Abstract 41: Platelet Dream Plays a Critical Role During Thrombogenesis in Mice

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Kyungho Kim ◽  
Alan Tseng ◽  
Andrew Barazia ◽  
Joseph E Italiano ◽  
Jaehyung Cho
Keyword(s):  
Thrombus Formation ◽  
Regulatory Element ◽  
Critical Role ◽  
Flow Chamber ◽  
Regulatory Function ◽  
Pi3k Activation ◽  
Granule Secretion ◽  
Phosphoinositide 3 Kinase

Downstream regulatory element antagonist modulator (DREAM), a transcriptional repressor, is known to modulate pain. Using intravital microscopy with DREAM-null mice and their bone marrow chimeras, we demonstrated that hematopoietic and endothelial cell DREAM are required for platelet thrombus formation following arteriolar injury. DREAM deletion also prolonged tail bleeding times. In vitro flow chamber assays and in vivo adoptive transfer experiments indicated the importance of platelet DREAM in thrombogenesis. We found that deletion of platelet DREAM does not alter ultrastructural features but significantly impaired aggregation and ATP secretion induced by collagen-related peptide (CRP), ADP, or A23187, but not thrombin or U46619. Biochemical studies showed that platelet DREAM is required for phosphoinositide 3-kinase (PI3K) activation induced by GPVI-, A23187-, or integrin-mediated signaling. Studies using DREAM-null platelets and isoform-specific PI3K inhibitors revealed that platelet DREAM positively regulates granule secretion, Ca2+ mobilization, and aggregation induced by CRP or A23187 through PI3K class Iβ (PI3K-Iβ) activity. Genetic and pharmacological studies in megakaryoblastic MEG-01 cells showed that DREAM regulates A23187-induced Ca2+ mobilization and that the regulatory function of DREAM requires Ca2+ binding and PI3K-Iβ activity. Taken together, we have identified platelet DREAM as a novel regulator of PI3K-Iβ activity during thrombus formation.

STIM1 Deficiency Results In Impaired Platelet Procoagulant Activity and Protection From Arterial Thrombosis

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 485-485
Author(s):  
Firdos Ahmad ◽  
Lucia Stefanini ◽  
Timothy Daniel Ouellette ◽  
Teshell K Greene ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Critical Role ◽  
Phosphatidyl Serine ◽  
Flow Chamber ◽  
Procoagulant Activity ◽  
Static Conditions

Abstract Abstract 485 Platelet activation is a central event in thrombosis and hemostasis. We recently demonstrated that most aspects of platelet activation depend on synergistic signaling by two signaling modules: 1) Ca2+/CalDAG-GEFI/Rap1 and 2) PKC/P2Y12/Rap1. The intracellular Ca2+ concentration of platelets is regulated by Ca2+ release from the endoplasmic reticulum (ER) and store-operated calcium entry (SOCE) through the plasma membrane. Stromal interaction molecule 1 (STIM1) was recently identified as the ER Ca2+ sensor that couples Ca2+ store release to SOCE. In this study, we compared the activation response of platelets lacking STIM1−/− or CalDAG-GEFI−/−, both in vitro and in vivo. To specifically investigate Ca2+-dependent platelet activation, some of the experiments were performed in the presence of inhibitors to P2Y12. The murine Stim1 gene was deleted in the megakaryocyte/platelet lineage by breeding Stim flox/flox mice with PF4-Cre mice (STIM1fl/fl). STIM1fl/fl platelets showed markedly reduced SOCE in response to agonist stimulation. aIIbβ3 activation in STIM1fl/fl platelets was significantly reduced in the presence but not in the absence of the P2Y12 inhibitor, 2-MesAMP. In contrast, aIIbb3 activation was completely inhibited in 2-MesAMP-treated CalDAG-GEFI−/− platelets. Deficiency in STIM1, and to a lesser extent in CalDAG-GEFI, reduced phosphatidyl serine (PS) exposure in platelets stimulated under static conditions. PS exposure was completely abolished in both STIM1fl/fl and CalDAG-GEFI−/− platelets stimulated in the presence of 2-MesAMP. To test the ability of platelets to form thrombi under conditions of arterial shear stress, we performed flow chamber experiments with anticoagulated blood perfused over a collagen surface. Thrombus formation was abolished in CalDAG-GEFI−/− blood and WT blood treated with 2-MesAMP. In contrast, STIM1fl/fl platelets were indistinguishable from WT platelets in their ability to form thrombi. STIM1fl/fl platelets, however, were impaired in their ability to express PS when adhering to collagen under flow. Consistently, when subjected to a laser injury thrombosis model, STIM1fl/fl mice showed delayed and reduced fibrin generation, resulting in the formation of unstable thrombi. In conclusion, our studies indicate a critical role of STIM1 in SOCE and platelet procoagulant activity, but not in CalDAG-GEFI mediated activation of aIIbb3 integrin. Disclosures: No relevant conflicts of interest to declare.

