Inhibition of Rat Platelet Aggregation by Mycalolide-B, a Novel Inhibitor of Actin Polymerization with a Different Mechanism of Action from Cytochalasin-D

1998 ◽  
Vol 79 (03) ◽  
pp. 614-619 ◽  
Author(s):  
Fumitoshi Asai ◽  
Shinya Saito ◽  
Hiroshi Ozaki ◽  
Nobuhiro Fusetani ◽  
Hideaki Karaki ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Mechanism Of Action ◽  
Actin Polymerization ◽  
De Novo ◽  
Platelet Rich Plasma ◽  
Cytochalasin D ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Concentration Dependent Manner ◽  
Induced Aggregation

SummaryIn vitro effects of mycalolide-B (MB), isolated from marine sponge, were investigated with regard to the activation of rat platelets. Collagen-induced platelet aggregation in platelet-rich plasma (PRP) was slightly but significantly potentiated by lower concentrations of MB (0.3 and 1 μM) but was inhibited by higher concentrations (3 and 10 μM). ADP-induced platelet aggregation in PRP was also significantly prevented by MB (1-10 μM). Potentiation of ADP-induced aggregation by MB (0.3 μM) was hardly observed. G-actin contents, determined by DNase I inhibition assay, were increased in resting washed platelets incubated with MB (3 μM). In contrast, cytochalasin-D (CD) at 3 μM slightly reduced G-actin contents in resting platelets. After platelet aggregation with collagen (3 μg/ml) or ADP (10 μM), G-actin contents in platelets were reduced, indicating de novo actin polymerization. MB (3 μM) and CD (3 μM) abolished both ADP (10 μM)- and collagen (3 μg/ml)-induced platelet aggregation and actin polymerization in washed platelets. MB (1-10 μM) had no effects on intracellular Ca2+ concentrations in ADP (10 μM)-stimulated platelets. [125I]-fibrinogen binding to activated platelets with ADP (10 μM) was inhibited by MB (0.3-3 μM) in a concentration-dependent manner. Thrombin-induced platelet-fibrin clot retraction was inhibited by MB (1 and 10 μM). These results suggest that MB inhibits platelet activation by interfering with actin polymerization through a different mechanism of action from CD. MB may be a useful tool for studying the role of actin polymerization in various cells.

FUNDC2 regulates platelet activation through AKT/GSK-3β/cGMP axis

2018 ◽  
Vol 115 (11) ◽  
pp. 1672-1679 ◽  
Author(s):  
Qi Ma ◽  
Weilin Zhang ◽  
Chongzhuo Zhu ◽  
Junling Liu ◽  
Quan Chen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Mitochondrial Protein ◽  
Dependent Manner ◽  
Clot Retraction ◽  
Akt Phosphorylation ◽  
Gsk 3Β

Abstract Aims AKT kinase is vital for regulating signal transduction in platelet aggregation. We previously found that mitochondrial protein FUNDC2 mediates phosphoinositide 3-kinase (PI3K)/phosphatidylinositol-3,4,5-trisphosphate (PIP3)-dependent AKT phosphorylation and regulates platelet apoptosis. The aim of this study was to evaluate the role of FUNDC2 in platelet activation and aggregation. Methods and results We demonstrated that FUNDC2 deficiency diminished platelet aggregation in response to a variety of agonists, including adenosine 5′-diphosphate (ADP), collagen, ristocetin/VWF, and thrombin. Consistently, in vivo assays of tail bleeding and thrombus formation showed that FUNDC2-knockout mice displayed deficiency in haemostasis and thrombosis. Mechanistically, FUNDC2 deficiency impairs the phosphorylation of AKT and downstream GSK-3β in a PI3K-dependent manner. Moreover, cGMP also plays an important role in FUNDC2/AKT-mediated platelet activation. This FUNDC2/AKT/GSK-3β/cGMP axis also regulates clot retraction of platelet-rich plasma. Conclusion FUNDC2 positively regulates platelet functions via AKT/GSK-3β/cGMP signalling pathways, which provides new insight for platelet-related diseases.

scholarly journals Uridine Triphosphate Thio-analogues Inhibit Platelet P2Y12 Receptors and Aggregation

2016 ◽  
Author(s):  
Dursun Guenduez ◽  
Christian Tanislav ◽  
Daniel Sedding ◽  
Mariana Parahuleva ◽  
Sentot Santoso ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Inhibitory Activity ◽  
Platelet Rich Plasma ◽  
P2y12 Receptor ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Uridine Triphosphate ◽  
Ic50 Value ◽  
Adp Receptor ◽  
First Time

