scholarly journals Synthetic glycopolymers and natural fucoidans cause human platelet aggregation via PEAR1 and GPIbα

2019 ◽  
Vol 3 (3) ◽  
pp. 275-287 ◽  
Author(s):  
Caroline Kardeby ◽  
Knut Fälker ◽  
Elizabeth J. Haining ◽  
Maarten Criel ◽  
Madelene Lindkvist ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Physiological Role ◽  
Human Platelets ◽  
Minor Role ◽  
Phosphatidylinositol 3 Kinase ◽  
Akt Phosphorylation ◽  
Therapeutic Value ◽  
Direct Binding ◽  
A Minor ◽  
Blocking Antibodies

Abstract Fucoidans are sulfated fucose-based polysaccharides that activate platelets and have pro- and anticoagulant effects; thus, they may have therapeutic value. In the present study, we show that 2 synthetic sulfated α-l-fucoside-pendant glycopolymers (with average monomeric units of 13 and 329) and natural fucoidans activate human platelets through a Src- and phosphatidylinositol 3-kinase (PI3K)–dependent and Syk-independent signaling cascade downstream of the platelet endothelial aggregation receptor 1 (PEAR1). Synthetic glycopolymers and natural fucoidan stimulate marked phosphorylation of PEAR1 and Akt, but not Syk. Platelet aggregation and Akt phosphorylation induced by natural fucoidan and synthetic glycopolymers are blocked by a monoclonal antibody to PEAR1. Direct binding of sulfated glycopolymers to epidermal like growth factor (EGF)–like repeat 13 of PEAR1 was shown by avidity-based extracellular protein interaction screen technology. In contrast, synthetic glycopolymers and natural fucoidans activate mouse platelets through a Src- and Syk-dependent pathway regulated by C-type lectin-like receptor 2 (CLEC-2) with only a minor role for PEAR1. Mouse platelets lacking the extracellular domain of GPIbα and human platelets treated with GPIbα-blocking antibodies display a reduced aggregation response to synthetic glycopolymers. We found that synthetic sulfated glycopolymers bind directly to GPIbα, substantiating that GPIbα facilitates the interaction of synthetic glycopolymers with CLEC-2 or PEAR1. Our results establish PEAR1 as the major signaling receptor for natural fucose-based polysaccharides and synthetic glycopolymers in human, but not in mouse, platelets. Sulfated α-l-fucoside-pendant glycopolymers are unique tools for further investigation of the physiological role of PEAR1 in platelets and beyond.

scholarly journals TRAF6 Is Required for TRAF2-Dependent CD40 Signal Transduction in Nonhemopoietic Cells

2005 ◽  
Vol 25 (22) ◽  
pp. 9806-9819 ◽  
Author(s):  
Clare C. Davies ◽  
Tak W. Mak ◽  
Lawrence S. Young ◽  
Aristides G. Eliopoulos
Keyword(s):  
Signal Transduction ◽  
Rna Interference ◽  
Mutational Analysis ◽  
Specific Binding ◽  
Physiological Role ◽  
Multiple Roles ◽  
Minor Role ◽  
Wide Range ◽  
Direct Binding ◽  
A Minor

ABSTRACT The emerging role of CD40, a tumor necrosis factor (TNF) receptor family member, in immune regulation, disease pathogenesis, and cancer therapy necessitates the analysis of CD40 signal transduction in a wide range of tissue types. In this study we present evidence that the CD40-interacting proteins TRAF2 and TRAF6 play an important physiological role in CD40 signaling in nonhemopoietic cells. Using mutational analysis of the CD40 cytoplasmic tail, we demonstrate that the specific binding of TRAF2 to CD40 is required for efficient signaling on the NF-κB, Jun N-terminal protein kinase (JNK), and p38 axis. In fibroblasts lacking TRAF2 or in carcinoma cells in which TRAF2 has been depleted by RNA interference, the CD40-mediated activation of NF-κB and JNK is significantly reduced, and the activation of p38 and Akt is severely impaired. Interestingly, whereas the TRAF6-interacting membrane-proximal domain of CD40 has a minor role in signal transduction, studies utilizing TRAF6 knockout fibroblasts and RNA interference in epithelial cells reveal that the CD40-induced activation of NF-κB, JNK, p38, and Akt requires the integrity of TRAF6. Furthermore, we provide evidence that TRAF6 regulates CD40 signal transduction not only through its direct binding to CD40 but also indirectly via its association with TRAF2. These observations provide novel insight into the mechanisms of CD40 signaling and the multiple roles played by TRAF6 in signal transduction.

