scholarly journals AN IMPORTANT STIMULATORY ROLE FOR THE cGMP-DEPENDENT PROTEIN KINASE II IN PLATELET ACTIVATION, IN VIVO THROMBOSIS AND HAEMOSTASIS

2020 ◽  
Author(s):  
Zhenyu Li ◽  
Ying Liang ◽  
can wang ◽  
Guoying Zhang ◽  
Jens Schlossmann ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Atp Release ◽  
Downstream Signaling ◽  
Dependent Protein ◽  
Deficient Mice

Background and Purpose: The intracellular second messenger cGMP mediates signals by activating two types of cGMP-dependent protein kinases (PKG), PKG I and PKG II, differentially expressed in different cells. In platelets, cGMP mediates biphasic signals that stimulate and inhibit platelet activation, and the downstream signaling of cGMP is mediated by PKG I, the only PKG known to be expressed in platelets. However, functional defects of PKG I knockout platelets did not fully explain the roles of cGMP and the effect of PKG inhibitors on platelet activation. Experimental Approach: To determine if PKG II is present in platelets and plays a role in platelet activation, we performed RT-PCR and isolation of PKG II protein using cGMP-conjugated beads. We further determined platelet aggregation and ATP release in vitro, and FeCl3-injured carotid artery thrombosis as well as tail bleeding time in vivo. Key Results: PKG II is expressed in platelets and plays an important role in selectively stimulating platelet activation but not in the negative regulatory role of cGMP. Collagen-induced platelet aggregation and ATP secretion were reduced in PKG II-deficient mice but not PKG I-deficient mice. In contrast, low-dose thrombin-induced platelet activation depended on PKG I but not PKG II. Tail bleeding time and FeCl3-induced artery thrombus formation were significantly prolonged in PKG II knockout mice. Conclusion and Implication: PKG II-mediated cGMP signals are important in platelet activation, thrombosis and haemostasis in vitro and in vivo.

scholarly journals IκB kinase 2 is not essential for platelet activation

2020 ◽  
Vol 4 (4) ◽  
pp. 638-643
Author(s):  
Manuel Salzmann ◽  
Sonja Bleichert ◽  
Bernhard Moser ◽  
Marion Mussbacher ◽  
Mildred Haase ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Active Site ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Iκb Kinase ◽  
Specific Deletion

Abstract Platelets are small anucleate cells that release a plethora of molecules to ensure functional hemostasis. It has been reported that IκB kinase 2 (IKK2), the central enzyme of the inflammatory NF-κB pathway, is involved in platelet activation, because megakaryocyte/platelet-specific deletion of exons 6 and 7 of IKK2 resulted in platelet degranulation defects and prolonged bleeding. We aimed to investigate the role of IKK2 in platelet physiology in more detail, using a platelet-specific IKK2 knockout via excision of exon 3, which makes up the active site of the enzyme. We verified the deletion on genomic and transcriptional levels in megakaryocytes and were not able to detect any residual IKK2 protein; however, platelets from these mice did not show any functional impairment in vivo or in vitro. Bleeding time and thrombus formation were not affected in platelet-specific IKK2-knockout mice. Moreover, platelet aggregation, glycoprotein GPIIb/IIIa activation, and degranulation were unaltered. These observations were confirmed by pharmacological inhibition of IKK2 with TPCA-1 and BMS-345541, which did not affect activation of murine or human platelets over a wide concentration range. Altogether, our results imply that IKK2 is not essential for platelet function.

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.

