A novel role for endoplasmic reticulum protein 46 (ERp46) in platelet function and arterial thrombosis in mice

Blood ◽  
2021 ◽  
Author(s):  
Junsong Zhou ◽  
Yi Wu ◽  
Lubica Rauova ◽  
Gavin Koma ◽  
Lu Wang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Disulfide Bonds ◽  
Active Sites ◽  
Atp Release ◽  
Molecular Architecture ◽  
Clot Retraction ◽  
Platelet Surface ◽  
Endoplasmic Reticulum Protein ◽  
Platelet Spreading

Although several members of protein disulfide isomerase (PDI) family support thrombosis, other PDI family members with the CXYC motif remain uninvestigated. ERp46 has three CGHC redox-active sites and a radically different molecular architecture than other PDIs. Expression of ERp46 on the platelet surface increased with thrombin stimulation. An anti-ERp46 antibody inhibited platelet aggregation, ATP release, and αIIbβ3 activation. ERp46 protein potentiated αIIbβ3 activation, platelet aggregation and ATP release, while inactive ERp46 inhibited these processes. ERp46-knockout mice had prolonged tail-bleeding times, and decreased platelet accumulation in thrombosis models that was rescued by infusion of ERp46. ERp46-deficient platelets had decreased αIIbβ3 activation, platelet aggregation, ATP release and P-selectin expression. The defects were reversed by wild-type ERp46 and partially reversed by ERp46 containing any of the three active sites. Platelet aggregation stimulated by an αIIbβ3-activating peptide was inhibited by the anti-ERp46 antibody and was decreased in ERp46-deficient platelets. ERp46 bound tightly to αIIbβ3 by surface plasmon resonance but poorly to platelets lacking αIIbβ3, and physically associated with αIIbβ3 upon platelet activation. ERp46 mediated clot retraction and platelet spreading. ERp46 more strongly reduced disulfide bonds in the β3 subunit than other PDIs, and in contrast to PDI generated thiols in β3 independently of fibrinogen. ERp46 cleaved the Cys473-Cys503 disulfide bond in β3 implicating a target for ERp46. Finally, ERp46-deficient platelets have decreased thiols in β3 implying that ERp46 cleaves disulfide bonds in platelets. In conclusion, ERp46 is critical for platelet function and thrombosis and facilitates αIIbβ3 activation by targeting disulfide bonds.

Defect of Platelet Aggregation and Adhesion Induced by Autoantibodies Against Platelet Glycoprotein IIIa

1992 ◽  
Vol 68 (02) ◽  
pp. 208-213 ◽  
Author(s):  
Carlo L Balduini ◽  
Giampiera Bertolino ◽  
Patrizia Noris ◽  
Franco Piovella ◽  
Fabiola Sinigaglia ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Atp Release ◽  
Platelet Glycoprotein ◽  
Thrombocytopenic Purpura ◽  
Platelet Surface ◽  
Platelet Number ◽  
Glycoprotein Iiia ◽  
Subendothelial Matrix ◽  
Functional Defects

SummaryA young patient developed chronic idiopathic thrombocytopenic purpura. Prednisone therapy normalized platelet number, but bleeding symptoms did not disappear. Platelet function was severely impaired, since platelet aggregation, ATP release and adhesion to collagen and subendothelial matrix were significantly reduced. Plasma and purified immunoglobulins of the patient reproduced the functional defects in normal platelets. Immunoblotting revealed that patient’s plasma contained an antibody reacting with a component of platelets with the same electrophoretic mobility of glycoproteins IIIa of normal platelets. Moreover, patient’s plasma inhibited the binding of an anti-GPIIb/IIIa monoclonal antibody to platelet surface. Additional immunosuppressive therapy with prednisone and azathioprine normalized platelet function and induced the disappearance of bleeding symptoms.