Role of Thromboxane A2 as a Mediator of Platelet-Activating-Factor-Induced Aggregation of Human Platelets

1989 ◽  
Vol 77 (1) ◽  
pp. 99-103 ◽  
Author(s):  
R. K. McCulloch ◽  
J. Summers ◽  
R. Vandongen ◽  
I. L. Rouse
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activating Factor ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
Minor Role ◽  
A Minor ◽  
Induced Aggregation

1. At present it is unclear whether platelet-activating-factor (PAF)-induced aggregation is mediated by thromboxane. To obtain further information about this event we have compared the affects of aspirin on platelet aggregation and secretion induced by PAF and collagen. 2. Collagen and PAF induced aggregation and secretion in human platelets in a dose-related manner. 3. Aspirin inhibited the magnitude of both platelet aggregation and secretion induced by PAF and collagen, but the degree of inhibition was much greater for collagen. 4. Aspirin strongly inhibited the aggregation rate of collagen-induced platelet aggregation, but had no measurable effect on the rate of PAF-induced aggregation. 5. Inconsistencies reported in previous studies of the effect of aspirin on PAF-induced platelet aggregation may be explained, in part, by the doses of PAF used and the method of inactivating cyclo-oxygenase (in vitro compared with in vivo). 6. Our results suggest that the initial events of PAF-induced aggregation are independent of thromboxane A2 formation and that thromboxane A2 plays only a minor role in the later phase of PAF-induced aggregation.

scholarly journals The synthetic RGDS peptide inhibits the binding of fibrinogen lacking intact alpha chain carboxyterminal sequences to human blood platelets

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 950-952 ◽  
Author(s):  
EI Peerschke ◽  
DK Galanakis
Keyword(s):  
Platelet Aggregation ◽  
Human Blood ◽  
Blood Platelets ◽  
Adenosine Diphosphate ◽  
Competitive Inhibitor ◽  
Minor Role ◽  
Solution Ph ◽  
Alpha Chain ◽  
A Minor ◽  
Human Blood Platelets

Abstract The alpha chain 572–574 Arg-Gly-Asp sequence of fibrinogen appears to play only a minor role in platelet aggregation based on the ability of fibrinogen preparations lacking alpha chain carboxyterminal segments to support platelet aggregation, but synthetic Arg-Gly-Asp-Ser (RGDS) peptides are capable of inhibiting platelet aggregation and fibrinogen binding. The present study thus examined the ability of RGDS peptides to inhibit platelet interactions with a plasmic degradation product of fibrinogen (8D–50) that resembles an intermediate fragment X. Gel- filtered, human blood platelets suspended in 0.01 mol/L HEPES-buffered modified Tyrode's solution, pH 7.5, were stimulated with 20 mumol/L adenosine diphosphate and the binding of 125I-labeled 8D–50 or intact fibrinogen (0.01 to 0.6 mg/mL) assessed in the presence of 0 to 117 mumol/L RGDS. The data revealed that RGDS decreased the apparent affinity of 8D–50 and intact fibrinogen for platelets but did not affect the maximum number of binding sites. RGDS thus appears to be a competitive inhibitor not only of intact fibrinogen (Ki = 12 +/- 2 mumol/L) but also of 8D–50 (Ki = 15 +/- 3 mumol/L) (mean +/- SD, n = 3).

scholarly journals Xanthohumol, a Prenylated Flavonoid from Hops (Humulus lupulus), Prevents Platelet Activation in Human Platelets

