Original Article: Comparison of A23187 vs Ionomycin-induced Responses and Cytosolic Calcium Increases in Aequorin-loaded Human Platelets. Evidence for Ionophore-specific Differences in Intracellular Calcium Release

The dose-dependent induction of platelet aggregation, dense granule secretion and thromboxane formation by the divalent cation ionophores A23187 and ionomycin were compared in citrated platelet rich plasma (PRP), and measured simultaneously with the increases in cytosolic Ca(2+) levels in aequorin-loaded gel-filtered platelet (GFP). In citrated PRP, both ionophores induced similar extents of aggregation at comparable concentrations, whereas A23187 induced somewhat greater extents of secretion, and substantially greater extents of thromboxane B(2) (TxB(2)) formation, than ionomycin. When 5 mM EDTA or EGTA was added to PRP secretion and TxB(2) formation occurred only with A23187; ionomycin was inactive at all concentrations tested, up to 100 pM. In aequorin-loaded GFP containing 1 mM Ca(2+), 1 mM EGTA or 2 mM EDTA, ionomycin as well as A23187 induced these platelet responses, but the concentration dose-response curve for ionomycin was shifted to the right by approximately one order of magnitude relative to that for A23187. Simultaneous measurements of the cytosolic Ca(2+) ([Ca(2+)](1)) increases induced by the two ionophores showed that the increases produced by ionomycin were consistently 2040% less than those induced by A23187 at all ionophore concentrations tested. Analysis of the extents of secretion and TxB, formation obtained in EDTA- or EGTA-containing systems as a function of the [Ca(2+)](1) increases suggested that the data for both ionophores were described by the same [Ca(2+)](1) dose-response curve, indicating that the decreased extents of these responses seen with ionomycin vs A23187 were due primarily, if not solely, to the lower [Ca(2+)], increases produced by ionomycin. Since ionomycin is theoretically capable of transporting twice as much divalent cation as A23187, these findings in platelets, together with similar findings in certain other cell systems, provide evidence that factors associated with the intracellular environment may differentially affect the abilities of A23187 and ionomycin to induce cellular responses and, more specifically, to release intracellular Ca(2+) stores.     

Platelet secretion defect in patients with the attention deficit disorder and easy bruising

Platelet function was evaluated in 12 patients with the attention deficit disorder and lifelong history of easy bruising. Aggregation and 14C-serotonin secretion studies in platelet-rich plasma in response to adenosine diphosphate (ADP), epinephrine, and arachidonic acid did not reveal striking abnormalities. Secretion of adenosine triphosphate (ATP), ADP, beta-hexosaminidase, and beta-glucuronidase by gel-filtered platelets in response to the divalent cation ionophore A23187 and low concentrations of thrombin (less than or equal to 0.1 U/ml) was impaired in patients as compared to normals. The aggregation response to A23187 (4 microM) was absent in 8 of the 12 patients. The total stores of the secretable constituents, the retention of incorporated 14C-serotonin, and the arachidonate metabolism of the platelets were normal. Our findings suggest a new platelet disorder with impaired secretion mechanism, without storage pool deficiency or impaired arachidonate metabolism. The secretion defect in platelets represents a tissue disorder in a functional psychiatric disease. We refocus attention on the role of platelets as a model for neurons in functional disorders, with emphasis on secretion mechanisms rather than amine uptake, storage, and metabolism.     

Phospholipase A2 activity in platelets of patients with primary thrombocythemia

Patients with primary thrombocythemia (PT) have both, bleeding and thrombotic events. Although platelet aggregation tests are usually abnormal, synthesis of thromboxane B2 (TxB2) by platelets is increased. This feature could be the consequence of an increased phospholipase activity or a facilitated metabolism of arachidonate by prostaglandin synthetase pathway. We studied the activity of phospholipase A2 as well the arachidonate metabolism in platelets of patients suffering from PT. Eleven patients and 11 controls were included. Platelets were labelled with [14C]arachidonic acid ([14C]AA). Lost of radioactivity from phospholipids and new radioactive prostanoids were evaluated in calcium ionophore A23187 activated platelets, to explore phospholipase A2 activity. This assay was also carried out in aspirin-incubated platelets. We also studied the formation of prostanoids in platelets activated by radioactive free arachidonic acid. Platelet aggregation studies of patients were abnormal. [14C]AA incorporation in platelet phospholipids was normal. Ionophore activated platelets from patients and controls lost 26.1 +/- 8.3% and 24.1 +/- 10.5% of radioactivity, respectively, mainly from phosphatidylcholine. The main arachidonate metabolite was 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid (HETE), which comprised 14.1 +/- 5.1% of the radioactivity released from phospholipids in patients, and a similar amount in the controls (14.4 +/- 7.5%). Formation of TxB2 was also similar in patients (5.5 +/- 1.2%) and controls (4.9 +/- 2.9%). Formation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid (HHT) was also normal. Ionophore A23187 activation of aspirinized platelets of patients released 19.5 +/- 7.4% of radioactivity from phospholipids, which was completely metabolized to HETE. Formation of prostanoids HETE, HHT and TxB2 by arachidonic acid activated platelets of patients was normal. Phospholipase A2 activity as well both cyclooxygenase and lipoxygenase activities in platelets of patients with PT were found to be normal.     