Antithrombotic Effect of a Recombinant von Willebrand Factor, VCL, on Nitrogen Laser-Induced Thrombus Formation in Guinea Pig Mesenteric Arteries

1995 ◽  
Vol 73 (02) ◽  
pp. 318-323 ◽  
Author(s):  
K Azzam ◽  
L I Garfinkel ◽  
C Bal dit Sollier ◽  
M Cisse Thiam ◽  
L Drouet
Keyword(s):  
Platelet Aggregation ◽  
Bleeding Time ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Mesenteric Arteries ◽  
Antithrombotic Effects ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo assess the antithrombotic effectiveness of blocking the platelet glycoprotein (GP) Ib/IX receptor for von Willebrand factor (vWF), the antiaggregating and antithrombotic effects were studied in guinea pigs using a recombinant fragment of vWF, Leu 504-Lys 728 with a single intrachain disulfide bond linking residues Cys 509-Cys 695. The inhibitory effect of this peptide, named VCL, was tested in vitro on ristocetin- and botrocetin-induced platelet aggregation and compared to the ADP-induced platelet aggregation. In vivo, the antithrombotic effect of VCL was tested in a model of laser-injured mesentery small arteries and correlated to the ex vivo ristocetin-induced platelet aggregation. In this model of laser-induced thrombus formation, five mesenteric arteries were studied in each animal, and the number of recurrent thrombi during 15 min, the time to visualization and time to formation of first thrombus were recorded.In vitro, VCL totally abolished ristocetin- and botrocetin-induced platelet aggregation, but had no effect on ADP-induced platelet aggregation. Ex vivo, VCL (0.5 to 2 mg/kg) administered as a bolus i. v. injection inhibits ristocetin-induced platelet aggregation with a duration of action exceeding 1 h. The maximum inhibition was observed 5 min after injection of VCL and was dose related. The same doses of VCL had no significant effect on platelet count and bleeding time. In vivo, VCL (0.5 to 2 mg/kg) had no effect on the appearance of the thrombi formed but produced dose-dependent inhibition of the mean number of recurrent thrombi (the maximal effect was obtained at 5 min following i. v. injection of the highest dose: 0.8 ± 0.2 thrombi versus 4 ± 0.4 thrombi in controls). The three doses of VCL increased the time in which the first thrombus in a concentration-dependent manner was formed. However, the time to visualize the first thrombus was only prolonged in the higher dose-treated group.These in-vivo studies confirm that VCL induces immediate, potent, and transient antithrombotic effects. Most importantly, this inhibition was achieved without inducing thrombocytopenia nor prolongation of the bleeding time.

scholarly journals Annexin A7 is a critical regulator of Ca2+ mobilization and lipid metabolism during platelet activation and arterial thrombosis

2020 ◽  
Vol 41 (Supplement_2) ◽  
Author(s):  
O Borst ◽  
S Geue ◽  
M.C Manke ◽  
B Peng ◽  
P Muenzer ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Platelet Activation ◽  
Arterial Thrombosis ◽  
Bleeding Time ◽  
Thrombus Formation ◽  
Vascular Occlusion ◽  
Funding Source ◽  
Annexin A7

