scholarly journals Astilbe chinensis Modulates Platelet Function via Impaired MAPK and PLCγ2 Expression

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Bo-Ra Jeon ◽  
Muhammad Irfan ◽  
Seung Eun Lee ◽  
Jeong Hoon Lee ◽  
Man Hee Rhee
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Plant Extract ◽  
Signaling Molecules ◽  
Calcium Mobilization ◽  
Therapeutic Effects ◽  
Western Blots ◽  
Downstream Signaling ◽  
Fibrinogen Binding ◽  
Granule Secretion

Background. Platelets play major role in maintaining hemostasis while hyperactivation of platelets may lead to arterial thrombosis. Natural products and ethnomedicine have been shown to reduce the risk of cardiovascular diseases (CVDs). Astilbe chinensis is a perennial herb found in China, Korea, Russia, and Japan, which is also known for its medicinal effects, and has been used in Korean traditional medicine to treat inflammation, cancer, chronic bronchitis, and headache. We hypothesized that given herbal plant exhibits pharmacological activities against CVDs, and we specifically explored their effects on platelet function. Methodology. Platelet aggregation was evaluated using standard light-transmission aggregometry. Intracellular calcium mobilization was assessed using Fura-2/AM, and granule secretion (ATP release) was measured in a luminometer. Fibrinogen binding to integrin αIIbβ3 was assessed using flow cytometry. Phosphorylation of mitogen-activated protein kinase (MAPK) signaling molecules and activation of the phosphoinositide 3-kinase (PI3K)/Akt were assessed using western blots, and further, glycoprotein VI (GPVI) signaling components were studied using immunoprecipitation. Key Results. A. chinensis extracts potently and significantly inhibited platelet aggregation, calcium mobilization, granule secretion, and fibrinogen binding to integrin αIIbβ3. Moreover, it significantly inhibited MAPK phosphorylation and expression of GPVI downstream signaling molecules. Conclusion. A. chinensis extract inhibited platelet aggregation and granule secretion and attenuated GPVI downstream signaling, indicating the potential therapeutic effects of this plant extract on the cardiovascular system and platelet function. We suggest that given plant extract may be a potent candidate to treat platelet-related CVDs and to be used as antiplatelet agent.

scholarly journals Impaired function of platelet membrane glycoprotein IIb-IIIa in end-stage renal disease.

1994 ◽  
Vol 5 (1) ◽  
pp. 36-46
Author(s):  
M P Gawaz ◽  
G Dobos ◽  
M Späth ◽  
P Schollmeyer ◽  
H J Gurland ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Platelet Aggregation ◽  
Platelet Function ◽  
Uremic Toxin ◽  
Bleeding Tendency ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Uremic Patients ◽  
Stage Renal Disease

Impaired platelet function and a bleeding tendency are well-recognized complications of chronic renal failure. Because the fibrinogen receptor GPIIb-IIIa plays a central role in platelet aggregation and adhesion to the subendothelium, it was reasoned that a defect in this receptor may underlie the impaired platelet function in uremia. To test this hypothesis, the function of this receptor in the platelets of 11 uremic patients was studied. Aggregation studies were performed with flow cytometric techniques with anti-GPIIb-IIIa conformation-specific monoclonal antibodies (mAb) (anti-LIBS1 and anti-PMI-1). Antifibrinogen and antithrombospondin mAb were used to characterize fibrinogen binding to GPIIb-IIIa and the release of alpha-granules, respectively. Platelets from patients with chronic renal failure showed significantly decreased binding of conformation-dependent anti-LIBS1 mAb after ADP, phorbol myristate acetate, or RGD-peptide stimulation compared with normal controls, suggesting a defect related to the ability of the fibrinogen receptor to undergo a conformational change. Moreover, antifibrinogen and antithrombospondin binding to activated platelets were reduced in uremic patients, implying impairment of both ligand-binding and alpha-granule release. Hemodialysis partially restored GPIIb-IIIa function, which may account for the observed effects of this therapy in restoring platelet aggregation. These findings indicate that platelets of patients with chronic renal failure reveal an aggregation defect at least partially due to an intrinsic GPIIb-IIIa dysfunction and the presence of a putative uremic toxin that inhibits fibrinogen binding to GPIIb-IIIa.