Heterogeneous defects of platelet secretion and responses to weak agonists in patients with bleeding disorders

Eleven patients with mild bleeding disorders had as a common abnormality, impaired platelet aggregation and secretion with low concentrations (0.5-1.0 micrograms/ml) of collagen and, in most cases, an absence of second phase aggregation with epinephrine. Platelet granule contents were normal, ruling out storage pool deficiency. To characterize further the platelet abnormalities, we measured aggregation, 14C-5HT secretion, and TxB2 formation induced by a variety of platelet agonists. In eight of the 11 patients we observed decreased initial rates as well as extents of aggregation with one or more weak agonists (ADP, epinephrine, thromboxane A2 and the endoperoxide analogue U44069), i.e. agonists which induced secretion only as a result of aggregation, but normal responses to strong agonists such as arachidonate and high (10 micrograms/ml) concentrations of collagen, which can induce secretion in the presence or absence of aggregation. In all of these patients, TxB2 formation with arachidonate and all concentrations of collagen was normal. The platelet defects in these eight patients have been designated as weak agonist response defects (WARDs). In contrast, the initial aggregation responses to all weak agonists were normal in the three other patients, while secretion and TxB2 formation induced by strong agonists were impaired. Thus, in contrast to the eight patients above, the platelet defects in these three patients were characteristic of defects in the secretion response per se. The results obtained in the 11 patients studied indicate that these types of platelet disorders, previously referred to as primary secretion defects, include defects in the initial platelet responses which precede secretion (WARD) as well as defects in the secretory mechanism per se. Both groups of defects appear to be heterogeneous in nature.     

A new congenital defect of platelet secretion: impaired responsiveness of the platelets to cytoplasmic free calcium

Summary . A 16-year-old boy with a bleeding disorder since infancy has a long bleeding time, normal platelet count and morphology and normal plasma factor-VIII activities. His platelets undergo normal shape change and primary aggregation in response to ADP but show defective 5-hydroxytryptamine (5-HT) secretion and aggregation in response to adrenaline, sodium arachidonate, U44069, PAF-acether, A23187 and low concentrations of collagen. Thrombin and higher concentrations of collagen produce a normal response. Secretion of β-thromboglobulin and platelet factor 4 parallels that of 5-HT. Thromboxane B₂ is produced normally in response to exogenous arachidonate and to stimulation by thrombin, collagen and A23187 in all concentrations tested. The patient's endoperoxides and thromboxane A₂ aggregate aspirin-treated platelets, though his platelets are themselves unresponsive. Cyclic AMP is present at normal concentration in the patient's unstimulated platelet-rich plasma, and PGI₂ inhibits platelet aggregation by ADP and thrombin in a normal dose-related manner. Platelet ultrastructure, 5-HT uptake and content of adenine nucleotides, platelet factor 4 and β-thromboglobulin are all within normal limits. When the patient's platelets were loaded with the fluorescent dye quin 2, which serves as an indicator of cytoplasmic free calcium ions, their responses to thrombin, whether in the presence or virtual absence of extracellular Ca2+, were entirely normal in respect of free calcium ions, secretion, shape-change and aggregation. In response to ionomycin, however, a normal increase in free calcium ions was accompanied by normal shape-change but virtually no aggregation or 5-HT secretion. The platelet calmodulin content was normal. These findings show that the defect in this patient's platelets is of utilization of cytoplasmic Caz+ for secretion and aggregation, rather than of Ca2+ uptake or mobilization of Ca2+ from intracellular storage sites. It is suggested that the most likely site of the defect is the phosphorylation of one of the proteins concerned in the secretory mechanism.     

