GPIb-dependent platelet activation is dependent on Src kinases but not MAP kinase or cGMP-dependent kinase

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2601-2609 ◽  
Author(s):  
Stuart J. Marshall ◽  
Yotis A. Senis ◽  
Jocelyn M. Auger ◽  
Robert Feil ◽  
Franz Hofmann ◽  
...  
Keyword(s):  
Map Kinase ◽  
Platelet Activation ◽  
Map Kinases ◽  
Thrombus Formation ◽  
Critical Role ◽  
Rabbit Model ◽  
Guanosine Monophosphate ◽  
Src Kinases ◽  
Aggregate Formation ◽  
No Donor

Abstract Glycoprotein Ib-IX-V (GPIb-IX-V) mediates platelet tethering to von Willebrand factor (VWF), recruiting platelets into the thrombus, and activates integrin αIIbβ3 through a pathway that is dependent on Src kinases. In addition, recent reports indicate that activation of αIIbβ3 by VWF is dependent on protein kinase G (PKG) and mitogen-activated protein (MAP) kinases. The present study compares the importance of these signaling pathways in the activation of αIIbβ3 by GPIb-IX-V. In contrast to a recent report, VWF did not promote an increase in cyclic guanosine monophosphate (cGMP), while agents that elevate cGMP, such as the nitrous oxide (NO) donor glyco–SNAP-1 (N-(β-D-glucopyranosyl)-N2-acetyl-S-nitroso-D,L-penicillaminamide) or the type 5 phosphosdiesterase inhibitor, sildenafil, inhibited rather than promoted activation of αIIbβ3 by GPIb-IX-V and blocked aggregate formation on collagen at an intermediate rate of shear (800 s-1). Additionally, sildenafil increased blood flow in a rabbit model of thrombus formation in vivo. A novel inhibitor of the MAP kinase pathway, which is active in plasma, PD184161, had no effect on aggregate formation on collagen under flow conditions, whereas a novel inhibitor of Src kinases, which is also active in plasma, PD173952, blocked this response. These results demonstrate a critical role for Src kinases but not MAP kinases in VWF-dependent platelet activation and demonstrate an inhibitory role for cGMP-elevating agents in regulating this process.

scholarly journals Signal-mediated localization of Candida albicans pheromone response pathway components

2020 ◽  
Author(s):  
Anna Carolina Borges Pereira Costa ◽  
Raha Parvizi Omran ◽  
Chris Law ◽  
Vanessa Dumeaux ◽  
Malcolm Whiteway
Keyword(s):  
Candida Albicans ◽  
Map Kinase ◽  
Nuclear Localization ◽  
Map Kinases ◽  
Critical Role ◽  
Cell Periphery ◽  
Pheromone Response ◽  
Pheromone Response Pathway ◽  
Localization Signal ◽  
In Cells

Abstract Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or β subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.

LPS Stimulates Platelet Secretion and Promotes Platelet Aggregation Via TLR4/MyD88 and the cGMP-Dependent Protein Kinase Pathway.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3658-3658
Author(s):  
Guoying Zhang ◽  
Emily Welch ◽  
Asrar B. Malik ◽  
Xiaoping Du ◽  
Zhenyu Li
Keyword(s):  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Critical Role ◽  
Atp Release ◽  
Dependent Protein Kinase ◽  
Cgmp Dependent Protein Kinase ◽  
Dependent Protein ◽  
Platelet Secretion

Abstract Bacterial lipopolysaccharide (LPS) induces rapid thrombocytopenia, hypotension and sepsis. Although growing evidence suggests that platelet activation plays a critical role in LPS-induced thrombocytopenia and tissue damage, the mechanism of LPS-mediated platelet activation is unclear. Here we show that LPS stimulated platelet secretion of dense and alpha granules as indicated by ATP release and P-selectin expression, and thus enhanced platelet activation induced by low concentrations of platelet agonists. Platelets express components of the LPS receptor-signaling complex, including Toll-like receptor (TLR4), CD14, MD2, and MyD88. The effect of LPS on platelet activation was abolished by an anti-TLR4 blocking antibody or TLR4 knockout. Furthermore, LPS-induced potentiation of platelet aggregation and FeCl3-induced thrombus formation were abolished in MyD88 knockout mice. Importantly, TLR4 mediates LPS-induced cGMP elevation and the stimulatory effect of LPS on platelet aggregation was also abolished by inhibitors of nitric oxide synthase (NOS) and the cGMP-dependent protein kinase (PKG). Thus, LPS promotes platelet secretion and aggregation through a TLR4/MyD88 and cGMP/PKG-dependent pathway.