Negative Regulation of Gq-Mediated Pathways in Platelets by G12/13 Pathways through Fyn Kinase

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2854-2854
Author(s):  
Soochong Kim ◽  
Satya P. Kunapuli
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Calcium ◽  
Signaling Pathways ◽  
Platelet Function ◽  
Shape Change ◽  
Wild Type Mouse ◽  
Calcium Mobilization ◽  
Negative Regulator ◽  
Platelet Response ◽  
Calcium Response

Abstract Platelets contain high levels of Src family kinases (SFKs) suggesting an important role for these enzymes in platelet function. In this study, we have investigated the regulation of platelet function by SFKs downstream of G12/13 pathways. Calcium-independent platelet shape change induced by selective G12/13 stimulation with YFLLRNP was potentiated with a small mobilization of intracellular calcium in the presence of SFK inhibitors PP1 or PP2, which was abolished by the chelation of intracellular calcium, suggesting that SFKs downstream of G12/13 negatively regulate calcium mobilization in platelets. In addition, PP1 or PP2 caused a leftward shift of human platelet aggregation, secretion, and calcium response induced by low concentrations of agonists that activate platelets through G12/13 signaling such as PAR1-activating peptide SFLLRN and PAR4-activating peptide AYPGKF. However, 2-MeSADP-induced calcium response and platelet aggregation were not affected by the presence of PP1 or PP2, suggesting that SFKs downstream of G12/13, but not Gq/Gi, pathways are involved in this platelet response. Moreover, platelet aggregation and secretion caused by combined stimulation of G12/13 and Gi/Gz (YFLLRNP + 2-MeSADP with P2Y1 antagonist/epinephrine) were also potentiated in the presence of PP1 or PP2 confirming the contribution of SFKs downstream of G12/13 as a negative regulator to platelet activation. Potentiation of platelet aggregation in the presence of SFK inhibitors was not affected in the presence of GF109203X, while PP2 failed to potentiate platelet aggregation in the presence of 5,5′-dimethyl-BAPTA indicating that potentiation of cytosolic calcium may have an important role in this enhanced platelet responses by SFK inhibition. Moreover, PP1 or PP2 failed to potentiate platelet responses in the presence of Gq selective inhibitor YM-254890 or in Gq-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of Gq signaling pathways. Importantly, AYPGKF-induced platelet aggregation, secretion, and calcium response were potentiated in Fyn-, but not in Lyn-, deficient platelets compared to the wild-type mouse platelets, whereas 2-MeSADP-induced platelet response was not affected in these platelets. We conclude that SFKs activated downstream of G12/13, but not Gq/Gi, pathways negatively regulate platelet responses by inhibiting intracellular calcium mobilization in platelets through Gq signaling pathways. Specifically, we define that Fyn plays a major role in this negatively regulatory pathway.

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.

Absent Platelet Aggregation with Normal Fibrinogen Binding in Basset Hound Hereditary Thrombopathy

1989 ◽  
Vol 62 (03) ◽  
pp. 1011-1015 ◽  
Author(s):  
Wayne R Patterson ◽  
Douglas W Estry ◽  
Kenneth A Schwartz ◽  
Ronald D Borchert ◽  
Thomas G Bell
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Platelet Membrane ◽  
Membrane Glycoproteins ◽  
Glycoprotein Complex ◽  
Normal Platelet ◽  
Fibrinogen Binding ◽  
Rule Out