Platelet P2Y12 is an important ADP receptor that is involved in agonist-induced platelet aggregation and is a valuable target for the development of anti-platelet drugs. Here we characterise the effects of thio-analogues of uridine triphosphate (UTP) on ADP-induced platelet aggregation. Using human platelet-rich plasma we demonstrate that UTP inhibits P2Y12 but not P2Y1 receptors and antagonises 10 μM ADP-induced platelet aggregation in a concentration-dependent manner with an IC50 value of ~250 μM. An 8-fold higher platelet inhibitory activity was observed with a 2-thio analogue of UTP (2S-UTP), with an IC50 of 30 μM. The 4-thio analogue (4S-UTP) with an IC50 of 7.5 μM was 33-fold more effective. A 3-fold decrease in inhibitory activity, however, was observed by introducing an isobutyl group at the 4S- position. A complete loss of inhibition was observed with thio-modification of the γ phosphate of the sugar moiety, which yields an enzymatically stable analogue. The interaction of UTP analogues with P2Y12 receptors was verified by P2Y12 receptor binding and cAMP assays. These novel data demonstrate for the first time that 2- and 4-thio analogues of UTP are potent P2Y12 receptor antagonists that may be useful for therapeutic intervention.

scholarly journals Resolvin E1, an EPA-derived mediator in whole blood, selectively counterregulates leukocytes and platelets

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 848-855 ◽  
Author(s):  
Maria Dona ◽  
Gabrielle Fredman ◽  
Jan M. Schwab ◽  
Nan Chiang ◽  
Makoto Arita ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Lipid Mediator ◽  
Dependent Manner ◽  
Omega 3 ◽  
Concentration Dependent Manner ◽  
Chemokine Production ◽  
Thromboxane Receptor

Abstract Resolvin E1 (RvE1) is an omega-3 eicosapentaenoic acid (EPA)–derived lipid mediator generated during resolution of inflammation and in human vasculature via leukocyte-endothelial cell interactions. RvE1 possesses anti-inflammatory and proresolving actions. Here, we report that RvE1 in human whole blood rapidly regulates leukocyte expression of adhesion molecules. RvE1 in the 10- to 100-nM range stimulated L-selectin shedding, while reducing CD18 expression in both neutrophils and monocytes. When added to whole blood, RvE1 did not stimulate reactive oxygen species by either neutrophils or monocytes, nor did it directly stimulate cytokine/chemokine production in heparinized blood. Intravital microscopy (IVM) demonstrated that RvE1 rapidly reduced leukocyte rolling (∼ 40%) in venules of mice. In human platelet-rich plasma (PRP), RvE1 selectively blocked both ADP-stimulated and thromboxane receptor agonist U46619-stimulated platelet aggregation in a concentration-dependent manner. In contrast, Δ6,14-trans-RvE1 isomer was inactive. RvE1 did not block collagen-stimulated aggregation, and regulation of ADP-induced platelet aggregation was not further enhanced with aspirin treatment. These results indicate RvE1 is a potent modulator of leukocytes as well as selective platelet responses in blood and PRP, respectively. Moreover, the results demonstrate novel agonist-specific antiplatelet actions of RvE1 that are potent and may underlie some of the beneficial actions of EPA in humans.

Specific Pharmacologic Targeting of Rho GTPases Rac1, Cdc42 and RhoA Reveals Their Differential and Critical Roles In Regulation of Platelet Activation

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2019-2019 ◽  
Author(s):  
Huzoor Akbar ◽  
Xun Shang ◽  
Rehana Perveen ◽  
Kevin Funk ◽  
Mark Berryman ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Rho Gtpase ◽  
Conflicts Of Interest ◽  
Small Gtpases ◽  
Signaling Cascades ◽  
Dependent Manner ◽  
Exchange Reactions ◽  
Concentration Dependent Manner ◽  
Induced Aggregation