scholarly journals RanBP2 and SENP3 Function in a Mitotic SUMO2/3 Conjugation-Deconjugation Cycle on Borealin

2009 ◽  
Vol 20 (1) ◽  
pp. 410-418 ◽  
Author(s):  
Ulf R. Klein ◽  
Markus Haindl ◽  
Erich A. Nigg ◽  
Stefan Muller
Keyword(s):  
Mammalian Cells ◽  
Spindle Assembly Checkpoint ◽  
Critical Role ◽  
Specific Interaction ◽  
Regulatory Function ◽  
Mitotic Progression ◽  
Chromosome Congression ◽  
Chromosomal Passenger

The ubiquitin-like SUMO system controls cellular key functions, and several lines of evidence point to a critical role of SUMO for mitotic progression. However, in mammalian cells mitotic substrates of sumoylation and the regulatory components involved are not well defined. Here, we identify Borealin, a component of the chromosomal passenger complex (CPC), as a mitotic target of SUMO. The CPC, which additionally comprises INCENP, Survivin, and Aurora B, regulates key mitotic events, including chromosome congression, the spindle assembly checkpoint, and cytokinesis. We show that Borealin is preferentially modified by SUMO2/3 and demonstrate that the modification is dynamically regulated during mitotic progression, peaking in early mitosis. Intriguingly, the SUMO ligase RanBP2 interacts with the CPC, stimulates SUMO modification of Borealin in vitro, and is required for its modification in vivo. Moreover, the SUMO isopeptidase SENP3 is a specific interaction partner of Borealin and catalyzes the removal of SUMO2/3 from Borealin. These data thus delineate a mitotic SUMO2/3 conjugation–deconjugation cycle of Borealin and further assign a regulatory function of RanBP2 and SENP3 in the mitotic SUMO pathway.

Endothelial stroma programs hematopoietic stem cells to differentiate into regulatory dendritic cells through IL-10

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1189-1197 ◽  
Author(s):  
Hua Tang ◽  
Zhenhong Guo ◽  
Minghui Zhang ◽  
Jianli Wang ◽  
Guoyou Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Hematopoietic Stem Cells ◽  
Critical Role ◽  
Regulatory Function ◽  
Hematopoietic Stem ◽  
Regulatory Dendritic Cells

Abstract Regulatory dendritic cells (DCs) have been reported recently, but their origin is poorly understood. Our previous study demonstrated that splenic stroma can drive mature DCs to proliferate and differentiate into regulatory DCs, and their natural counterpart with similar regulatory function in normal spleens has been identified. Considering that the spleen microenvironment supports hematopoiesis and that hematopoietic stem cells (HSCs) are found in spleens of adult mice, we wondered whether splenic microenvironment could differentiate HSCs into regulatory DCs. In this report, we demonstrate that endothelial splenic stroma induce HSCs to differentiate into a distinct regulatory DC subset with high expression of CD11b but low expression of Ia. CD11bhiIalo DCs secreting high levels of TGF-β, IL-10, and NO can suppress T-cell proliferation both in vitro and in vivo. Furthermore, CD11bhiIalo DCs have the ability to potently suppress allo-DTH in vivo, indicating their preventive or therapeutic perspectives for some immunologic disorders. The inhibitory function of CD11bhiIalo DCs is mediated through NO but not through induction of regulatory T (Treg) cells or T-cell anergy. IL-10, which is secreted by endothelial splenic stroma, plays a critical role in the differentiation of the regulatory CD11bhiIalo DCs from HSCs. These results suggest that splenic microenvironment may physiologically induce regulatory DC differentiation in situ.