Abstract Background Platelet activation after contact to subendothelial collagen following atherosclerotic plaque rupture can lead to arterial thrombosis with acute thrombotic vascular occlusion. Annexin A7 (AnxA7) is an intracellular Ca2+- and phospholipid-binding protein that participates in the regulation of prostaglandin production in inflammatory diseases, but also in cell survival and tumor growth. Objective In the present study, we aimed to determine the role of AnxA7 for platelet Ca2+ signaling and lipid metabolism in platelet activation and arterial thrombosis in gene-targeted mice lacking annexin A7 (Anxa7−/−). Results AnxA7 is strongly expressed in platelets of platelet-rich human coronary thrombi aspirated from patients with acute ST elevation myocardial infarction. Functionally, platelet aggregation and dense granule secretion were significantly abrogated in Anxa7−/− platelets as compared to wildtype platelets (Anxa7+/+) after activation with collagen or collagen-related peptide (CRP), a specific agonist of the major platelet collagen receptor glycoprotein VI (GPVI). Further, in vitro thrombus formation on a collagen-coated surface under high arterial shear rates was significantly diminished in Anxa7-deficient platelets, and thrombotic vascular occlusion after FeCl3-induced injury in vivo was blunted in Anxa7−/−bone marrow chimeric mice, but no prolongation of bleeding time was observed. Moreover, Anxa7−/− platelets showed a significant reduction of IP3 production due to an abolished phospholipase C (PLC) gamma2 phosphorylation resulting in an abolished increase of [Ca2+]i after platelet activation with CRP. Moreover, we could show by quantitative lipidomics analysis that annexin A7 critically affects platelet oxylipid metabolism following activation of GPVI-dependent platelet signalling since Anxa7−/− platelets showed a significant reduction of the bioactive metabolites thromboxane A2 and 12(S)-hydroxy-eicosatetraenoic acid (12(S)-HETE) levels as well as significantly reduced levels of several other prostaglandins following stimulation with collagen or CRP. Finally, defective PLCgamma2 phosphorylation, IP1 production and blunted increase of [Ca2+]i in Anxa7−/− platelets could be rescued by exogenous addition of 12(S)-HETE indicating that AnxA7 is a critical regulator of the platelet oxygenase 12-lipoxygenase (12-LOX) in GPVI-dependent platelet Ca2+ signalling during arterial thrombosis following activation by collagen. Conclusions The present study reveals annexin A7 as a critical regulator of oxylipid metabolism and Ca2+ signaling in GPVI-dependent platelet activation. Anxa7-deficiency further results in decreased in vitro and in vivo thrombus formation, but does not affect bleeding time. In conclusion, annexin A7 plays an important role in platelet signaling during arterial thrombosis and thus, may reflect a promising target for novel antiplatelet strategies. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (Deutsche Forschungsgemeinschaft, DFG)

scholarly journals Impaired platelet activation and cAMP homeostasis in MRP4-deficient mice

Blood ◽  
2015 ◽  
Vol 126 (15) ◽  
pp. 1823-1830 ◽  
Author(s):  
Benoit Decouture ◽  
Elise Dreano ◽  
Tiphaine Belleville-Rolland ◽  
Orjeta Kuci ◽  
Blandine Dizier ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Key Points ◽  
Platelet Signaling ◽  
Deficient Mice ◽  
Kinase A

Key PointsIn vivo and in vitro thrombus formation is altered in MRP4-deficient mice. MRP4 modulates the cAMP–protein kinase A platelet signaling pathway.

scholarly journals An Antithrombotic Strategy by Targeting Phospholipase D in Human Platelets

2018 ◽  
Vol 7 (11) ◽  
pp. 440 ◽  
Author(s):  
Wan Lu ◽  
Chi Chung ◽  
Ray Chen ◽  
Li Huang ◽  
Li Lien ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Phospholipase D ◽  
Thrombus Formation ◽  
Human Platelets ◽  
Platelet Activity ◽  
Clot Retraction ◽  
First Time

Phospholipase D (PLD) is involved in many biological processes. PLD1 plays a crucial role in regulating the platelet activity of mice; however, the role of PLD in the platelet activation of humans remains unclear. Therefore, we investigated whether PLD is involved in the platelet activation of humans. Our data revealed that inhibition of PLD1 or PLD2 using pharmacological inhibitors effectively inhibits platelet aggregation in humans. However, previous studies have showed that PLD1 or PLD2 deletion did not affect mouse platelet aggregation in vitro, whereas only PLD1 deletion inhibited thrombus formation in vivo. Intriguingly, our data also showed that the pharmacological inhibition of PLD1 or PLD2 does not affect mouse platelet aggregation in vitro, whereas the inhibition of only PLD1 delayed thrombus formation in vivo. These findings indicate that PLD may play differential roles in humans and mice. In humans, PLD inhibition attenuates platelet activation, adhesion, spreading, and clot retraction. For the first time, we demonstrated that PLD1 and PLD2 are essential for platelet activation in humans, and PLD plays different roles in platelet function in humans and mice. Our findings also indicate that targeting PLD may provide a safe and alternative therapeutic approach for preventing thromboembolic disorders.