scholarly journals Coenzyme Q10 Attenuates Platelet Integrin αIIbβ3 Outside-in Signaling through Targeting cAMP/PKA Pathway and Inhibits Atherosclerosis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2423-2423
Author(s):  
Yan Yang ◽  
Xiaohong Ruby Xu ◽  
Heyu Ni ◽  
Liping Ma ◽  
Wenhua Ling ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Clot Retraction ◽  
Integrin Αiibβ3 ◽  
Granule Secretion ◽  
Platelet Spreading ◽  
Pka Pathway ◽  
Inside Out

Abstract Introduction: Platelet integrin αIIbβ3 outside-in signaling is crucial for platelet adhesion and aggregation, and contributes to atherogenesis. Coenzyme Q10 (CoQ10) has been implicated as a protective factor against cardiovascular diseases (CVDs), particularly atherosclerosis. However, whether CoQ10 attenuates atherosclerosis through inhibiting platelet function and αIIbβ3 outside-in signaling is unknown. The aim of this study was to explore whether CoQ10 affects platelet function and αIIbβ3 outside-in signalling and thus inhibiting the progress of atherosclerosis in vivo and the underlying mechanisms in vitro. Methods: In vitro study, The murine platelet rich plasma (PRP) from C57BL/6J wild-type (WT) mice or human PRP and gel-filtered platelets were incubated with different concentrations (1, 10 or 100 μM) of CoQ10 or the vehicle control for 50 min. Platelet aggregation, spreading on fibrinogen (Fg) and clot retraction were determined. In addition, the effects of CoQ10 on platelet integrin αIIbβ3 inside-out signalling (e.g., talin-1 and kindlin-3 binding to integrin β3) were determined by immunoprecipitation, and outside-in signalling (e.g., phosphorylation of sarcoma tyrosine-protein kinase (c-Src), focal adhesion kinase (FAK), and β3 cytoplasmic tail, myosin light chain (MLC)) were determined by Western blotting. The levels of platelet ATP and cAMP were measured by ELISA assays. In vivo study, male homozygous apolipoprotein E-deficient (apoE-/-) mice (C57BL/6 genetic background) were fed either a standard normal AIN-93G diet (NC group), a Western-type diet (HFD group) or a Western-type diet supplemented with CoQ10 (1800 mg/kg diet) (CoQ10 group) for 12 weeks. Platelet aggregation, granule secretion, platelet spreading, clot retraction, integrin αIIbβ3 outside-in signalling, platelet-leukocyte interactions and carotid artery plaque area were also examined. In our randomized, double-blind, placebo-controlled trial, 101 hypercholesterolemic subjects were randomly administrated to 120 mg CoQ10 or placebo daily for 24 weeks. Platelet intracellular CoQ10 levels, platelet aggregation in PRP, platelet platelet factor 4 (PF-4) and C-C motif ligand 5 (CCL5) release, and platelet integrin αIIbβ3 outside-in signalling were also evaluated before and after 24 weeks of intervention. Results: We found that CoQ10 inhibited human and WT mouse platelet aggregation, platelet spreading, granule secretion, and clot retraction in vitro and apoE-/- mice on a high fat diet. CoQ10 also reduced atherosclerosis and platelet-monocyte aggregation in apoE-/- mice. The inhibitory effects of CoQ10 is mediated by attenuated αIIbβ3 outside-in signalling pathway (e.g., attenuation of phosphorylation of c-Src, FAK, and β3 cytoplasmic tail, and MLC in thrombin-activated platelets or platelets exposed to immobilized Fg), which requires up-regulation of the cAMP/PKA pathway, where CoQ10 inhibited phosphodiesterase 3A activity and activated the A2A adenosine receptor. However, CoQ10 did not affect platelet integrin αIIbβ3 inside-out signalling pathway, platelet cellular ATP, or platelet apoptosis (the mitochondrial membrane potential and phosphatidylserine exposure). Moreover, our clinical trial in dyslipidemic patients demonstrated that CoQ10 supplementation attenuated platelet aggregation, which was positively correlated with the increased platelet CoQ10 concentrations, inhibited αIIbβ3 outside-in signalling and decreased platelet PF-4 and CCL5 secretion. Conclusions: We present new data to suggest that CoQ10 plays a novel role in attenuating platelet function and integrin αIIbβ3 outside-in signalling though targeting cAMP/PKA signalling cascade and thus inhibiting the progress of atherosclerosis. CoQ10 is therefore a promising agent for the prevention and/or treatment for cardiovascular disease. Disclosures No relevant conflicts of interest to declare.

