Phase Of ADP-induced Aggregation Research Articles

Platelet Aggregation Increased by Advanced Glycated Hemoglobin

Introduction: Many studies have provided strong evidence for an association between diabetes complications and an increase in platelet (PLT) reactivity. Though some metabolic abnormalities have been reported as the major causes of this reactivity and malfunction, the precise mechanism has not been fully elucidated. Aim: The aim of this study is to investigate the effect of advanced glycation end product of hemoglubin (AGE-Hb) on human PLT reactivity and malfunction. Materials and Methods: A solution of bovine hemoglobin was prepared with fructose in phosphate buffer. The solution was then sterilized and incubated under sterile conditions at 37°C in the dark. The control solution was prepared in the same way, but without fructose. Human PLTs were isolated and prepared from 15 healthy volunteers, men and women. Blood was collected in the morning from fasting healthy subjects using a 21-gauge needle with 117 mM sodium citrate (1:9 v/v), as an anticoagulant. Fluorescence measurements were performed using a Hitachi F-4500 spectrofluorometer. PLT aggregation was measured using the photometric system Packs-4 aggregometer. Results: The relative fluorescence intensity increased in Hb samples but not in controls that incubated with fructose. PLTS aggregation did not change in controls while after incubation with 10, 22, 30-days fructose-glycated Hb decreased by 10, 12, 30%, respectively. Taken together our data show that PLT secondary phase of ADP-induced aggregation gradually affected with advanced increase in Hb glycation.

Pharmacological actions of ICI 180080, a novel thromboxane receptor antagonist.

ICI 180080 (5(Z)-7-[2,2-dimethyl-4-(2-hydroxyphenyl)-1,3-dioxan-cis-5-yl] heptenoic acid) potently inhibited contractions of rat and rabbit aortae and guinea-pig trachea elicited by 11,9-epoxymethano PGH2 (U-46619). This antagonism was selective because contractions of aortae to noradrenaline and 5-hydroxytryptamine and trachea to histamine were not antagonized by ICI 180080. Schild analysis of data obtained from experiments on rabbit aortae indicated that this thromboxane receptor antagonism was competitive (pA2 = 7.50, slope = 1.07). Addition of ICI 180080 to human platelet-rich plasma caused dose-related inhibition of U-46619-induced platelet aggregation. This modification of platelet aggregation was not associated with inhibition of thromboxane synthetase, cyclo-oxygenase or lipoxygenase. ICI 180080 did not modify the primary phase of ADP-induced aggregation of human platelets neither did it affect the platelet inhibitory activity of prostacyclin. When dosed orally to anaesthetized guinea-pigs, ICI 180080 (5-50 mg kg-1) caused dose-related inhibition of U-46619-evoked bronchoconstriction. We conclude that ICI 180080 is a potent, selective, competitive, orally active thromboxane antagonist.

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: Direct measurement of percent release of β‐thromboglobulin and assessment by flow cytometry of P‐selectin expression

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of α-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radio-immunoassay of β-thromboglobulin (βTG), and expression on the platelet surface of the α-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 ± 8.3% of βTG and 54.1 ± 4.6% of 14C-serotonin were released (means ± SEM, n = 13); aspirin treatment reduced these values to 6.0 ± 1.2 and 1.0 ± 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 ± 1.7% release of βTG and 2.1 ± 0.4% release of 14C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggregation in PRP anticoagulated with FPRCH2Cl (PPACK), only 4.7 ± 0.9% release of βTG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 μM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2–10 μM), the mean fluorescence intensity due to P-selectin was <14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2??? concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both α-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP. © 1996 Wiley-Liss, Inc.

Conditions influencing release of granule contents from human platelets in citrated plasma induced by ADP or the thrombin receptor activating peptide SFLLRN: direct measurement of percent release of beta-thromboglobulin and assessment by flow cytometry of P-selectin expression.