SummaryPlatelets from dogs with Basset Hound Hereditary Thrombopathy (BHT) initially displayed a thrombasthenia-like aggregation defect but have been shown to have normal amounts of platelet membrane glycoproteins lib and Ilia (GPIIb-IIIa), and therefore are more accurately described as thrombopathic. The presence of normal quantities of GPIIb-IIIa, however, did not rule out the possibility of a functionally abnormal glycoprotein complex which would be unable to bind radio-labeled fibrinogen. Therefore, fibrinogen binding in BHT platelets was evaluated. Platelets from BHT and normal dogs were activated with 1 × 10−5 M ADP in the presence of 125I-fibrinogen and the surface-bound radioactivity was quantitated. The amount of fibrinogen bound by BHT dog platelets was not significantly different than that bound by normal dog platelets. Platelets from dogs with BHT bound 30,282 ± 3,133 and normal dog platelets bound 31,664 ± 2,772 molecules of fibrinogen per platelet. The quantitatively normal GPIIb-IIIa complex binds fibrinogen in normal amounts and does not seem to represent the abnormality responsible for the aggregation defect in BHT platelets. Therefore, other factors central to normal platelet function and related to platelet aggregation must be considered.

Fibrinogen Binding Is Independent of an Increase in Intracellular Calcium Concentration in Thrombin Degranulated Platelets

1995 ◽  
Vol 73 (02) ◽  
pp. 304-308 ◽  
Author(s):  
Fabio M Pulcinelli ◽  
James L Daniel ◽  
Silvia Riondino ◽  
Pier Paolo Gazzaniga ◽  
Leon Salganicoff
Keyword(s):  
Platelet Aggregation ◽  
Intracellular Calcium ◽  
Binding Sites ◽  
Calcium Concentration ◽  
Calcium Release ◽  
Shape Change ◽  
Cytosolic Calcium ◽  
Calcium Mobilization ◽  
Induce Platelet Aggregation ◽  
Fibrinogen Binding

SummaryIn a suspension of thrombin degranulated platelets (TDP), ADP and epinephrine can induce platelet aggregation, whereas the synthetic agonist of the thromboxane/endoperoxide receptor U46619 causes only shape change. However, U46619 can enhance platelet aggregation induced by ADP and epinephrine. In this paper, we have measured fibrinogen binding in relation to phospholipase C(PLC) activation and calcium mobilization in TDP activated by ADP, epinephrine and U46619.ADP caused fibrinogen binding in TDP but neither activated PLC nor caused a calcium mobilization. The requirement for ADP in inducing exposure of fibrinogen binding sites was not absolute since the combination of epinephrine and U46619 produced an increase in fibrinogen binding. U46619 caused significant PLC activation and cytosolic calcium release but not fibrinogen binding. These results suggest that in TDP the exposure of fibrinogen binding sites, after agonist activation, is independent of both PLC activation and calcium mobilization.

scholarly journals Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins.

1990 ◽  
Vol 111 (6) ◽  
pp. 3117-3127 ◽  
Author(s):  
A Golden ◽  
J S Brugge ◽  
S J Shattil
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Dense Granule ◽  
Platelet Response ◽  
Induce Platelet Aggregation ◽  
Fibrinogen Binding ◽  
Molecular Masses ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Platelet Proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.

scholarly journals From Discovery of Snake Venom Disintegrins to A Safer Therapeutic Antithrombotic Agent

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 372 ◽  
Author(s):  
Yu-Ju Kuo ◽  
Ching-Hu Chung ◽  
Tur-Fu Huang
Keyword(s):  
Platelet Aggregation ◽  
Tumor Cells ◽  
Platelet Function ◽  
Platelet Aggregation Inhibitors ◽  
Platelet Glycoprotein ◽  
Antiangiogenic Agents ◽  
Antithrombotic Agent ◽  
Induce Platelet Aggregation ◽  
Migration Effect ◽  
Fibrinogen Binding