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.

Activation of p38 MAP Kinase and JNK But Not ERK Is Required for Erythropoietin-Induced Erythroid Differentiation

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 1859-1869 ◽  
Author(s):  
Yuka Nagata ◽  
Noriko Takahashi ◽  
Roger J. Davis ◽  
Kazuo Todokoro
Keyword(s):  
Cell Growth ◽  
Map Kinase ◽  
Antisense Oligonucleotides ◽  
Map Kinases ◽  
Physiological Function ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
P38 Map Kinase ◽  
American Society

p38 MAP kinase (p38) and JNK have been described as playing a critical role in the response to a variety of environmental stresses and proinflammatory cytokines. It was recently reported that hematopoietic cytokines activate not only classical MAP kinases (ERK), but also p38 and JNK. However, the physiological function of these kinases in hematopoiesis remains obscure. We found that all MAP kinases examined, ERK1, ERK2, p38, JNK1, and JNK2, were rapidly and transiently activated by erythropoietin (Epo) stimulation in SKT6 cells, which can be induced to differentiate into hemoglobinized cells in response to Epo. Furthermore, p38-specific inhibitor SB203580 but not MEK-specific inhibitor PD98059 significantly suppressed Epo-induced differentiation and antisense oligonucleotides of p38, JNK1, and JNK2, but neither ERK1 nor ERK2 clearly inhibited Epo-induced hemoglobinization. However, in Epo-dependent FD-EPO cells, inhibition of either ERKs, p38, or JNKs suppressed cell growth. Furthermore, forced expression of a gain-of-function MKK6 mutant, which specifically activated p38, induced hemoglobinization of SKT6 cells without Epo. These results indicate that activation of p38 and JNKs but not of ERKs is required for Epo-induced erythroid differentiation of SKT6 cells, whereas all of these kinases are involved in Epo-induced mitogenesis of FD-EPO cells. © 1998 by The American Society of Hematology.

scholarly journals Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis

2017 ◽  
Vol 114 (11) ◽  
pp. 2964-2969 ◽  
Author(s):  
Yiwen Zhang ◽  
Jian Zhang ◽  
Rong Yan ◽  
Jingluan Tian ◽  
Yang Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Critical Role ◽  
In Vivo Model ◽  
Clot Retraction ◽  
Interacting Protein ◽  
Bone Marrow Derived Cells

Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3−/−) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3−/− bone marrow-derived cells had longer occlusion times than RIP3−/− mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3−/− platelets. Moreover, RIP3 interacted with Gα13. Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

Involvement of endogenous nitric oxide in angiotensin II-induced activation of vascular mitogen-activated protein kinases

2007 ◽  
Vol 293 (4) ◽  
pp. H2403-H2408 ◽  
Author(s):  
Guo-Xing Zhang ◽  
Yukiko Nagai ◽  
Toshitaka Nakagawa ◽  
Hiroshi Miyanaka ◽  
Yoshihide Fujisawa ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Angiotensin Ii ◽  
Map Kinase ◽  
Map Kinases ◽  
No Synthase ◽  
Dependent Manner ◽  
Ang Ii ◽  
No Donor ◽  
Mitogen Activated Protein ◽  
Kinase Phosphorylation

Angiotensin II (ANG II) is a powerful activator of mitogen-activated protein (MAP) kinase cascades in cardiovascular tissues through a redox-sensitive mechanism. Nitric oxide (NO) is considered to antagonize the vasoconstrictive and proarteriosclerotic actions of ANG II. However, the role of endogenous NO in ANG II-induced redox-sensitive signal transduction is not yet clear. In this study using catheterized, conscious rats, we found that acute intravenous administration of NG-nitro-l-arginine methyl ester (l-NAME; 5 mg/kg) enhanced phosphorylation of aortic MAP kinases extracellular signal regulated kinase (ERK) 1/2 and p38, which were suppressed only partially by a superoxide dismutase mimetic (Tempol), whereas ANG II-induced MAP kinase phosphorylation was markedly suppressed by Tempol. FK409, a NO donor, had little effect on vascular MAP kinase phosphorylation. On the other hand, acute exposure to a vasoconstrictor dose of ANG II (200 ng·kg−1·min−1 iv) failed to enhance phosphorylation of aortic MAP kinases in the chronically l-NAME-treated rats, whereas the vasoconstrictor effect of ANG II was not affected by l-NAME treatment. Furthermore, three different inhibitors of NO synthase suppressed, in a dose-dependent manner, ANG II-induced MAP kinase phosphorylation in rat vascular smooth muscle cells, which was closely linked to superoxide generation in cells. These results indicate the involvement of endogenous NO synthase in ANG II-induced signaling pathways, leading to activation of MAP kinase, and that NO may have dual effects on the vascular MAP kinase activation associated with redox sensitivity.