scholarly journals Platelet interaction with activated endothelium: mechanistic insights from microfluidics

Blood ◽  
2017 ◽  
Vol 130 (26) ◽  
pp. 2819-2828 ◽  
Author(s):  
Daniëlle M. Coenen ◽  
Tom G. Mastenbroek ◽  
Judith M. E. M. Cosemans
Keyword(s):  
Molecular Mechanisms ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Coagulation System ◽  
Intercellular Adhesion Molecule ◽  
Platelet Glycoprotein ◽  
Intercellular Adhesion Molecule 1 ◽  
Chamber Studies

Abstract Traditionally, in vitro flow chamber experiments and in vivo arterial thrombosis studies have been proved to be of vital importance to elucidate the mechanisms of platelet thrombus formation after vessel wall injury. In recent years, it has become clear that platelets also act as modulators of inflammatory processes, such as atherosclerosis. A key element herein is the complex cross talk between platelets, the coagulation system, leukocytes, and the activated endothelium. This review provides insight into the platelet-endothelial interface, based on in vitro flow chamber studies and cross referenced with in vivo thrombosis studies. The main mechanisms of platelet interaction with the activated endothelium encompass (1) platelet rolling via interaction of platelet glycoprotein Ib-IX-V with endothelial-released von Willebrand factor with a supporting role for the P-selectin/P-selectin glycoprotein ligand 1 axis, followed by (2) firm platelet adhesion to the endothelium via interaction of platelet αIIbβ3 with endothelial αvβ3 and intercellular adhesion molecule 1, and (3) a stimulatory role for thrombin, the thrombospondin-1/CD36 axis and cyclooxygenase 1 in subsequent platelet activation and stable thrombus formation. In addition, the molecular mechanisms underlying the stimulatory effect of platelets on leukocyte transendothelial migration, a key mediator of atheroprogression, are discussed. Throughout the review, emphasis is placed on recommendations for setting up, reporting, interpreting, and comparing endothelial-lined flow chamber studies and suggestions for future studies.

BCR activation of PI3K is Vav-independent in murine B cells

2004 ◽  
Vol 32 (5) ◽  
pp. 781-784 ◽  
Author(s):  
E. Vigorito ◽  
E. Clayton ◽  
M. Turner
Keyword(s):  
B Cells ◽  
Tyrosine Kinases ◽  
Adaptor Proteins ◽  
Pleckstrin Homology Domain ◽  
Pi3k Activation ◽  
Lipid Product ◽  
Lipid Kinases ◽  
Phosphoinositide 3 Kinase

BCR (B-cell antigen receptor)-induced Ca2+ signalling is initiated by activation of tyrosine kinases, which in concert with adaptor proteins and lipid kinases regulate PLC (phospholipase C) γ2 activation. Vav and PI3K (phosphoinositide 3-kinase) are required for optimal Ca2+ responses, although it has not been established, in primary B-cells, if both proteins are components of the same pathway. In vitro evidence suggests that binding of the PI3K lipid product PIP3 to Vav pleckstrin homology domain contributes to Vav activation. However, pharmacological inhibition of PI3K by wortmannin or deletion of the p110δ catalytic subunit has no effect on Vav activation in response to BCR engagement, suggesting that this mechanism does not operate in vivo. We also show that PI3K recruitment to phosphorylated-tyrosine-containing complexes is Vav-independent. Taken together with our previous observation that protein kinase B phosphorylation is normal in Vav-deficient B-cells, we suggest that PI3K activation is Vav-independent in response to strong signals delivered by multivalent cross-linking.