Contrary to a recent report [Rinder et al.: Blood 82:505, 1993], aspirin does inhibit the release of alpha-granule contents as well as inhibiting the release of dense granule contents by human platelets during ADP-induced aggregation in citrated platelet-rich plasma (PRP). Measurements were: percent release of 14C-serotonin from prelabeled platelets, radio-immunoassay of beta-thromboglobulin (beta TG), and expression on the platelet surface of the alpha-granule constituent, P-selectin, by flow cytometry. During the second phase of ADP-induced aggregation, 69.0 +/- 8.3% of beta TG and 54.1 +/- 4.6% of 14C-serotonin were released (mean +/- SEM, n = 13); aspirin treatment reduced these values to 6.0 +/- 1.2 and 1.0 +/- 0.3%, respectively. In contrast, incubation of platelets with ADP without stirring caused only 6.7 +/- 1.7% release of beta TG and 2.1 +/- 0.4% release of 14C-serotonin; these low values were not appreciably affected by aspirin. During ADP-induced primary aggregation in PRP anticoagulated with FPRCH2CI (PPACK), only 4.7 +/- 0.9% release of beta TG and no detectable release of 14C-serotonin occurred; aspirin had no effect. In both stirred and unstirred PRP, the thrombin receptor activating peptide, SFLLRN (50 microM), caused at least 75% release of the contents of both granules, which was partially inhibited by aspirin. Upon incubation of platelets with ADP (2-10 microM), the mean fluorescence intensity due to P-selectin was < 14% of that induced by SFLLRN. In this unstirred system used for flow cytometry, aspirin treatment caused no significant inhibition of P-selectin expression. Thus, under conditions in which ADP does not cause secondary aggregation (physiological Ca2+ concentration or unstirred citrated PRP) release of the contents of both types of granules is less than 7% and aspirin is not inhibitory; the P-selectin expression associated with this low percent release is also unaffected by aspirin. However, aspirin does strongly inhibit the extensive release of both alpha-granule and dense granule contents during ADP-induced secondary aggregation in citrated PRP.

Inhibition of platelet retention on artificial microvascular grafts with monoclonal antibodies and a high-affinity peptide directed against platelet membrane glycoproteins.

Rapid occlusion of artificial microvascular grafts (AMGs; less than or equal to 2-mm diameter) by platelet-rich thrombi prevents the clinical use of AMGs in cardiac, vascular, and plastic surgery. Since present antiplatelet agents are unable to assure AMG patency, we have studied the role of specific platelet membrane-glycoprotein blockade on platelet retention by AMGs. In a customized in vitro perfusion chamber, retention on polytetrafluoroethylene (PTFE) AMGs (1.0-mm i.d.) of indium-111-labeled platelets in human whole blood was measured in the presence and absence of inhibitors. Specific blockade of platelet membrane glycoprotein (Gp) IIb/IIIa was achieved using monoclonal antibody 10E5 (10 micrograms/ml) and the peptide GRGDS (Gly-Arg-Gly-Asp-Ser, 0.75 mM). These inhibited 98% and 35%, respectively, of platelet retention under circumstances in which aspirin (7 mM) and dextran (4 mg/ml) inhibited 19% and 18%, respectively, of platelet retention. Nonspecific immunoglobulin G F(ab')2 (10 micrograms/ml) and nonspecific peptide (GGDA; Gly-Gly-Asp-Ala, 0.75 mM), used as control reagents, were ineffective in this setting. Monoclonal antibody 6D1 (10 micrograms/ml), which blocks platelet membrane GpIb, prevented 82% of platelet retention on PTFE. These doses of 10E5 and GRGDS completely inhibited platelet aggregation in response to 20 microM ADP, and the dose of 6D1 completely inhibited ristocetin-induced platelet agglutination. The aspirin dose prevented the second phase of ADP-induced aggregation. These data indicate that not only does initial platelet adhesion to PTFE require GpIIb/IIIa but also that GpIb plays a significant role in the early stages of platelet retention on PTFE AMGs.

Blockade of ADP-induced Ca 2+-signal and platelet aggregation by lipoxygenase inhibitors

Stimulation of platelets results in the liberation of arachidonic acid (AA) which is further metabolized via the cyclooxygenase or lipoxygenase (LPG) pathway. We have examined the effect of inhibition of LPG on (i) the ADP-induced increase of cytoplasmic Ca 2+ concentration and (ii) platelet aggregation. Lipoxygenase inhibitors, nordigidroguaiaretic acid (NDGA) and BW-755C, both suppressed ADP-induced Ca 2+-signals and aggregation in a dose-dependent manner, with an IC 50 value of 1–2 μM for NDGA. Qualitatively the same effect was obtained with 4-bromophenylacyl bromide, the inhibitor of phospholipases A 2 and C. By contrast, cyclooxygenase inhibitor indomethacin had only a negligible effect on Ca 2+-signals and suppressed only the second phase of ADP-induced aggregation. It is concluded that the LPG pathway of AA metabolism in platelets might play a crucial role in ADP-induced Ca 2+-signal generation and platelet aggregation.