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Ye-Ming Lee ◽  
Kuo-Hsien Hsieh ◽  
Wan-Jung Lu ◽  
Hsiu-Chu Chou ◽  
Duen-Suey Chou ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Protein Kinase ◽  
Platelet Activation ◽  
Therapeutic Potential ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Humulus Lupulus ◽  
Mitogen Activated Protein Kinase ◽  
Akt Phosphorylation ◽  
Mitogen Activated Protein

Xanthohumol is the principal prenylated flavonoid in the hop plant (Humulus lupulusL.). Xanthohumol was found to be a very potent cancer chemopreventive agent through regulation of diverse mechanisms. However, no data are available concerning the effects of xanthohumol on platelet activation. The aim of this paper was to examine the antiplatelet effect of xanthohumol in washed human platelets. In the present paper, xanthohumol exhibited more-potent activity in inhibiting platelet aggregation stimulated by collagen. Xanthohumol inhibited platelet activation accompanied by relative [Ca2+]imobilization, thromboxane A2formation, hydroxyl radical (OH●) formation, and phospholipase C (PLC)γ2, protein kinase C (PKC), mitogen-activated protein kinase (MAPK), and Akt phosphorylation. Neither SQ22536, an inhibitor of adenylate cyclase, nor ODQ, an inhibitor of guanylate cyclase, reversed the xanthohumol-mediated inhibitory effect on platelet aggregation. Furthermore, xanthohumol did not significantly increase nitrate formation in platelets. This study demonstrates for the first time that xanthohumol possesses potent antiplatelet activity which may initially inhibit the PI3-kinase/Akt, p38 MAPK, and PLCγ2-PKC cascades, followed by inhibition of the thromboxane A2formation, thereby leading to inhibition of [Ca2+]iand finally inhibition of platelet aggregation. Therefore, this novel role of xanthohumol may represent a high therapeutic potential for treatment or prevention of cardiovascular diseases.

scholarly journals cAMP- and cGMP-elevating agents inhibit GPIbα-mediated aggregation but not GPIbα-stimulated Syk activation in human platelets

2019 ◽  
Vol 17 (1) ◽  
Author(s):  
Stephanie Makhoul ◽  
Katharina Trabold ◽  
Stepan Gambaryan ◽  
Stefan Tenzer ◽  
Daniele Pillitteri ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Light Transmission ◽  
Thrombus Formation ◽  
Coagulation Factors ◽  
Human Platelets ◽  
Induce Platelet Aggregation ◽  
Akt Phosphorylation ◽  
Camp And Cgmp

Abstract Background The glycoprotein (GP) Ib-IX-V complex is a unique platelet plasma membrane receptor, which is essential for platelet adhesion and thrombus formation. GPIbα, part of the GPIb-IX-V complex, has several physiological ligands such as von Willebrand factor (vWF), thrombospondin and distinct coagulation factors, which trigger platelet activation. Despite having an important role, intracellular GPIb-IX-V signaling and its regulation by other pathways are not well defined. Our aim was to establish the intracellular signaling response of selective GPIbα activation in human platelets, in particular the role of the tyrosine kinase Syk and its regulation by cAMP/PKA and cGMP/PKG pathways, respectively. We addressed this using echicetin beads (EB), which selectively bind to GPIbα and induce platelet aggregation. Methods Purified echicetin from snake Echis carinatus venom was validated by mass spectrometry. Washed human platelets were incubated with EB, in the presence or absence of echicetin monomers (EM), Src family kinase (SFK) inhibitors, Syk inhibitors and the cAMP- and cGMP-elevating agents iloprost and riociguat, respectively. Platelet aggregation was analyzed by light transmission aggregometry, protein phosphorylation by immunoblotting. Intracellular messengers inositolmonophosphate (InsP1) and Ca2+i were measured by ELISA and Fluo-3 AM/FACS, respectively. Results EB-induced platelet aggregation was dependent on integrin αIIbβ3 and secondary mediators ADP and TxA2, and was antagonized by EM. EB stimulated Syk tyrosine phosphorylation at Y352, which was SFK-dependent and Syk-independent, whereas Y525/526 phosphorylation was SFK-dependent and partially Syk-dependent. Furthermore, phosphorylation of both Syk Y352 and Y525/526 was completely integrin αIIbβ3-independent but, in the case of Y525/526, was partially ADP/TxA2-dependent. Syk activation, observed as Y352/ Y525/Y526 phosphorylation, led to the phosphorylation of direct substrates (LAT Y191, PLCγ2 Y759) and additional targets (Akt S473). PKA/PKG pathways inhibited EB-induced platelet aggregation and Akt phosphorylation but, surprisingly, enhanced Syk and LAT/PLCγ2 tyrosine phosphorylation. A similar PKA/PKG effect was confirmed with convulxin−/GPVI-stimulated platelets. EB-induced InsP1 accumulation/InsP3 production and Ca2+-release were Syk-dependent, but only partially inhibited by PKA/PKG pathways. Conclusion EB and EM are specific agonists and antagonists, respectively, of GPIbα-mediated Syk activation leading to platelet aggregation. The cAMP/PKA and cGMP/PKG pathways do not inhibit but enhance GPIbα−/GPVI-initiated, SFK-dependent Syk activation, but strongly inhibit further downstream responses including aggregation. These data establish an important intracellular regulatory network induced by GPIbα. Graphical abstract