scholarly journals SREBP-1c/MicroRNA 33b Genomic Loci Control Adipocyte Differentiation

2016 ◽  
Vol 36 (7) ◽  
pp. 1180-1193 ◽  
Author(s):  
Nathan L. Price ◽  
Brandon Holtrup ◽  
Stephanie L. Kwei ◽  
Martin Wabitsch ◽  
Matthew Rodeheffer ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Target Genes ◽  
Adipocyte Differentiation ◽  
Regulatory Element ◽  
Critical Role ◽  
Peroxisome Proliferator ◽  
Peroxisome Proliferator Activated Receptor ◽  
The Impact

White adipose tissue (WAT) is essential for maintaining metabolic function, especially during obesity. The intronic microRNAs miR-33a and miR-33b, located within the genes encoding sterol regulatory element-binding protein 2 (SREBP-2) and SREBP-1, respectively, are transcribed in concert with their host genes and function alongside them to regulate cholesterol, fatty acid, and glucose metabolism. SREBP-1 is highly expressed in mature WAT and plays a critical role in promotingin vitroadipocyte differentiation. It is unknown whether miR-33b is induced during or involved in adipogenesis. This is in part due to loss of miR-33b in rodents, precludingin vivoassessment of the impact of miR-33b using standard mouse models. This work demonstrates that miR-33b is highly induced upon differentiation of human preadipocytes, along withSREBP-1. We further report that miR-33b is an important regulator of adipogenesis, as inhibition of miR-33b enhanced lipid droplet accumulation. Conversely, overexpression of miR-33b impaired preadipocyte proliferation and reduced lipid droplet formation and the induction of peroxisome proliferator-activated receptor γ (PPARγ) target genes during differentiation. These effects may be mediated by targeting of HMGA2, cyclin-dependent kinase 6 (CDK6), and other predicted miR-33b targets. Together, these findings demonstrate a novel role of miR-33b in the regulation of adipocyte differentiation, with important implications for the development of obesity and metabolic disease.

scholarly journals Thymosin β4 is essential for thrombus formation by controlling the G-actin/F-actin equilibrium in platelets

Haematologica ◽  
2021 ◽  
Author(s):  
Inga Scheller ◽  
Sarah Beck ◽  
Vanessa Göb ◽  
Carina Gross ◽  
Raluca A. I. Neagoe ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Critical Role ◽  
Actin Dynamics ◽  
Thymosin Β4 ◽  
Clot Retraction ◽  
And Function ◽  
External Signals

Coordinated rearrangements of the actin cytoskeleton are pivotal for platelet biogenesis from megakaryocytes (MKs) but also orchestrate key functions of peripheral platelets in hemostasis and thrombosis, such as granule release, the formation of filopodia and lamellipodia, or clot retraction. Along with profilin (Pfn) 1, thymosin β4 (encoded by Tmsb4x) is one of the two main G-actin sequestering proteins within cells of higher eukaryotes, and its intracellular concentration is particularly high in cells that rapidly respond to external signals by increased motility, such as platelets. Here, we analyzed constitutive Tmsb4x knockout (KO) mice to investigate the functional role of the protein in platelet production and function. Thymosin β4 deficiency resulted in a macrothrombocytopenia with only mildly increased platelet volume and an unaltered platelet life span. MK numbers in the bone marrow (BM) and spleen were unaltered, however, Tmsb4x KO MKs showed defective proplatelet formation in vitro and in vivo. Thymosin β4 deficient platelets displayed markedly decreased G-actin levels and concomitantly increased F-actin levels resulting in accelerated spreading on fibrinogen and clot retraction. Moreover, Tmsb4x KO platelets showed activation defects and an impaired immunoreceptor tyrosine-based activation motif (ITAM) signaling downstream of the activating collagen receptor glycoprotein (GP) VI. These defects translated into impaired aggregate formation under flow, protection from occlusive arterial thrombus formation in vivo and increased tail bleeding times. In summary, these findings point to a critical role of thymosin β4 for actin dynamics during platelet biogenesis, platelet activation downstream of GPVI and thrombus stability.