Snake venoms affect blood coagulation and platelet function in diverse ways. Some venom components inhibit platelet function, while other components induce platelet aggregation. Among the platelet aggregation inhibitors, disintegrins have been recognized as unique and potentially valuable tools for examining cell–matrix and cell–cell interactions and for the development of antithrombotic and antiangiogenic agents according to their anti-adhesive and anti-migration effect on tumor cells and antiangiogenesis activities. Disintegrins represent a family of low molecular weight, cysteine-rich, Arg-Gly-Asp(RGD)/Lys-Gly-Asp(KGD)-containing polypeptides, which inhibit fibrinogen binding to integrin αIIbβ3 (i.e., platelet glycoprotein IIb/IIIa), as well as ligand binding to integrins αvβ3, and α5β1 expressed on cells (i.e., fibroblasts, tumor cells, and endothelial cells). This review focuses on the current efforts attained from studies using disintegrins as a tool in the field of arterial thrombosis, angiogenesis, inflammation, and tumor metastasis, and briefly describes their potential therapeutic applications and side effects in integrin-related diseases. Additionally, novel R(K)GD-containing disintegrin TMV-7 mutants are being designed as safer antithrombotics without causing thrombocytopenia and bleeding.

scholarly journals Adhesive ligand binding to integrin alpha IIb beta 3 stimulates tyrosine phosphorylation of novel protein substrates before phosphorylation of pp125FAK

1993 ◽  
Vol 122 (2) ◽  
pp. 473-483 ◽  
Author(s):  
MM Huang ◽  
L Lipfert ◽  
M Cunningham ◽  
JS Brugge ◽  
MH Ginsberg ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Actin Polymerization ◽  
Protein Tyrosine Kinase ◽  
Free Calcium ◽  
Dependent Manner ◽  
Integrin Receptors ◽  
Fibrinogen Binding ◽  
Granule Secretion

Tyrosine phosphorylation of multiple platelet proteins is stimulated by thrombin and other agonists that cause platelet aggregation and secretion. The phosphorylation of a subset of these proteins, including a protein tyrosine kinase, pp125FAK, is dependent on the platelet aggregation that follows fibrinogen binding to integrin alpha IIb beta 3. In this report, we examined whether fibrinogen binding, per se, triggers a process of tyrosine phosphorylation in the absence of exogenous agonists. Binding of soluble fibrinogen was induced with Fab fragments of an anti-beta 3 antibody (anti-LIBS6) that directly exposes the fibrinogen binding site in alpha IIb beta3. Proteins of 50-68 KD and 140 kD became phosphorylated on tyrosine residues in a fibrinogen-dependent manner. This response did not require prostaglandin synthesis, an increase in cytosolic free calcium, platelet aggregation or granule secretion, nor was it associated with tyrosine phosphorylation of pp125FAK. Tyrosine phosphorylation of the 50-68-kD and 140-kD proteins was also observed when (a) fibrinogen binding was stimulated by agonists such as epinephrine, ADP, or thrombin instead of by anti-LIBS6; (b) fragment X, a dimeric plasmin-derived fragment of fibrinogen was used instead of fibrinogen; or (c) alpha IIb beta 3 complexes were cross-linked by antibodies, even in the absence of fibrinogen. In contrast, no tyrosine phosphorylation was observed when the ligand consisted of monomeric cell recognition peptides derived from fibrinogen (RGDS or gamma 400-411). Fibrinogen-dependent tyrosine phosphorylation was inhibited by cytochalasin D. These studies demonstrate that fibrinogen binding to alpha IIb beta 3 initiates a process of tyrosine phosphorylation that precedes platelet aggregation and the phosphorylation of pp125FAK. This reaction may depend on the oligomerization of integrin receptors and on the state of actin polymerization, organizational processes that may juxtapose tyrosine kinases with their substrates.

scholarly journals Artocarpesin Prevents Collagen Induced Platelet Aggregation and Clot Retraction Through Cyclic Nucleotides and Dephosphorylation of MAPKs  