Activation of p38 MAP Kinase and JNK But Not ERK Is Required for Erythropoietin-Induced Erythroid Differentiation

Blood ◽  
1998 ◽  
Vol 92 (6) ◽  
pp. 1859-1869 ◽  
Author(s):  
Yuka Nagata ◽  
Noriko Takahashi ◽  
Roger J. Davis ◽  
Kazuo Todokoro
Keyword(s):  
Cell Growth ◽  
Map Kinase ◽  
Antisense Oligonucleotides ◽  
Map Kinases ◽  
Physiological Function ◽  
Critical Role ◽  
Specific Inhibitor ◽  
Erythroid Differentiation ◽  
P38 Map Kinase ◽  
American Society

Abstract p38 MAP kinase (p38) and JNK have been described as playing a critical role in the response to a variety of environmental stresses and proinflammatory cytokines. It was recently reported that hematopoietic cytokines activate not only classical MAP kinases (ERK), but also p38 and JNK. However, the physiological function of these kinases in hematopoiesis remains obscure. We found that all MAP kinases examined, ERK1, ERK2, p38, JNK1, and JNK2, were rapidly and transiently activated by erythropoietin (Epo) stimulation in SKT6 cells, which can be induced to differentiate into hemoglobinized cells in response to Epo. Furthermore, p38-specific inhibitor SB203580 but not MEK-specific inhibitor PD98059 significantly suppressed Epo-induced differentiation and antisense oligonucleotides of p38, JNK1, and JNK2, but neither ERK1 nor ERK2 clearly inhibited Epo-induced hemoglobinization. However, in Epo-dependent FD-EPO cells, inhibition of either ERKs, p38, or JNKs suppressed cell growth. Furthermore, forced expression of a gain-of-function MKK6 mutant, which specifically activated p38, induced hemoglobinization of SKT6 cells without Epo. These results indicate that activation of p38 and JNKs but not of ERKs is required for Epo-induced erythroid differentiation of SKT6 cells, whereas all of these kinases are involved in Epo-induced mitogenesis of FD-EPO cells. © 1998 by The American Society of Hematology.

Crosstalk between Platelet and Bacteria: A Therapeutic Prospect

2019 ◽  
Vol 25 (38) ◽  
pp. 4041-4052
Author(s):  
Vivek K. Yadav ◽  
Pradeep K. Singh ◽  
Vishnu Agarwal ◽  
Sunil K. Singh
Keyword(s):  
Platelet Activation ◽  
Molecular Mechanisms ◽  
Thrombus Formation ◽  
Critical Role ◽  
Direct Interaction ◽  
Vascular Wall ◽  
Cardiovascular Complications ◽  
Bacterial Cells ◽  
Platelet Surface ◽  
Wide Range

Platelets are typically recognized for their roles in the maintenance of hemostasis and vascular wall repair to reduce blood loss. Beyond hemostasis, platelets also play a critical role in pathophysiological conditions like atherosclerosis, stroke, thrombosis, and infections. During infection, platelets interact directly and indirectly with bacteria through a wide range of cellular and molecular mechanisms. Platelet surface receptors such as GPIbα, FcγRIIA, GPIIbIIIa, and TLRs, etc. facilitate direct interaction with bacterial cells. Besides, the indirect interaction between platelet and bacteria involves host plasma proteins such as von Willebrand Factor (vWF), fibronectin, IgG, and fibrinogen. Bacterial cells induce platelet activation, aggregation, and thrombus formation in the microvasculature. The activated platelets induce the Neutrophil Extracellular Traps (NETs) formation, which further contribute to thrombosis. Thus, platelets are extensively anticipated as vital immune modulator cells during infection, which may further lead to cardiovascular complications. In this review, we cover the interaction mechanisms between platelets and bacteria that may lead to the development of thrombotic disorders. Platelet receptors and other host molecules involved in such interactions can be used to develop new therapeutic strategies to combat against infection-induced cardiovascular complications. In addition, we highlight other receptor and enzyme targets that may further reduce infection-induced platelet activation and various pathological conditions.