Platelet aggregation and thromboxane release induced by arachidonic acid, collagen, ADP and platelet-activating factor following low dose acetylsalicylic acid in man

The present study was undertaken in order to characterize the dose-dependent nature of acetylsalicylic acid (ASA) on platelet aggregation and plasma thromboxane B 2 (TXB 2) release in healthy volunteers. Volunteers received either 25, 50, 100 or 500 mg daily for five consecutive days. At the end of the five day period, all dosages of ASA were capable of completely suppressing TXB 2 production and arachidonic acid-induced platelet aggregation. At that time, the second phase of ADP-induced aggregation was also blocked. However, while the inhibition following 500 mg ASA was complete after 24 hours, total inhibition with 100, 50 and 25 mg was attained only after two, three and four days, respectively, indicating the cumulative effect of ASA on platelets. Aggregation induced by collagen was also inhibited dose-dependently- yet slower and at no time complete. ASA had no inhibitory effect on aggregation by platelet-activating factor (PAF). It is concluded that a daily dose of 50 mg ASA would suffice in blocking platelet TXA 2 production and aggregation induced by most physiological agents.

Thromboxane A2 and prostaglandin endoperoxide receptors in platelets and vascular smooth muscle.

9,11-Dimethylmethano-11,12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15 alpha beta-omega-tetranor-TXA2 (I-PTA-OH), a recently synthesized thromboxane (TX) A2/prostaglandin (PG) H2 receptor antagonist, was shown to be a competitive antagonist of human platelet aggregation induced by the stable endoperoxide analog U46619. This antagonism was due to competitive blockade of the platelet TXA2/PGH2 receptor since I-PTA-OH did not antagonize the first phase of ADP-induced aggregation which is TXA2/PGH2 independent, nor did it inhibit TXA2 synthesis. In addition, analysis of dose-response curves to U46619 (0.1 to 40 microM) in the presence of increasing concentrations of I-PTA-OH (0.5 to 10 microM) showed that I-PTA-OH produced a parallel rightward shift of the dose-response curve. Further analysis of the data in the form of a Schild plot yielded a straight line with a slope (m = 1.03) not significantly different from -1. These results are consistent with the notion that I-PTA-OH acts as a competitive antagonist of the TXA2/PGH2 receptor.

Antibodies against a 23Kd heparin binding fragment of thrombospondin inhibit platelet aggregation

Antiserum against a 23Kd heparin binding fragment of thrombospondin inhibits the aggregation of platelets in response to ADP, collagen or thrombin. The antiserum inhibits the secretion-dependent second phase, but not the primary phase of aggregation of platelets responding to ADP. Although immune serum added during the second phase of ADP-induced aggregation causes some inhibition of secretion, it also causes reversal of aggregation to the level produced during primary aggregation. Since thrombospondin is the endogenous lectin of human platelets, these results support the conclusion that the endogenous lectin mediates, at least in part, the secretion-dependent aggregation of platelets. Our data suggest that the region of thrombospondin which contains the heparin binding domain(s) present in the 23Kd fragment play(s) a critical role in secretion-dependent aggregation of platelets.