2021 ◽  
Author(s):  
Jung-Hae Shin ◽  
Muhammad Irfan ◽  
Yuan Yee Lee ◽  
Man Hee Rhee ◽  
Hyuk-Woo Kwon
Keyword(s):  
Platelet Aggregation ◽  
Cardiovascular Diseases ◽  
Thrombus Formation ◽  
Signaling Molecules ◽  
Serotonin Release ◽  
Calcium Mobilization ◽  
Clot Retraction ◽  
Natural Substances ◽  
Mitogen Activated Protein

Abstract Background The cardiovascular diseases (CVDs) are becoming a critical threat to our lives in these years. It is now widely accepted that platelets play an important role in cardiovascular disease as they have a fundamental role in thrombosis. Therefore, many drugs or natural substances have been developed to treat CVDs. Cudrania tricuspidata (C. tricuspidata) is a regional plant containing various flavonoids and xanthones, and various physiological activities have been reported. Therefore, we evaluated antiplatelet effects using artocarpesin isolated from C. tricuspidata. Methods The in vitro effects of artocarpesin on platelets was assessed using measurement of calcium mobilization and serotonin release, glycoprotein IIb/IIIa activation, clot retraction and phosphorylation of signaling molecules. Results Artocarpesin inhibited human platelet aggregation, calcium mobilization, glycoprotein IIb/IIIa activation and thrombin-induced clot retraction through the regulation of associated signaling molecules such as vasodilator-stimulated phosphoprotein (VASP) and inositol 1, 4, 5-triphosphate receptor I (IP3RI), and on the dephosphorylation of cytosolic phospholipase A2 (cPLA2), mitogen-activated protein kinases p38, JNK and phosphoinositide 3-kinase (PI3K)/Akt. Conclusions This study highlights that artocarpesin has inhibitory effects on platelet activities and thrombus formation and has potential value for preventing platelet-induced cardiovascular diseases.

Difference of [Ca2+]i Movements in Platelets Stimulated by Thrombin and TRAP: the Involvement of αIIbβ3-Mediated TXA2 Synthesis

1998 ◽  
Vol 79 (06) ◽  
pp. 1184-1190 ◽  
Author(s):  
Yoshiaki Tomiyama ◽  
Shigenori Honda ◽  
Kayoko Senzaki ◽  
Akito Tanaka ◽  
Mitsuru Okubo ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Fibrinogen Binding ◽  
Adp Release ◽  
The Difference ◽  
Txa2 Receptor Antagonist ◽  
High Level ◽  
Inhibition Studies ◽  
Peak Increase ◽  
Second Peak ◽  
Assay Conditions

SummaryThis study investigated the difference of [Ca2+]i movement in platelets in response to thrombin and TRAP. The involvement of αIIbβ3 in this signaling was also studied. Stimulation of platelets with thrombin at 0.03 U/ml caused platelet aggregation and a two-peak increase in [Ca2+]i. The second peak of [Ca2+]i, but not the first peak was abolished by the inhibition of platelet aggregation with αIIbβ3 antagonists or by scavenging endogenous ADP with apyrase. A cyclooxygenase inhibitor, aspirin, and a TXA2 receptor antagonist, BM13505, also abolished the second peak of [Ca2+]i but not the first peak, although these regents did not inhibit aggregation. Under the same assay conditions, measurement of TXB2 demonstrated that αIIbβ3 antagonists and aspirin almost completely inhibited the production of TXB2. In contrast to thrombin-stimulation, TRAP caused only a single peak of [Ca2+]i even in the presence of platelet aggregation, and a high level of [Ca2+]i increase was needed for the induction of platelet aggregation. The inhibition of aggregation with αIIbβ3 antagonists had no effect on [Ca2+]i change and TXB2 production induced by TRAP. Inhibition studies using anti-GPIb antibodies suggested that GPIb may be involved in the thrombin response, but not in the TRAP. Our findings suggest that low dose thrombin causes a different [Ca2+]i response and TXA2 producing signal from TRAP. Endogenous ADP release and fibrinogen binding to αIIbβ3 are responsible for the synthesis of TXA2 which results in the induction of the second peak of [Ca2+]i in low thrombin- but not TRAP-stimulated platelets.

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