Abstract 391: Platelet CD40L Affects Thrombus Formation via Phosphatidylinositol 3-kinase ß, But Not Via CD40 or IKKα

2015 ◽  
Vol 35 (suppl_1) ◽  
Author(s):  
Marijke J Kuijpers ◽  
Nadine J Mattheij ◽  
Lina Cipolla ◽  
Johanna P van Geffen ◽  
Toby Lawrence ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Aggregate Formation ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Soluble Cd40l ◽  
Granule Secretion ◽  
Collagen Receptor

Objective: To investigate the roles and signaling pathways of CD40L and CD40 in platelet activation and thrombus formation under atherothrombotic conditions. Approach and Results: Mouse platelets lacking CD40L (Cd40lg -/- Apoe -/- ) showed diminished αIIbβ3 activation and α-granule secretion in response to collagen receptor (GPVI) stimulation, while CD40 deficient platelets (Cd40 -/- Apoe -/- ) showed increased responses. ADP- or thrombin-evoked activation was unaffected. In both Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- mice, formation of multi-layered thrombi was decreased on both atherosclerotic plaque material and collagen, in comparison to controls. Addition of CD40L prior to perfusion over collagen or plaque material enhanced dense aggregate formation in Apoe -/- , Cd40lg -/- Apoe -/- and Cd40 -/- Apoe -/- blood. CD40L or low GPVI stimulation separately did not cause platelet aggregation. But when combined, aggregation was potentiated, even in the absence of CD40. This potentiation was antagonized by inhibiting PI3Kβ, as well as in platelets from Pik3cb R/R mice. CD40L enhanced Akt phosphorylation at low GPVI stimulation, which was again antagonized by PI3Kβ inhibition and absent in platelets from Pik3cb R/R mice. Finally, Chuk1 A/A Apoe -/- mice, deficient in IKKα, displayed no differences in platelet aggregation - with or without CD40L - nor in thrombus formation in whole blood, indicating that these effects are not mediated via IKKα/NFkB. Conclusions: Under atherothrombotic conditions, CD40L enforces collagen-dependent platelet activation, by supporting integrin αIIbβ3 activation, secretion and dense thrombus formation via PI3Kβ, but not IKKα. Since shedding of CD40L starts minutes after activation, these results point to a joint role of both platelet-bound and soluble CD40L in controlling the size of rapidly formed thrombi.

Platelet-specific markers are associated with monocyte-platelet aggregate formation and thrombin generation potential in advanced atherosclerosis

2016 ◽  
Vol 115 (03) ◽  
pp. 615-621 ◽  
Author(s):  
Cihan Ay ◽  
Julia Riedl ◽  
Christoph W. Kopp ◽  
Beate Eichelberger ◽  
Renate Koppensteiner ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Thrombin Generation ◽  
Thrombus Formation ◽  
Cd40 Ligand ◽  
Aggregate Formation ◽  
Soluble Cd40 Ligand ◽  
Platelet Factor ◽  
Platelet Aggregate ◽  
Angioplasty And Stenting

SummaryPlatelet activation and thrombin generation are crucial steps in primary and secondary haemostasis. However, both also play major roles in intravascular thrombus formation and therefore in the development of adverse cardiovascular events. In the current study, we first sought to investigate the associations of the platelet biomarkers platelet factor (PF)-4, thrombospondin (TSP)-1, soluble CD40 ligand (sCD40L), and soluble P-selectin (sP-selectin) with each other and with monocyte-platelet aggregate (MPA) formation in 316 patients undergoing angioplasty and stenting. To better understand the interplay between platelet activation and thrombin generation, we subsequently investigated the associations of the platelet biomarkers with thrombin generation potential. The mostly platelet-specific markers PF-4, TSP-1 and sCD40L correlated strongly with each other (all p < 0.001), and the best correlation was observed between PF-4 and TSP-1 (r=0.91). In contrast, sP-selectin, which derives from platelets and endothelial cells, correlated rather poorly with TSP-1 (r=0.12, p=0.04), and did not correlate with PF-4 and sCD40L. While PF-4, TSP-1 and sP-selectin correlated significantly with in vivo MPA formation (all p < 0.001), no such association was found between sCD40L and MPA formation. PF-4, TSP-1 and sCD40L correlated strongly with peak thrombin generation (all p < 0.001) with the best correlation between PF-4 and peak thrombin generation (r=0.55), whereas sP-selectin did not correlate with peak thrombin generation. Likewise, PF-4, TSP-1 and sCD40L correlated significantly with the area under the thrombin generation curve (AUC; all p< 0.01), whereas sP-selectin did not correlate with the AUC. In conclusion, platelet-specific markers are associated with MPA formation and thrombin generation potential in patients with advanced atherosclerosis.

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