Abstract Abstract 2019 Rac1, Cdc42 and RhoA, members of the Rho family of small GTPases, play critical roles in reorganization of actin cytoskeleton and aggregation in platelets. Although they have been implicated in regulation of platelet activation, the unique and redundant roles of each of the Rho GTPase in various signaling cascades and the resulting functional outcomes have yet to be clearly defined. In this study we compared their roles in several aspects of platelet activation by utilizing three small molecule inhibitors, NSC23766, CASIN, and GO4, that specifically suppress endogenous Rac1, Cdc42, and RhoA activities, respectively. These novel pharmacological inhibitors are active in direct binding to their specific GTPase substrates, i.e. NSC23766 to Rac1, CASIN to Cdc42, and G04 to RhoA, and in interfering with the GTP loading exchange reactions of each Rho GTPase catalyzed by respective guanine nucleotide exchange factors at 5–50 uM concentration range. First, effector-domain pull down assays confirmed that treatment of platelets with NSC23766 (30 uM), CASIN (10 uM) or GO4 (30-50 uM) specifically blocked collagen induced Rac1-GTP, Cdc42-GTP, and RhoA-GTP formations, respectively. Incubation of platelets with NSC23766 (30 uM) or CASIN (10 uM) effectively inhibited collagen-induced phosphorylation of the Rac/Cdc42 effector, PAK1. Addition of GO4 (30 uM) to platelets prior to stimulation with thrombin blocked RhoA/ROCK mediated phosphorylation of myosin light chain (MLC). Second, incubation of aspirin treated platelets containing apyrase (3 U/ml) with CASIN (10 uM), but not NSC23766 (30 uM) or GO4 (30 uM), inhibited filopodia formation on immobilized fibrinogen or collagen-related peptide (CRP), a GPVI agonist. On the other hand, treatment of platelets with CASIN (10 uM) or GO4 (30 uM), but not with NSC23766 (30 uM), inhibited spreading of platelets on immobilized fibrinogen in the presence of aspirin and apyrase. Third, NSC23766 (3-30 uM), CASIN (3-10 uM), and GO4 (5-50 uM) all inhibited secretion from platelet granules and secretion-dependent aggregation induced by threshold concentration of ADP, collagen, CRP, or thrombin in a concentration-dependent manner. However, while CASIN (10 uM) or GO4 (30 uM) completely blocked collagen or CRP induced aggregation in aspirin treated platelets containing apyrase, NSC23766 (30 uM) showed no effect. Fourth, while pre-incubation of platelets with 5 uM CASIN or 10 uM G04 alone only partially (15%) inhibited CRP induced platelet aggregation in aspirin treated samples, CASIN at 10 uM or a combination of 5 uM CASIN and 5 uM G04 were able to inhibit platelet aggregation by 90%. Fifth, GO4 (30 uM) but not CASIN (10 uM) inhibited thrombin stimulated phosphorylation of p38-MAPK (137%) in aspirin treated platelets in the presence of apyrase. Addition of GO4 (30 uM) or CASIN (10 uM) to aspirin treated platelets containing apyrase inhibited CRP induced phosphorylation of ERK1/2 by 94% and 53% respectively, However, in the absence of aspirin and apyrase GO4 (30 uM), but not CASIN (10 uM), completely inhibited CRP induced phosphorylation of ERK1/2. Finally, although both GO4 (30 uM) and CASIN (10 uM) completely inhibited CRP induced phosphorylation of MLC in aspirin treated platelets containing apyrase, GO4 (30 uM) maximally (94%) while CASIN (10 uM) partially (36%) inhibited phosphorylation of MLC in the absence of aspirin and apyrase. Taken together, these data suggest that: (a) Cdc42 is involved in integrin alphaIIbbeta3 and GPVI mediated filopodia formation, RhoA is involved in regulation of integrin alphaIIbbeta3 induced platelet spreading, whereas Rac1 is critical in secondary mediators (ADP/TXA2) mediated lamellipodia formation; (b) Cdc42 and RhoA regulate platelet aggregation in parallel pathways, possibly by affecting the RhoA/ROCK-MAPK-dependent and -independent phosphorylation of MLC; and (c) the crosstalk among Cdc42, Rac1 and RhoA plays an important role in signaling cascades involved in platelet activation. Disclosures: No relevant conflicts of interest to declare.