scholarly journals The transmembrane protein disulfide isomerase TMX1 negatively regulates platelet responses

Blood ◽  
2019 ◽  
Vol 133 (3) ◽  
pp. 246-251 ◽  
Author(s):  
Zhenzhen Zhao ◽  
Yi Wu ◽  
Junsong Zhou ◽  
Fengwu Chen ◽  
Aizhen Yang ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Protein Disulfide Isomerase ◽  
Transmembrane Protein ◽  
Protein A ◽  
Atp Release ◽  
Platelet Surface ◽  
Knockout Mouse Model ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Secreted platelet protein disulfide isomerases, PDI, ERp57, ERp5, and ERp72, have important roles as positive regulators of platelet function and thrombosis. Thioredoxin-related transmembrane protein 1 (TMX1) was the first described transmembrane member of the protein disulfide isomerase family of enzymes. Using a specific antibody, the recombinant extracellular domain of TMX1 (rTMX1) protein, a knockout mouse model, and a thiol-labeling approach, we examined the role of TMX1 in platelet function and thrombosis. Expression of TMX1 on the platelet surface increased with thrombin stimulation. The anti-TMX1 antibody increased platelet aggregation induced by convulxin and thrombin, as well as potentiated platelet ATP release. In contrast, rTMX1 inhibited platelet aggregation and ATP release. TMX1-deficient platelets had increased aggregation, ATP release, αIIbβ3 activation, and P-selectin expression, which were reversed by addition of rTMX1. TMX1-knockout mice had increased incorporation of platelets into a growing thrombus in an FeCl3-induced mesenteric arterial injury model, as well as shortened tail-bleeding times. rTMX1 oxidized thiols in the αIIbβ3 integrin and TMX1-deficient platelets had increased thiols in the β3 subunit of αIIbβ3, consistent with oxidase activity of rTMX1 against αIIbβ3. Thus, TMX1 is the first identified extracellular inhibitor of platelet function and the first disulfide isomerase that negatively regulates platelet function.

PLATELET FUNCTION AND LIPOPROTEINS IN PATIENTS WITH HYPOTHYROIDISM

1987 ◽  
Author(s):  
S Fujii ◽  
T Kariya
Keyword(s):  
Platelet Aggregation ◽  
Thyroid Hormones ◽  
Platelet Function ◽  
Gel Electrophoresis ◽  
Atp Release ◽  
Serum Lipoprotein ◽  
Platelet Factor ◽  
Cholesterol Levels ◽  
Gradient Gel Electrophoresis ◽  
Hdl Metabolism

Platelet function and serum lipoprotein levels were studied in ten patients (two males and eight females) with hypothyroidism. Platelet aggregation and ATP release were determined by Lumi-aggregometer using ADP , collagen and epinephrine as stimulants. Platelet factor 4 (PF4) and thromboxane B2 (TXB2) were determined by radioimmunoassay. High density lipoproteincholesterol (HDL-C) was determined by heparin-manga-nese method. HDL subfractions were separated by gradient gel electrophoresis (PAA 4/30). Apolipopro-teins were measured by single radial immunodiffusion. Platelet aggregation increased in those patients at stimulating by epinephrine. ATP release also increased at stimulating by epinephrine. PF4 increased at stimulating by epinephrine. TXB2 increased at stimu-lating--by ADP or epinephrine significantly (p<0.05), respectively. Platelet aggregation was not correlated with thyroid hormones or total cholesterol levels.But it had a positive correlation tendency with HDL-C or HDL2-C and a negative one with HDL3-C levels.These results suggested some relationships between platelet function and HDL metabolism in patients with hypothyroidism.