Endothelial nitric oxide synthase plays a minor role in inhibition of arterial thrombus formation

2005 ◽  
Vol 93 (06) ◽  
pp. 1161-1167 ◽  
Author(s):  
Burcin Özüyaman ◽  
Susanne Küsters ◽  
Elisabeth Kirchhoff ◽  
Rüdiger Scharf ◽  
Jürgen Schrader ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Thrombus Formation ◽  
Surface Expression ◽  
No Synthase ◽  
Minor Role ◽  
Endothelial No Synthase ◽  
Tail Tip ◽  
A Minor

SummaryEndothelial NO synthase (eNOS) expressed in the vascular en-dothelium or formed within platelets was postulated to inhibit platelet activation and aggregation. We have assessed the role of eNOS in platelet aggregation in vitro and in vivo by comparison of WT and eNOS-/- mice. Aggregometer studies revealed that collagen over a concentration range of 0.36–10 µg aggregated WT and eNOS-/- platelets to the same extent (10 µg: WT 86.7±4.7%, eNOS-/- 91±12%, n=6). Collagen treatment did not result in a significant increase in cGMP formation and VASP phosphorylation. Thrombin-induced P-selectin surface expression was unchanged in eNOS-/- platelets. In line with these findings no eNOS protein was detectable within the platelets of WT mice. In vivo, bleeding time after tail tip resection tended to be shorter in eNOS/- mice (WT: 116±35 s; eNOS-/- 109±37 s, n.s). Similarly, time to occlusion of the A.carotis after focal induction of thrombosis was 501±76 s (WT) and 457±95 s (eNOS-/-) (n.s.). These data demonstrate that eNOS-deficiency minimally affects platelet aggregation and is not associated with accelerated arterial thrombosis in vivo. Thus, in the mouse endothelial NO synthase does not play a major role in the autocrine modulation of platelet function and in thrombosis of conduit vessels in vivo.

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.

Metabolic and haemodynamic effects of α2-adrenoceptor stimulation and antagonism in man

1985 ◽  
Vol 68 (s10) ◽  
pp. 137s-139s ◽  
Author(s):  
M. J. Brown ◽  
A. D. Struthers ◽  
L. Di Silvio ◽  
T. Yeo ◽  
M. Ghatei ◽  
...  
Keyword(s):  
Physiological Role ◽  
Fold Increase ◽  
Plasma Noradrenaline ◽  
Minor Role ◽  
Hormone Release ◽  
Plasma Growth Hormone ◽  
Similar Reduction ◽  
Α2 Adrenoceptor ◽  
Baseline Values ◽  
A Minor