In vitro Effects of Aprosulate sodium, a Novel Anticoagulant, On Platelet Activation: Possible Mechanism for Antiplatelet Action

1996 ◽  
Vol 76 (05) ◽  
pp. 786-790 ◽  
Author(s):  
Atsuhiro Sugidachi ◽  
Norbert Breiter ◽  
Taketoshi Ogawa ◽  
Fumitoshi Asai ◽  
Hiroyuki Koike
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Anticoagulant Activity ◽  
Acid Amide ◽  
Human Platelets ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free System ◽  
Induced Aggregation

SummaryAprosulate sodium, a bis-lactobionic acid amide derivative, is a novel synthetic polyanion with potent anticoagulant activities. In the present study, the effects of aprosulate on platelet aggregation were investigated in a plasma-free system. Aprosulate inhibited thrombin (0.03-0.3 U/ml)-induced aggregation in rat washed platelets in a concentration-dependent manner, with an IC50 value of 0.38 Μg/ml. In contrast, aprosulate, at up to 10 Μg/ml, did not affect collagen (1 Μg/ml) - or ADP (3 ΜM)-induced aggregation. In fura 2-loaded platelets, aprosulate (1-10 Μg/ml) inhibited intracellular Ca2+ mobilization induced by thrombin, but not that by ADP. Protamine, a highly basic protein, abolished aprosulate-mediated inhibition of thrombin-induced platelet aggregation, suggesting that the observed inhibition is primarily due to the negative charge contained on the aprosulate molecule. In human platelets, aprosulate inhibited thrombin-induced aggregation, but failed to inhibit platelet aggregation induced by SFLLRN, a synthetic tethered ligand of a thrombin receptor. Antiplatelet profiles of aprosulate were largely similar to those of heparin, although heparin inhibited both thrombin- and collagen-induced aggregation. These in vitro studies indicate that aprosulate is capable of inhibiting thrombin-induced platelet activation and that this effect is independent of its anticoagulant activity. These results suggest that the polyanionic feature of aprosulate plays an essential role in promoting its antiplatelet activities, and that a plausible mechanism to explain the observed inhibition conferred by this agent, would be one which involves blocking the platelet-thrombin interaction.

scholarly journals Chlorin e6 Prevents ADP-Induced Platelet Aggregation by Decreasing PI3K-Akt Phosphorylation and Promoting cAMP Production

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Ji Young Park ◽  
Hyun Dong Ji ◽  
Bo Ra Jeon ◽  
Eun Ju Im ◽  
Young Min Son ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Underlying Mechanism ◽  
Serotonin Release ◽  
Intracellular Camp ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Akt Phosphorylation ◽  
Mitogen Activated Protein ◽  
Induced Aggregation ◽  
Camp Levels

A number of reagents that prevent thrombosis have been developed but were found to have serious side effects. Therefore, we sought to identify complementary and alternative medicinal materials that are safe and have long-term efficacy. In the present studies, we have assessed the ability of chlorine e6 (CE6) to inhibit ADP-induced aggregation of rat platelets and elucidated the underlying mechanism. CE6 inhibited platelet aggregation induced by 10 µM ADP in a concentration-dependent manner and decreased intracellular calcium mobilization and granule secretion (i.e., ATP and serotonin release). Western blotting revealed that CE6 strongly inhibited the phosphorylations of PI3K, Akt, c-Jun N-terminal kinase (JNK), and different mitogen-activated protein kinases (MAPKs) including extracellular signal-regulated kinase 1/2 (ERK1/2) as well as p38-MAPK. Our study also demonstrated that CE6 significantly elevated intracellular cAMP levels and decreased thromboxane A2formation in a concentration-dependent manner. Furthermore, we determined that CE6 initiated the activation of PKA, an effector of cAMP. Taken together, our findings indicate that CE6 may inhibit ADP-induced platelet activation by elevating cAMP levels and suppressing PI3K/Akt activity. Finally, these results suggest that CE6 could be developed as therapeutic agent that helps prevent thrombosis and ischemia.

scholarly journals Daphnoretin, a new protein kinase C activator isolated from Wikstroemia indica C.A. Mey

1993 ◽  
Vol 295 (1) ◽  
pp. 321-327 ◽  
Author(s):  
F N Ko ◽  
Y L Chang ◽  
Y H Kuo ◽  
Y L Lin ◽  
C M Teng
Keyword(s):  
Protein Kinase C ◽  
Platelet Aggregation ◽  
Protein Kinase ◽  
Atp Release ◽  
Biologically Active ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Rabbit Platelets ◽  
Induced Aggregation