The Effects Of Chemical Alterations Of The Benzoic Acid Nucleus On Platelet Function And Prostacyclin Activity In The Arterial Wall

1981 ◽  
Author(s):  
B A Killackey ◽  
J J Killackey ◽  
R B Philp
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Benzoic Acid ◽  
Platelet Function ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Rabbit Aorta ◽  
Arachidonic Acid Metabolism ◽  
Second Phase ◽  
Benzoic Acid Derivatives

The effects of a series of benzoic acid derivatives (ASA analogs) on prostacyclin (PGI2) synthesis by rabbit aorta rings and on human platelet function were examined to determine if antiplatelet activity could be separated from anti-PGI2 activity.Rings of rabbit aorta were incubated with or without drugs in Tris 0.05 M, pH 7.5 for 6 m at room temperature (R.T.). Supernatant was then transferred to platelet-rich plasma incubated at 37°C for 3 m. ADP was added 60 s later and aggregation was measured and compared to controls. Rings were also incubated with 14C-arachidonic acid (14C-AA) for 60 m at R.T. in Tris with or without drugs. Products were extracted and measured by radio-T.L.C. along with known standards. Platelet aggregation and release of ATP were measured using a ChronoLog Lumi aggregometer. The effects of these agents on PGI2 activity were similar to their effects on platelet aggregation. ASA however did not exhibit the marked inhibitory potency that it had on the second phase of platelet aggregation and ATP release. Changing the 2-acetoxy group of A.S.A. to a 2-acetyl or 3-propionyloxy resulted in a loss of inhibitory activity in both systems. 2-Propionyloxy substitution resulted in a similar spectrum of activity to ASA. The effects of these agents on the metabolism of 14C-AA by rabbit aorta rings generally confirmed the bioassay results although some of the agents had novel effects on blood vessel arachidonic acid metabolism.Despite potential species differences, this study demonstrates an inability to separate antiplatelet and anti-PGI2 effects with this series of benzoic acid derivatives. Further study of the effects of these agents on the metabolism of 14C-AA by rings of rabbit aorta may lead to a better understanding of PGI2 formation.

Pyridoxal phosphate inhibition of platelet function

1980 ◽  
Vol 238 (1) ◽  
pp. H54-H60 ◽  
Author(s):  
E. Kornecki ◽  
H. Feinberg
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Pyridoxal Phosphate ◽  
Shape Change ◽  
Serotonin Release ◽  
Platelet Surface ◽  
Partial Inhibition ◽  
Induced Aggregation

The effect of pyridoxal phosphate (PLP) on human platelet function in vitro was studied. PLP inhibited adenosine diphosphate (ADP)-induced shape change, aggregation, and the potentiation by ADP of arachidonic acid-induced aggregation. This inhibition could easily be reversed by increasing concentrations of ADP or by removing PLP. The addition of sodium borohydride to PLP-treated platelets produced an irreversible inhibition of ADP aggregation. Thus it is possible that PLP inhibited ADP-induced platelet function by forming a Schiff base with platelet-surface amino groups. PLP also produced a partial inhibition of platelet aggregation to epinephrine, arachidonic acid, A23187, and a dose-dependent inhibition of [14C]serotonin release to epinephrine and arachidonic acid. PLP did not inhibit [14C]serotonin release to A23187, nor did it suppress arachidonic acid-induced malondialdehyde production. The conclusion is drawn that the partial inhibition by PLP of platelet aggregation observed to epinephrine, arachidonic acid, and A23187 resulted from PLP's inhibition of the effect of released ADP.

scholarly journals Effect of alteplase on platelet function and receptor expression

