Ho
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Regulation Of Gene Expression ◽  
Transgenic Models ◽  
Altered Expression ◽  
Artificial Chromosomes ◽  
New Development ◽  
Yeast Artificial Chromosomes ◽  
The Impact

Transgenic models provide a means of understanding the molecular mechanisms for the temporal, spatial, and stimulus-responsive regulation of gene expression in vivo and importantly the pathophysiological consequences of the altered expression of a normal or mutated gene. To facilitate the application of transgenic models in lung research, this review describes several practical considerations in generation of transgenic mice. The potential of transgenic models in lung research is also illustrated by depicting the current models in lung research including those for understanding lung gene regulation, tumorigenesis, mutation detection, antioxidant defense, emphysema, fibrosis, and hypertension. The impact of important new development of producing transgenic mice carrying large fragments of DNA contained in yeast artificial chromosomes to achieve proper control of transgene expression and gene targeting technology is also discussed. It is anticipated that transgenic models will provide invaluable information in future lung research.

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