Tetrahydrocurcumin Downregulates MAPKs/cPLA2 Signaling and Attenuates Platelet Thromboxane A2 Generation, Granule Secretion, and Thrombus Growth

2021 ◽  
Author(s):  
Weiqi Li ◽  
Yongjie Ma ◽  
Chunmei Zhang ◽  
Binlin Chen ◽  
Xiandan Zhang ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Biological Activities ◽  
Thromboxane A2 ◽  
Mapk Signaling ◽  
Inhibitory Effects ◽  
Platelet Factor ◽  
Thrombosis Model ◽  
Granule Secretion

AbstractPlatelet granule secretion plays a key role in atherothrombosis. Curcumin, a natural polyphenol compound derived from turmeric, exerts multiple biological activities. The current study sought to investigate the efficacy of tetrahydrocurcumin (THC, the major active metabolite of curcumin) on platelet granule secretion in vitro and thrombus formation in vivo. We found that THC significantly attenuated agonist-induced granule secretion in human gel-filtered platelets in vitro, including CD62P and CD63 expression and platelet factor 4, CCL5, and adenosine triphosphate release. These inhibitory effects of THC were partially mediated by the attenuation of cytosolic phospholipase A2 (cPLA2) phosphorylation, leading to a decrease in thromboxane A2 (TxA2) generation. Moreover, the MAPK (Erk1/2, JNK1/2, and p38 MAPK) signaling pathways were downregulated by THC treatment, resulting in reduced cPLA2 activation, TxA2 generation, and granule secretion. Additionally, THC and curcumin attenuated murine thrombus growth in a FeCl3-induced mesenteric arteriole thrombosis model in C57BL/6J mice without prolonging the tail bleeding time. THC exerted more potent inhibitory effects on thrombosis formation than curcumin. Through blocking cyclooxygenase-1 activity and thus inhibiting platelet TxA2 synthesis and granule secretion with aspirin, we found that THC did not further decrease the inhibitory effects of aspirin on thrombosis formation. Thus, through inhibiting MAPKs/cPLA2 signaling, and attenuating platelet TxA2 generation, granule secretion, and thrombus formation, THC may be a potent cardioprotective agent.

Agonist-Induced Binding of the Regulatory Scaffold Protein Spinophilin to Protein Phosphatase-1 In Platelets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2029-2029
Author(s):  
Andrew Sinnamon ◽  
Peisong Ma ◽  
Lawrence F. Brass
Keyword(s):  
Platelet Activation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Tyrosine Phosphatase ◽  
Critical Role ◽  
Scaffold Protein ◽  
Western Blots ◽  
Co Precipitation

Abstract Abstract 2029 Platelet regulation plays a critical role in hemostasis. Underactivation can result in failure to stop bleeding, whereas inappropriate platelet activation can cause thrombus formation. The 130-kDa scaffold protein spinophilin (SPL) has recently been shown to play a role in preventing platelet overactivation by forming a complex with the proteins RGS10, RGS18, and the tyrosine phosphatase SHP-1. This complex dissociates when platelet are activated by thrombin or thromboxane A2 and evidence from spinophilin knockout mice suggests that this regulates platelet activation in vitro and in vivo. Spinophilin was originally isolated as a binding partner for the serine/threonine phosphatase, PP-1, in neurons. Here we asked whether PP-1 forms a complex with spinophilin in human platelets and, if so, whether the complex is affected by platelet activation. The approaches that we used to answer this question included Western blotting with antibodies to PP-1 and spinophilin, and co-precipitation studies looking for an association between spinophilin and PP-1. The results of the Western blots confirm the presence of PP-1 in platelets. The initial co-precipitation studies show that little, if any, PP-1 is associated with spinophilin in resting platelets, but there is a time-dependent increase in the SPL/PP-1 complex when platelets are activated with the PAR1 (thrombin receptor) activating peptide, SFLLRN. Thus it appears that within approximately the same time frame that the SPL/RGS/SHP-1 complex is decaying in activated platelets, the SPL/PP-1 complex is forming. Targets for PP1 have not been fully identified in platelets, but it is known that spinophilin localizes to the plasma membrane upon platelet activation. Since spinophilin is thought to direct PP1 targeting in neurons, it is reasonable to propose that it may be directing PP1 to targets in platelets in a similar manner. The studies described in this abstract were supported in part by a 2010 ASH Trainee Research Award to Andrew Sinnamon, who is a first year medical student at the University of Pennsylvania. Disclosures: No relevant conflicts of interest to declare.