2019 ◽  
Vol 47 (4) ◽  
pp. 1731-1739 ◽  
Author(s):  
Jun Lu ◽  
Peng Hu ◽  
Guangyu Wei ◽  
Qi Luo ◽  
Jianlin Qiao ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Function ◽  
Adenosine Diphosphate ◽  
Receptor Expression ◽  
Light Transmittance ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Platelet Receptors

Objective To investigate the role of alteplase, a widely-used thrombolytic drug, in platelet function. Methods Human platelets were incubated with different concentrations of alteplase followed by analysis of platelet aggregation in response to adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid or epinephrine using light transmittance aggregometry. Platelet activation and surface levels of platelet receptors GPIbα, GPVI and αIIbβ3 were analysed using flow cytometry. The effect of alteplase on clot retraction was also examined. Results This study demonstrated that alteplase significantly inhibited platelet aggregation in response to ADP, collagen and epinephrine in a dose-dependent manner, but it did not affect ristocetin- or arachidonic acid-induced platelet aggregation. Alteplase did not affect platelet activation as demonstrated by no differences in P-selectin levels and PAC-1 binding being observed in collagen-stimulated platelets after alteplase treatment compared with vehicle. There were no changes in the surface levels of the platelet receptors GPIbα, GPVI and αIIbβ3 in alteplase-treated platelets. Alteplase treatment reduced thrombin-mediated clot retraction. Conclusions Alteplase inhibits platelet aggregation and clot retraction without affecting platelet activation and surface receptor levels.

Pioglitazone Inhibits Platelet Function and Potentiates the Effects of Aspirin

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1228-1228
Author(s):  
John P. Mongan ◽  
Hanna Mieszczanska ◽  
Richard P. Phipps ◽  
Charles W. Francis
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Blood Sample ◽  
Platelet Function ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Thromboxane B2 ◽  
Pparγ Agonists

Abstract Abstract 1228 BACKGROUND: Thiazolidinediones (TZDs) are agonists of PPARγ which favorably modify metabolic parameters and markers of atherosclerosis among type 2 diabetics. Enucleate platelets express PPARγ protein, and PPARγ agonists blunt release of CD40L and thromboxane B2 (TXB2) from thrombin-activated platelets. (Abbiyik, F et al, Human bone marrow megakaryocytes and platelets express PPARγ and PPARγ agonists blunt platelet release of CD40 ligand and thromboxanes. Blood, 2004 104(5):p.1361–8.) Diabetic subjects disproportionately experience arterial thromboses despite aspirin therapy. We assessed platelet function after pioglitazone in two risk groups in the presence and absence of aspirin to characterize its range of antiplatelet effect. SUBJECTS: 20 diabetic and 20 non-diabetic subjects were enrolled in a prospective study. Exclusion criteria among all subjects included current use of antiplatelet, anticoagulants, or pioglitazone, bleeding disorder, renal or liver disease, congestive heart failure, pregnancy or hypersensitivity to aspirin or pioglitazone. Non-diabetic subjects were excluded for BMI > 30 kg/m2, cardiovascular disease or risk factors. All subjects previously on aspirin underwent a 7 day minimum “wash-out” period. METHODS: Four separate blood samples from each subject were collected on 2 separate days separated by a 7 day interval. On day 1, a baseline blood sample was obtained followed by a second blood sample 3 hours after ingestion of 30 mg pioglitazone. Subjects returned 1 week later after having taken a single 81 mg aspirin 2–3 hours before arrival. Samples 3 and 4 were collected in the same manner as during week 1. Platelet rich plasma (PRP) was immediately prepared and platelet aggregation performed by the turbidometric method of Born with simultaneous measurement of ATP release. ADP (5M and 10M), arachidonic acid (0.5mM) and collagen (2g/mL) were used as agonists. PRP was activated with 0.8 unit/ mL thrombin for subsequent ELISA assays of TXB2 (Thromboxane B2), TGF-β (Transforming Growth Factor-Beta) and CD40L (CD 40 Ligand). RESULTS: By Diabetic Status: a.) Baseline platelet aggregations, ATP release and ELISAs were similar between diabetic and non-diabetic subjects, with the exception of platelet aggregation using 5 uM and 10uM as agonist. b.) Mean maximum platelet aggregation after aspirin alone was 20% higher among diabetic subjects. lp;&0.5qAmong all Subjects: a.) Mean TXB2 release among all subjects was reduced from a baseline of 42,075 ± 4,479 pg/ml to 32,719 ± 3,589pg/ml after pioglitazone alone (p = 0.0004). b.) Mean TXB2 release after aspirin alone was 20,829 ± 2,753 pg/ml which was reduced to 9,569 ± 1,653 pg/ml after the addition of pioglitazone (p = 0.0001). (Figure 1) c.) Twenty-five of 40 subjects (63%) had aggregation of greater than 20% using arachidonic acid as agonist despite ingestion of 81 mg aspirin. This decreased to 11 /40 (28%) after the administration of 30 mg of pioglitazone (p < 0.0001). (Figure 2) No significant effects were observed on release of CD40L or TGFβ. Conclusion: Pioglitazone has a direct platelet stabilizing effect and potentiates the effect of aspirin irrespective of underlying cardiovascular risk. Disclosures: Francis: Takeda Pharmaceuticals North America, Inc.: Research Funding.