Endotoxin-induced production of thromboxane and prostacyclin by equine peritoneal macrophages

Equine peritoneal macrophages were isolated and cultured in vitro to assess their ability to produce thromboxane (TxA2) and prostacyclin (PGI2) in response to endotoxin. Peritoneal macrophages (2.5 x 10(6)/ml) were incubated in tissue culture media, containing 1) no additive (nonstimulated control), 2) endotoxin (0.5 to 100 ng/ml) or 3) the calcium ionophore, A23187 (0.95 microM) for two and six h. Concentrations of the stable metabolites of TxA2 and PGI2 thromboxane B2 (TxB2) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in the incubation media were determined by radioimmunoassay. The concentrations of both metabolites increased from two to six h incubation. Endotoxin increased the production of TxA2 and PGI2 over the nonstimulated control values at both two and six h and endotoxin-induced concentrations of 6-keto-PGF1 alpha were higher at six than at two h. The response of macrophages to A23187 was similar to endotoxin. Mean eicosanoid concentrations did not differ among the range of endotoxin concentrations at either time; however there was significant curvilinear regression between endotoxin concentration and TxB2 at both times, and between endotoxin and 6-keto-PGF1 alpha at two h. The results indicate that equine macrophages may be a significant source of TxA2 and PGI2 during endotoxemia.     

Pathogenetic analysis of three cases with a bleeding disorder characterized by defective platelet aggregation induced by Ca2+ ionophores

We report three cases of platelet dysfunction characterized by defective Ca2+ ionophore-induced platelet aggregation without impaired production of thromboxane A2 (TXA2). The patients had mild to moderate bleeding tendencies, and their platelet aggregation and secretion induced by ADP, collagen, arachidonic acid, stable TXA2 (STA2) and Ca2+ ionophore A23187 was defective or much reduced. However, ristocetin- or thrombin-induced platelet aggregation was normal. The analysis of second messenger formation showed that inositol 1,4,5-triphosphate formation or Ca2+ mobilization induced by thrombin, STA2 or A23187 was normal. Furthermore, the phosphorylation of 47 kDa protein (pleckstrin) and 20 kDa protein (myosin light chain, MLC) in response to those agonists was normal. These findings suggest that the defective site in the patients' platelets lies in the process distal to or independent of protein kinase C activation, Ca2+ mobilization and MLC phosphorylation.     

Phospholipase A2 activity in platelets of patients with uremia

Platelets of patients with uremia develop a defective platelet function and have a decreased production of thromboxane B2 (TxB2). Activated platelets generate thromboxane from free arachidonate that is previously released from the membrane phospholipids (PLs) by phospholipases. Phospholipase A2 (PLA2) release up to 70% of the arachidonate in normal platelets, and to date, the activity of this enzyme in uremia is unknown. This work studied the PLA2 activity in the platelets of nine uremic patients and nine healthy volunteers. Washed platelets were labelled with [(14)C]arachidonic acid and activated with calcium ionophore A-23187 (4 microgr/ml). Lipids were resolved by TLC and identified by autoradiography. The distribution of [(14)C]arachidonic acid in the five major platelet phospholipids was found to be normal. Uremic platelets released more radioactivity than normal platelets (19.0 +/- 5.2% versus 11.3 +/- 1.6%, P = 0.001). The production of both, radioactive thromboxane B2 and hydroxyheptadecatrienoic acid was normal (2.6 +/- 1.2% and 3.5 +/- 1.6% of total radioactivity respectively), but the formation of the lipoxygenase metabolite hydroxyeicosatetraenoic acid was increased with respect to the controls (12.9 +/- 4.6% vs 7.0 +/- 1.3% of total radioactivity, P = 0002). In conclusion, platelets of patients with uremia have an increased activity of phospholipase A2 and produce increased amounts of hydroxyeicosatetraenoic acid, an inhibitor of the platelet function.     

Thrombin and ionophore A23187-induced dense granule secretion in storage pool deficient platelets: evidence for impaired nucleotide storage as the primary dense granule defect

The secretion of the dense granule constituents ATP, ADP, calcium, pyrophosphate (PPi), and orthophosphate (Pi), and the release of magnesium induced by thrombin and the divalent cation ionophore A23187 have been quantitated directly in gel-filtered platelets from patients with storage pool deficiency (SPD). Both the contents and the maximal amounts of the dense granule constituents secretable by thrombin were decreased in all the patients studied, while the nonsecretable, retained amounts of these substances were identical in SPD and normal platelets. In response to both thrombin and A23187, the amounts of secretable ATP and ADP were strongly correlated in the platelets of individual patients; in contrast, secretable calcium showed no correlation with the nucleotides, and significant amounts of calcium were secreted in the total absence of nucleotide secretion in the platelets of several patients. The contents of magnesium were normal in all patients, and approximately 12% of platelet magnesium was liberated by thrombin in both SPD and normal platelets. A23187 induced the release of up to 70% of the magnesium content of normal platelets, but released significantly less (46%) magnesium from SPD platelets. Platelet aggregation induced by A23187 in platelet-rich plasma was also markedly decreased in SPD platelets. The correlations among secretable dense granule constituents suggest the presence in SPD platelets of abnormal dense granule structures that sequester calcium and other constituents but little or no adenine nucleotides, and are thus consistent with a hypothesis that impaired nucleotide transport and/or storage may be the primary dense granule defect in this disorder. In addition, these results demonstrate that certain responses to A23187 are impaired in SPD platelets.     