FcγRIIa Enhances Thrombus Growth in Vitro and in Vivo

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 191-191
Author(s):  
Huiying Zhi ◽  
Lubica Rauova ◽  
Vincent M Hayes ◽  
Jimmy Crockett ◽  
Cunji Gao ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Platelet Function ◽  
Whole Blood ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Wild Type ◽  
Integrin Αiibβ3 ◽  
Platelet Thrombus

Abstract Abstract 191 Outside-in signal transduction is one of several autocrine amplification loops that platelets employ to stabilize and consolidate a platelet thrombus following their adhesion to each other or to components of the extracellular matrix. Binding of soluble fibrinogen to activated integrin αIIbβ3 on the platelet surface, or binding of αIIbβ3 to platelet-immobilized fibrinogen, initiates an outside-in signaling cascade that results in the activation of integrin β3-associated Src family kinases, which in turn phosphorylate tyrosine residues within the cytoplasmic domain of the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor protein, FcγRIIa. “Activation” of FcγRIIa sets off a cascade of events that result in the assembly and activation of other key signaling intermediates, including the tyrosine kinase Syk and phospholipase Cγ2(PLCγ2), through its lipase activity, generates lipid products that support a multitude of cellular activation responses, including cytoskeletal rearrangements leading to platelet shape change and spreading, secretion of platelet granules, and activation of additional cell surface integrins. We have previously shown that either antibody-mediated or genetic disruption of the functional interaction between integrin αIIbβ3 and FcγRIIa blocks tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2, and inhibits platelet spreading on immobilized fibrinogen. The physiological significance of FcγRIIa in supporting platelet thrombus formation, however, remains unknown. To further explore the importance of FcγRIIa in platelet function, we compared the relative ability of wild-type FcγRIIa-negative and transgenic FcγRIIa-positive (FcγRIIaTGN) murine platelets to support thrombosis and hemostasis in a number of well-accepted models of platelet function. FcγRIIaTGN platelets exhibited increased tyrosine phosphorylation of Syk and PLCγ2 and increased spreading upon interaction with immobilized fibrinogen. FcγRIIaTGN platelets also retracted a fibrin clot faster than did wild-type FcγRIIa-negative platelets. When anti-coagulated whole blood was perfused over a collagen-coated flow chamber under conditions of arterial shear, the rate and extent of adhesion, aggregation, and thrombus formation was significantly increased for FcγRIIaTGN platelets compared to their wild-type murine counterparts. Addition of Fab fragments specific for FcγRIIa to whole blood derived from either humans or FcγRIIaTGN mice strongly inhibited thrombus formation in the arterial in vitro flow chamber assay. Finally, to examine the in vivo relevance of FcγRIIa, mice were subjected to two models of vascular injury: electrolytic injury of the femoral vein and laser injury of cremaster arterioles. In both in vivo models, FcγRIIaTGN mice displayed increased thrombus formation compared with their wild-type, FcγRIIa-negative counterparts. Taken together, these data establish FcγRIIa as a physiologically-important functional conduit for αIIbβ3–mediated outside-in signaling, and suggest that modulating the activity of this novel integrin/ITAM pair might be effective in controlling thrombosis. Disclosures: No relevant conflicts of interest to declare.

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