scholarly journals VAS2870 and VAS3947 attenuate platelet activation and thrombus formation via a NOX-independent pathway downstream of PKC

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Wan Jung Lu ◽  
Jiun Yi Li ◽  
Ray Jade Chen ◽  
Li Ting Huang ◽  
Tzu Yin Lee ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Thrombus Formation ◽  
Therapeutic Agents ◽  
Independent Manner ◽  
Downstream Signaling ◽  
Nox Inhibitors ◽  
Normal Hemostasis ◽  
Granule Release

AbstractNADPH oxidase (NOX) enzymes are involved in a various physiological and pathological processes such as platelet activation and inflammation. Interestingly, we found that the pan-NOX inhibitors VAS compounds (VAS2870 and its analog VAS3947) exerted a highly potent antiplatelet effect. Unlike VAS compounds, concurrent inhibition of NOX1, 2, and 4 by treatment with ML171, GSK2795039, and GKT136901/GKT137831 did not affect thrombin and U46619-induced platelet aggregation. These findings suggest that VAS compounds may inhibit platelet aggregation via a NOX-independent manner. Thus, we aimed to investigate the detailed antiplatelet mechanisms of VAS compounds. The data revealed that VAS compounds blocked various agonist-induced platelet aggregation, possibly via blocking PKC downstream signaling, including IKKβ and p38 MAPK, eventually reducing platelet granule release, calcium mobilization, and GPIIbIIIa activation. In addition, VAS compounds inhibited mouse platelet aggregation-induced by collagen and thrombin. The in vivo study also showed that VAS compounds delayed thrombus formation without affecting normal hemostasis. This study is the first to demonstrate that, in addition to inhibiting NOX activity, VAS compounds reduced platelet activation and thrombus formation through a NOX-independent pathway downstream of PKC. These findings also indicate that VAS compounds may be safe and potentially therapeutic agents for treating patients with cardiovascular diseases.

Reversible Inhibition of Human Platelet Activation by Hypothermia In Vivo and In Vitro

1994 ◽  
Vol 71 (05) ◽  
pp. 633-640 ◽  
Author(s):  
Alan D Michelson ◽  
Hollace MacGregor ◽  
Marc R Barnard ◽  
Anita S Kestin ◽  
Michael J Rohrer ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Whole Blood ◽  
Peripheral Blood ◽  
Bleeding Time ◽  
Human Platelet ◽  
Platelet Surface ◽  
Shed Blood

SummaryA hypothermia-induced hemorrhagic diathesis is associated with cardiopulmonary bypass, major surgery, and multiple trauma, but its pathophysiological basis is not well understood. We examined the hypothesis that hypothermia reversibly inhibits human platelet activation in vitro and in vivo. Platelet activation was studied in normal volunteers by whole blood flow cytometric analysis of modulation of platelet surface GMP-140 and the glycoprotein (GP) Ib-IX complex in: a) shed blood emerging from a standardized in vivo bleeding time wound; b) peripheral blood activated in vitro with either thrombin (in the presence of gly-pro-arg-pro, an inhibitor of fibrin polymerization) or the stable thromboxane (TX) A2 analogue U46619. Platelets in peripheral whole blood were activated at temperatures between 22° C and 37° C. the forearm skin temperature was maintained at temperatures between 22° C and 37° C prior to and during the bleeding time incision. Platelet aggregation was studied in shed blood by flow cytometry and in peripheral blood by aggregometry. Generation of TXB 2 (the stable metabolite of TXA 2) was determined by radioimmunoassay. In vitro, hypothermia inhibited both thrombin- and U46619-induced upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregation, and TXB2 generation. These inhibitory effects of hypothermia were all completely reversed by rewarming the blood to 37° C. In vivo, platelet activation was inhibited by hypothermia as shown by 5 independent assays of shed blood: upregulation of GMP-140, downregulation of the GPIb-IX complex, platelet aggregate formation, TXB 2 ggeneration, and the bleeding time. In summary, by a combination of immunologic, biochemical, and functional assays, we demonstrate that hypothermia inhibits human platelet activation in whole blood in vitro and in vivo. Rewarming hypothermic blood completely reverses the activation defect. These results suggest that maintaining normothermia or rewarming a hypothermic bleeding patient may reduce the need for platelet transfusions.