Effects of ketanserin and mepyramine on platelet aggregation and on the uptake of 5-hydroxytryptamine into platelets

We have determined the effects of ketanserin, a 5HT 2 antagonist, and of mepyramine, a histamine H 1 antagonist, on the aggregation induced in platelet rich plasma by 5-hydroxytryptamine (5HT), adenosine diphosphate (ADP), and by combinations of 5HT and ADP. Both ketanserin and mepyramine inhibited 5HT-induced aggregation and the 5HT component of the aggregation induced by combinations of 5HT and ADP. Both agents also inhibited the second phase of the aggregation induced by ADP alone but higher concentrations of the agents were required. On a molar basis, ketanserin was always a more potent inhibitor of aggregation than mepyramine. Ketanserin did not consistently inhibit the ADP-induced release reaction nor did it inhibit ADP-induced aggregation in aspirin - treated platelets that were unable to undergo a release reaction. The results suggest that 5HT 2 receptors are involved in 5HT-induced aggregation and that the platelets' endogenous 5HT may contribute to the second phase of ADP-induced aggregation. We have also compared the effects of ketanserin and mepyramine on 5HT uptake into platelets. In this case mepyramine was a much more potent inhibitor than ketanserin. The different effectiveness of ketanserin and mepyramine as inhibitors of aggregation and 5HT uptake provides further evidence for different 5HT receptors for these two processes, and indicates that those involved in the 5HT uptake may be more akin to histamine H 1 receptors.

Effect of the Calcium-Entry Blocking Agent Nifedipine on Activation of Human Platelets and Comparison with Verapamil

The effects of the calcium-entry blocking agent nifedipine on the activation of human platelets by various agonists has been studied and compared with verapamil. Like verapamil, nifedipine inhibited platelet aggregation and secretion caused by collagen, the second phase of ADP-induced aggregation, and aggregation caused by the ionophore A23187. Both agents inhibited the formation of TXB2 from endogenous arachidonate, whereas only nifedipine inhibited platelet aggregation and decreased TXB2 formation caused by exogenous arachidonate without inhibiting uptake. These results indicate that both calcium-blocking agents may be inhibiting the release of arachidonate in platelets by phospholipases, and that nifedipine also inhibits the formation and action of thromboxane A2 in platelets. Epinephrine-induced aggregation was inhibited by low concentrations of verapamil while nifedipine only inhibited aggregation by epinephrine at much higher concentrations. It is suggested that low concentrations of verapamil inhibit epinephrine-induced aggregation by interacting with platelet alpha-adrenergic receptors, and that higher concentrations of both calcium-blocking agents inhibit platelet responses to other aggregating agents by preventing intracellular calcium mobilization.

Effects of Ticlopidine, a new platelet function suppressing drug in patients with prosthetic heart valves

The effect of ticlopidine (fig. 1), a new platelet aggregation inhibitor, was studied in 15 patients with Björk-Shiley prosthetic heart valves (6 aortic, 6 mitral, 3 aortic and mitral). The patients were 9 men and 6 women, aged 22 to 55 years old, who had had valve prostheses placed 2 to 43 months (mean 23.7 months) prior to entry into the study. They received 200mg of ticlopidine, divided into two oral doses, daily, and were followed up for more than 3 months. Platelet adhesiveness and aggregation were examined 1, 2, and 4 weeks, and 3 months after the treatment, and the values were compared to those obtained before the treatment. Platelet adhesiveness was determined by a modified technique of Salzman. A Sienco dual sample aggregation meter was used to measure changes of platelet aggregation through light transmission of citrated platelet-rich plasma to which adenosine diphosphate (ADP) had been added. The platelet count of the plasma sample was adjusted to a range of 250-350, 000/mm3. Student's paired t-test was used for statistical significance.Platelet adhesiveness decreased significantly at 1 week, 2 weeks, and 3 months after the treatment, but no significant change was found at 4 weeks (fig. 2).In every patient, platelet aggregation induced by 4μM ADP (final concentration) showed an obvious reduction one week after the treatment, and this effect continued for more than 3 months (fig. 3). A similar reduction in the aggregation caused by 20μM ADP (final concentration) was observed. In many cases, the aggregation caused by 20μM ADP, which showed the second phase of aggregation, changed after the treatment to show only the first phase of aggregation (fig. 4). Ticlopidine inhibited both the first and the second phase of ADP-induced aggregation. This dosage seemed to cause a reduction in platelet aggregation about 3 days after the start of the treatment.We did not observe any interaction between ticlopidine and warfarin in this study.None of the patients had clinical symptoms or side effects due to ticlopidine and no episodes of thromboembolism occurred during the examination period. Bleeding time increased slightly at 4 weeks after the treatment. No abnormal alteration was observed in the white blood cell counts, coagulation time, fibrinogen level, fasting blood sugar, serum electrolites, total protein, hepatic and renal function tests at 4 weeks and 3 months after the treatment.Ticlopidine seems to be a useful platelet function suppressing drug for patients with prosthetic heart valves.