Daphnoretin, a biologically active principle isolated from Wikstroemia indica C.A. Mey., caused platelet aggregation in washed rabbit platelets, platelet-rich plasma and whole blood. The aggregation of and ATP release from platelets induced by daphnoretin were similar to phorbol ester- and diacylglycerol-induced aggregation and release. The EC50 values of daphnoretin-, phorbol 12,13-dibutyrate (PDBu)- and 1-oleoyl-2-acetylglycerol (OAG)-induced platelet aggregation in washed rabbit platelets were 17.2 +/- 2.8 microM, 20.6 +/- 2.1 nM and 38.6 +/- 1.7 microM respectively. Platelet aggregation induced by daphnoretin and PDBu was not inhibited by indomethacin, BN52021 or sodium nitroprusside. ADP-scavenging systems, apyrase and phosphocreatine/creatine kinase, showed weak inhibition of the aggregation, and EGTA, triflavin, verapamil and prostaglandin E1 markedly inhibited the aggregation. Staurosporine, a potent protein kinase C inhibitor, suppressed daphnoretin-, PDBu- and OAG-induced aggregation and ATP release in a concentration-dependent manner. The IC50 values of staurosporine on daphnoretin (50 microM)-, PDBu (100 nM)- and OAG (50 microM)-induced aggregation were 37.7 +/- 8.3, 52.2 +/- 6.3 and 42.8 +/- 8.9 nM respectively. Daphnoretin did not cause significant thromboxane B2 formation in rabbit platelets. Neither daphnoretin nor PDBu caused [3H]inositol monophosphate formation or an increase in intracellular Ca2+ concentration in myo-[3H]inositol-labelled and Fura-2-loaded platelets. Platelet cytosolic protein kinase C was activated by daphnoretin and PDBu in a concentration-dependent manner with an EC50 of 12.4 +/- 1.2 microM and 18.7 +/- 1.4 nM respectively. Membrane-associated protein kinase C activity was increased by either daphnoretin or PDBu. [3H]PDBu binding to washed rabbit platelets was inhibited by daphnoretin in a concentration-dependent manner with an IC50 value of 45.2 +/- 5.2 microM. These results indicate that daphnoretin is a protein kinase C activator in rabbit platelets.

The Relationship between the Production of Thromboxane B2 and Malondialdehyde by Human Blood Platelets

1980 ◽  
Vol 59 (2) ◽  
pp. 131-135 ◽  
Author(s):  
L. C. Best ◽  
P. B. B. Jones ◽  
R. G. G. Russell
Keyword(s):  
Platelet Rich Plasma ◽  
Blood Platelets ◽  
Thromboxane B2 ◽  
Ionophore A23187 ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Thromboxane Synthetase ◽  
Malondialdehyde Formation ◽  
Induced Aggregation ◽  
The Relationship

1. The formation of thromboxane B2 and malondialdehyde was studied in human platelet-rich plasma, in gel-filtered platelets and in bovine platelet microsomes. 2. Exogenous sodium arachidonate was converted into thromboxane B2 and malondialdehyde in a concentration-dependent manner. Pre-incubation of platelets with aspirin inhibited the production of both thromboxane B2 and malondialdehyde, although malondialdehyde could apparently be detected in the absence of thromboxane B2. 3. The aggregating agents, thrombin, collagen and the ionophore A23187 also caused production of thromboxane B2 and malondialdehyde. ADP and adrenaline produced a smaller rise whilst the endoperoxide analogue U 46619 had only a slight influence on thromboxane and malondialdehyde, even though they all induced aggregation. 4. Pre-incubation of platelets with imidazole or 1-N-butylimidazole, which inhibit thromboxane synthetase, resulted in an inhibition of both thromboxane B2 and malondialdehyde formation in response to collagen. 5. The results indicate that thromboxane B2 and malondialdehyde are formed in parallel, in comparable quantities. However, under the conditions used in these studies, the apparent amounts of malondialdehyde exceed those of thromboxane B2, especially in the presence of exogenous arachidonate. Thus the thiobarbiturate reaction used to assay malondialdehyde may detect other products of lipid peroxidation. 6. Platelet thromboxane B2 concentrations did not always relate to the extent of aggregation. In particular, platelet aggregation could occur in the absence of detectable thromboxane B2 production.

scholarly journals NPP4 is a procoagulant enzyme on the surface of vascular endothelium

Blood ◽  
2012 ◽  
Vol 120 (22) ◽  
pp. 4432-4440 ◽  
Author(s):  
Ronald A. Albright ◽  
William C. Chang ◽  
Donna Robert ◽  
Deborah L. Ornstein ◽  
Wenxiang Cao ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Vascular Endothelium ◽  
Platelet Rich Plasma ◽  
Dense Granule ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Phosphodiesterase 4 ◽  
Adp Receptors