scholarly journals Antiplatelet and Antithrombotic Effects of Isaridin E Isolated from the Marine-Derived Fungus via Downregulating the PI3K/Akt Signaling Pathway

Marine Drugs ◽  
2021 ◽  
Vol 20 (1) ◽  
pp. 23
Author(s):  
Ni Pan ◽  
Zi-Cheng Li ◽  
Zhi-Hong Li ◽  
Sen-Hua Chen ◽  
Ming-Hua Jiang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Signaling Pathway ◽  
Platelet Activation ◽  
Bleeding Time ◽  
Akt Signaling ◽  
Akt Signaling Pathway ◽  
Risk Of Bleeding ◽  
Antithrombotic Effects

Isaridin E, a cyclodepsipeptide isolated from the marine-derived fungus Amphichorda felina (syn. Beauveria felina) SYSU-MS7908, has been demonstrated to possess anti-inflammatory and insecticidal activities. Here, we first found that isaridin E concentration-dependently inhibited ADP-induced platelet aggregation, activation, and secretion in vitro, but did not affect collagen- or thrombin-induced platelet aggregation. Furthermore, isaridin E dose-dependently reduced thrombosis formation in an FeCl3-induced mouse carotid model without increasing the bleeding time. Mechanistically, isaridin E significantly decreased the ADP-mediated phosphorylation of PI3K and Akt. In conclusion, these results suggest that isaridin E exerts potent antithrombotic effects in vivo without increasing the risk of bleeding, which may be due to its important role in inhibiting ADP-induced platelet activation, secretion and aggregation via the PI3K/Akt pathways.

Anti-Human VWF Monoclonal Antibody SZ-123 Prevents Arterial Thrombus Formation by Inhibiting VWF–collagen and VWF-Platelet Interactions in Rhesus Monkeys

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 5194-5194
Author(s):  
Yiming Zhao ◽  
Changgeng Ruan
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Bleeding Time ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Thrombus Formation ◽  
Significant Prolongation ◽  
Arterial Thrombus

Abstract Abstract 5194 Objective: To investigate the in vivo antithrombotic efficacy of an anti-VWF monoclonal antibody SZ-123, and its potential underlying mechanism. Methods and Results: Cyclic flow reductions (CFRs) were measured in the femoral artery of monkeys before and after intravenous administration of SZ-123. Ex vivo VWF binding to collagen, platelet aggregation, platelet count and template bleeding time were performed as measurements of antithrombotic activity. In addition, plasma VWF, SZ-123 levels, and VWF occupancy were measured by ELISA. Administration of 0. 1, 0. 3, and 0. 6 mg/kg SZ-123 resulted in 45. 3%, 78. 2%, and 100% reduction in CFRs, respectively. When 0. 3 and 0. 6 mg/kg SZ-123 were administrated, 100% of VWF was occupied by the antibody. Moreover, 100% ex vivo inhibition of VWF-collagen binding and 60–95% inhibition of platelet aggregation were observed from 15 min to 1h. None of the doses resulted in significant prolongation of bleeding time. In vitro experiment also revealed that SZ-123 not only blocks collagen-VWF A3 interaction but also inhibits indirectly VWF A1 binding to GPIba induced by ristocetin. Conclusions: SZ-123 prevents in vivo arterial thrombus formation under high shear conditions by inhibiting VWF A3–collagen and VWF A1-platelet interactions and does not prolong bleeding time. Disclosures: No relevant conflicts of interest to declare.

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