β-Blocking Agents and Platelet Aggregation

To the Editor.— There is general agreement that substances that inhibit platelet aggregation tend to be associated with a rise in platelet cyclic adenosine monophosphate (cAMP), and those that stimulate platelet-clumping with the reverse. 1 Epinephrine reduces platelet cAMP by inhibition of adenyl cyclase. Like epinephrine-induced platelet aggregation, this phenomenon is an α-adrenergic effect, being subject to blockade by phentolamine. 2 Hampton et al 3 discovered that propranolol inhibited noradrenaline-induced platelet aggregation. Propranolol proved also to be a powerful inhibitor of adenosine diphosphate (ADP)-induced aggregation and of potentiating effect of epinephrine on the second phase of ADP-induced aggregation. 4 It seems that this effect would be not related to β-blocking activity but to a stabilizing action on biological membranes like imipramine and related compounds. 5 In regard to its cardiac effects, propranolol appears to have a more evident activity on the inhibition of platelet aggregation.

Secretory mechanisms. Behaviour of adenine nucleotides during the platelet release reaction induced by adenosine diphosphate and adrenaline.

1. Platelets containing adenine nucleotides labelled with (3)H and (14)C in vitro were aggregated biphasically with ADP and adrenaline. Amounts of ATP and ADP as well as the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine and adenine were determined in platelets and plasma at different stages of aggregation. 2. ATP and ADP were released during the second aggregation phase and had a low specific radioactivity compared with the ATP and ADP retained by the cells. The specific radioactivity of intracellular nucleotides increased during release. The parameters observed with ADP and adrenaline as release inducers were the same as for collagen and thrombin. 3. Release induced by all four inducers was accompanied by conversion of cellular [(3)H]ATP into extracellular [(3)H]-hypoxanthine. By variation of temperature, inducer concentration, time after blood withdrawal and use of acetylsalicylic acid, the aggregation pattern caused by adrenaline and ADP could be made mono- or bi-phasic. Release or second-phase aggregation was intimately connected with the ATP-hypoxanthine conversion, whereas first phase aggregation was not. 4. The [(3)H]ATP-hypoxanthine conversion started immediately after ADP addition. With adrenaline it usually started with the appearance of the second aggregation phase. The conversion was present during first phase of ADP-induced aggregation only if a second phase were to follow. 5. When secondary aggregation took place while radioactive adenine was being taken up by the platelets, increased formation of labelled hypoxanthine still occurred, but there was either no change or an increase in the concentration of labelled ATP. 6. Biphasically aggregated platelets converted [(3)H]adenine more rapidly into [(3)H]-ATP and -hypoxanthine than non-aggregated platelets. Addition of [(3)H]adenine at different stages of biphasic aggregation showed that more [(3)H]hypoxanthine was formed during than after the release step. 7. We conclude that ADP and adrenaline, like thrombin and collagen, cause extrusion of non-metabolic granula-located platelet adenine nucleotides. During release metabolic ATP breaks down to hypoxanthine, and this process might reflect an ATP-requiring part of the release reaction.

The Effect of Neuraminidase on Platelet Aggregation Induced by ADP, Norepinephrine, Collagen or Serotonin

SummaryIncubation of human platelet-rich plasma (PRP) with neuraminidase enhanced platelet aggregation induced by adenosine diphosphate (ADP), norepinephrine, collagen or serotonin. Both first and second phases of ADP- and norepinephrine -induced aggregation were enhanced. In two plasmas a second phase of ADP-induced aggregation occurred after incubation with neuraminidase, but not in the control preparations. In one plasma a second phase of serotonin-induced platelet aggregation occurred after incubation with neuraminidase. The incubation of PRP with exogenous N-acetylneuraminic acid (a product of the action of neuraminidase) had no effect on platelet aggregation. A possible explanation of the observed enhancement of platelet aggregation by neuraminidase is that the enzyme may have released sialic acid from platelet membranes and thereby reduced their net negative surface charge.