Abstract Ap3A is a platelet-dense granule component released into the extracellular space during the second wave of platelet aggregation on activation. Here, we identify an uncharacterized enzyme, nucleotide pyrophosphatase/phosphodiesterase-4 (NPP4), as a potent hydrolase of Ap3A capable of stimulating platelet aggregation and secretion. We demonstrate that NPP4 is present on the surface of vascular endothelium, where it hydrolyzes Ap3A into AMP and ADP, and Ap4A into AMP and ATP. Platelet aggregation assays with citrated platelet-rich plasma reveal that the primary and secondary waves of aggregation and dense granule release are strongly induced by nanomolar NPP4 in a concentration-dependent manner in the presence of Ap3A, while Ap3A alone initiates a primary wave of aggregation followed by rapid disaggregation. NPP2 and an active site NPP4 mutant, neither of which appreciably hydrolyzes Ap3A, have no effect on platelet aggregation and secretion. Finally, by using ADP receptor blockade we confirm that NPP4 mediates platelet aggregation via release of ADP from Ap3A and activation of ADP receptors. Collectively, these studies define the biologic and enzymatic basis for NPP4 and Ap3A activity in platelet aggregation in vitro and suggest that NPP4 promotes hemostasis in vivo by augmenting ADP-mediated platelet aggregation at the site of vascular injury.

scholarly journals Effect of prostaglandin E1 alone and in combination with theophylline or aspirin on collagen-induced platelet aggregation and on platelet nucleotides including adenosine 3′:5′-cyclic monophosphate

1970 ◽  
Vol 120 (4) ◽  
pp. 709-718 ◽  
Author(s):  
G. Ball ◽  
G. G. Brereton ◽  
Mary Fulwood ◽  
D. M. Ireland ◽  
Patricia Yates
Keyword(s):  
Platelet Aggregation ◽  
Cyclic Amp ◽  
Prostaglandin E1 ◽  
Platelet Rich Plasma ◽  
Dependent Manner ◽  
Intracellular Changes ◽  
Induced Aggregation ◽  
Dose Dependent ◽  
Aggregation Of Platelets ◽  
Collagen Treatment

1. Human platelet nucleotides were labelled by incubating platelet-rich plasma with [U-14C]adenine. With such platelets, the effects of prostaglandin E1, theophylline and aspirin were determined on collagen-induced platelet aggregation and release of platelet ATP and ADP. Intracellular changes of platelet radioactive nucleotides, particularly 3′:5′-cyclic AMP, were also determined both with and without collagen treatment. 2. Prostaglandin E1, theophylline and aspirin inhibited collagen-induced aggregation of platelets in a dose-dependent manner. Collagen-induced release of ATP and ADP and breakdown of radioactive ATP were also inhibited in a dose-dependent manner. 3. Prostaglandin E1 stimulated the formation of platelet radioactive 3′:5′-cyclic AMP in a dose-dependent manner. With a given dose of prostaglandin E1, maximum formation of radioactive 3′:5′-cyclic AMP occurred by 10–30s and thereafter the concentrations declined. The degree of inhibition of aggregation produced by prostaglandin E1, however, increased with its time of incubation in platelet-rich plasma before addition of collagen, so that there was an inverse relationship between the radioactive 3′:5′-cyclic AMP concentration measured at the time of collagen addition and the subsequent degree of inhibition of aggregation obtained. 4. Neither theophylline nor aspirin at a concentration in platelet-rich plasma of 1.7mm altered platelet radioactive 3′:5′-cyclic AMP contents. In the presence of prostaglandin E1, theophylline increased the concentration of radioactive 3′:5′-cyclic AMP over that noted with prostaglandin E1 alone, but aspirin did not. 5. Mixtures of prostaglandin E1 and theophylline had a synergistic effect on inhibition of platelet aggregation. The same was true to a lesser extent with mixtures of prostaglandin E1 and aspirin. Such mixtures also inhibited collagen-induced release of platelet ATP and ADP and breakdown of platelet radioactive ATP. 6. Certain concentrations of either theophylline or aspirin and mixtures of small concentrations of prostaglandin E1 with either theophylline or aspirin caused little or no increase of radioactive 3′:5′-cyclic AMP at the time of collagen addition, but inhibited aggregation to a marked degree, whereas higher concentrations of prostaglandin E1 alone caused a much greater increase of radioactive 3′:5′-cyclic AMP at the time of collagen addition but inhibited aggregation to a lesser extent. With these compounds there does not appear to be a correlation between these parameters.

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