1. Six healthy volunteers received a 60 min infusion of guanfacine (α2-agonist) on two occasions, preceded by either idazoxan (α2-antagonist) or vehicle. 2. Idazoxan elevated blood pressure by 8/7 mmHg, but there was no change on either day during guanfacine infusion. 3. Guanfacine reduced plasma noradrenaline by approximately 30%, and this was not antagonized by idazoxan. By contrast, the 30-fold increase in plasma growth hormone caused by guanfacine was almost completely blocked by idazoxan. 4. Guanfacine caused a two- to three-fold increase in plasma glucagon and a similar reduction in plasma insulin. Only the latter was antagonized by idazoxan. No consistent changes in plasma ACTH were observed after either idazoxan or guanfacine. 5. Idazoxan itself elevated plasma noradrenaline up to twice baseline values, but did not affect the other metabolic measurements. 6. α2-Adrenoceptor stimulation plays a minor role in control of hormone release but has a greater physiological role in regulating release of the neurotransmitter, noradrenaline.

scholarly journals Bidirectional transport of glutamine across the cell membrane in rat liver

1983 ◽  
Vol 216 (2) ◽  
pp. 401-408 ◽  
Author(s):  
P Fafournoux ◽  
C Demigné ◽  
C Rémésy ◽  
A Le Cam
Keyword(s):  
Cell Membrane ◽  
Physiological Role ◽  
Minor Role ◽  
Facilitated Diffusion ◽  
Dependent Component ◽  
Glutamine Transport ◽  
Diffusion Component ◽  
Bidirectional Transport ◽  
A Minor

Hepatocytes isolated from fed rats were used to investigate glutamine transport. Glutamine transport appears as a composite process involving at least two saturable components. The Na+-dependent component probably represents the entry through the N system. The Na+-independent component was also inhibited by histidine and exhibited trans-stimulation, suggestive of a facilitated diffusion process. Kinetic parameters for both systems suggest that facilitated diffusion only plays a minor role in glutamine influx. In contrast, the Km for glutamine efflux was consistent with a physiological role of the facilitated-diffusion component in glutamine release. In Na+ medium, relatively constant distribution ratios (about 8) between intra- and extra-cellular concentrations were observed, with external glutamine ranging from 0.5 to 5 mM. The present observations suggest that glutamine influx might largely be mediated by the N system, whereas facilitated diffusion allows hepatocytes to release glutamine when intracellular concentrations are elevated. The physiological consequences of this bidirectional transfer of glutamine across the liver cell membrane is discussed.

scholarly journals The synthetic RGDS peptide inhibits the binding of fibrinogen lacking intact alpha chain carboxyterminal sequences to human blood platelets

Blood ◽  
1987 ◽  
Vol 69 (3) ◽  
pp. 950-952
Author(s):  
EI Peerschke ◽  
DK Galanakis
Keyword(s):  
Platelet Aggregation ◽  
Human Blood ◽  
Blood Platelets ◽  
Adenosine Diphosphate ◽  
Competitive Inhibitor ◽  
Minor Role ◽  
Solution Ph ◽  
Alpha Chain ◽  
A Minor ◽  
Human Blood Platelets

The alpha chain 572–574 Arg-Gly-Asp sequence of fibrinogen appears to play only a minor role in platelet aggregation based on the ability of fibrinogen preparations lacking alpha chain carboxyterminal segments to support platelet aggregation, but synthetic Arg-Gly-Asp-Ser (RGDS) peptides are capable of inhibiting platelet aggregation and fibrinogen binding. The present study thus examined the ability of RGDS peptides to inhibit platelet interactions with a plasmic degradation product of fibrinogen (8D–50) that resembles an intermediate fragment X. Gel- filtered, human blood platelets suspended in 0.01 mol/L HEPES-buffered modified Tyrode's solution, pH 7.5, were stimulated with 20 mumol/L adenosine diphosphate and the binding of 125I-labeled 8D–50 or intact fibrinogen (0.01 to 0.6 mg/mL) assessed in the presence of 0 to 117 mumol/L RGDS. The data revealed that RGDS decreased the apparent affinity of 8D–50 and intact fibrinogen for platelets but did not affect the maximum number of binding sites. RGDS thus appears to be a competitive inhibitor not only of intact fibrinogen (Ki = 12 +/- 2 mumol/L) but also of 8D–50 (Ki = 15 +/- 3 mumol/L) (mean +/- SD, n = 3).

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