Platelet ERp57 Is Required for Hemostasis and Thrombosis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2168-2168
Author(s):  
Lu Wang ◽  
Yi Wu ◽  
Junsong Zhou ◽  
Syed S. Ahmad ◽  
Bulent Mutus ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Protein Disulfide Isomerase ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Disulfide Isomerase ◽  
Protein Disulfide

Abstract Abstract 2168 Several members of the protein disulfide isomerase family of enzymes are important in platelet function and in thrombosis. Platelet protein disulfide isomerase (PDI) has been shown to have an important role in platelet function but is reported to not be required for thrombus formation in vivo. A novel platelet PDI called ERp57 mediates platelet aggregation but its role in thrombus formation is unknown. To determine the specific role of platelet-derived ERp57 in hemostasis and thrombosis we generated a megakaryocyte/platelet specific knockout. Despite normal platelet counts and platelet glycoprotein expression, mice with ERp57-deficient platelets had prolonged tail-bleeding times and thrombus occlusion times, and defective activation of the αIIbβ3 integrin and platelet aggregation. The aggregation defect was corrected by addition of exogenous ERp57 implicating surface ERp57 in platelet aggregation. Platelet surface ERp57 protein and activity increased substantially with platelet activation. We conclude that platelet-derived ERp57 is required for hemostasis and thrombosis and platelet function. Disclosures: No relevant conflicts of interest to declare.

Effects of Single and Multiple Moxibustions on Activity of Platelet Function, Blood Coagulation and Fibrinolysis in Mice

1990 ◽  
Vol 18 (01n02) ◽  
pp. 77-85 ◽  
Author(s):  
Masako Okazaki ◽  
Hideharu Sakamoto ◽  
Makoto Suzuki ◽  
Katsuji Oguchi
Keyword(s):  
Platelet Aggregation ◽  
Blood Coagulation ◽  
Host Defense ◽  
Platelet Function ◽  
Fibrinolytic Activity ◽  
Defense Mechanism ◽  
Atp Release ◽  
Host Defense Mechanism ◽  
Coagulation And Fibrinolysis ◽  
Platelet Functions

The effects of single and multiple moxibustions on platelet function, blood coagulation and fibrinolytic activity in ddY mice were studied. The increase in platelet aggregation and ATP-release after a single moxibustion was dependent on moxa weight and the kind of platelet stimulus. Blood coagulative activity tended to increase in the early phase after a single moxibustion. However, multiple moxibustions maintained the homeostasis on blood coagulation and fibrinolytic activiity. This investigation suggests that the effects of moxibustion on platelet functions and coagulative and fibrinolytic activities cause an enhancement of the phagocytic activity in the host defense mechanism.

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