A defect of platelet release reaction in a patient with SLE: impaired platelet aggregation induced by phorbol ester with a normal phosphorylation of 40K protein

A 37-year-old female who suffered from SLE had a bleeding disorder. At the time of initial evaluation, the main disease demonstrated was a delta-storage pool deficiency. After this improved, a marked decrease of aggregation still remained, when induced by either ADP, epinephrine, collagen, A23187, thrombin, or PAF-acether. Although arachidonate-induced aggregation was slightly decreased, thromboxane B2 was produced normally in response to exogenous arachidonate. The patient's endoperoxides and/or thromboxane A2 aggregated aspirin-treated platelets, though her platelets were themselves unresponsive. Impaired aggregability induced by TPA (12-0-tetradecanoylphorbol-13-acetate) or OAG (1-oleoyl-2-acetyl-glycerol) was also found. However, the phosphorylation of P43 and P20 induced by several stimulators including CA++ ionophore was normal, using 32P-labelled platelets. It is suggested that TPA or OAG-induced platelet aggregation requires not only the phosphorylation of those proteins, but also another unknown mechanism after the phosphorylation, and that the platelet dysfunction of this patient was due to a defect of some mechanism involving Ca++ uptake or mobilization of cytoplasmic Ca++ from intracellular storage sites.     

Studies on the role of calcium in the control of thromboxane B2 production

5-hydroxytryptamine (5-ht) secretion and thromboxane B2 (TxB2) production by platelet-rich plasma in response to the Ionophore A23187 or the sulphydryl group reagent N-ethyl maleimide (NEM) were potentiated in the presence of EDTA. No such potentiation by EDTA was observed using aspirin-treated platelets. The addition of Ca2+ and also Mg2+ to suspensions of washed human platelets reduced the rate and extent of TxB2 formation in response to thrombin, A23187, collagen or NEM. In contrast, Sr2+ resulted in spontaneous TxB2 production and 5-HT secretion.     

Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation.

Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.     

Modulation of platelet function by prostaglandins: characterization of platelet receptors for stimulatory prostaglandins and the role of arachidonate metabolites in platelet degranulation responses.

The effects of PGG2 and PGH2 on platelets are mimicked by synthetic PG analogues in which the nature and specificity of the substituents on carbons 11 and 15 (or 16) are important determinants of reactivity. Arachidonic acid and stimulatory PGs induce secretion of platelet dense granule and alpha granule constituents, but not lysomal constituents, although arachidonate metabolism is necessary for collagen-induced release of lysosomal enzymes. NO164 acts on platelets as an endoperoxide antagonist: Trimethoquinol acts as an endoperoxide and TxA2 antagonist. PGs induce platelet aggregation by combining with a specific (endoperoxide) receptor.     

Inhibition of human platelet aggregation and cytoplasmic calcium response by calcium antagonists: studies with aequorin and quin2

Calcium antagonists inhibit platelet aggregation, but whether this action is due to inhibition of the effect of agonists on cytoplasmic ionized calcium concentration is unknown. We studied this problem by loading gel-filtered platelets with either quin2 or aequorin and stimulating them with epinephrine, arachidonate, thrombin, the calcium ionophore A23187, 1-oleoyl-2-acetyl glycerol, or adenosine diphosphate in media with or without extracellular calcium. In response to all of these agonists, aequorin indicated an increase in cytoplasmic calcium that accompanied or preceded platelet aggregation. In calcium-containing media, verapamil, nifedipine, and diltiazem inhibited these effects in a concentration-dependent fashion, except for those produced by thrombin and A23187. Removal of extracellular calcium with EGTA reduced the calcium response to arachidonate, adenosine diphosphate, and 1-oleoyl-2-acetyl glycerol, and the calcium response and aggregation were further inhibited by the calcium antagonists. In general, strong inhibition of the aequorin cytoplasmic calcium signal by approximately 100 microM concentrations of nifedipine, verapamil, and diltiazem was correlated with inhibition of platelet aggregation, but high concentrations of the inhibitors were required. Since inhibition by the calcium antagonists of the cytoplasmic calcium response and aggregation exceeded the effect of simple removal of extracellular calcium, these drugs may affect internal redistribution of calcium in human platelets.     

Output of prostanoids from rat lung following endotoxin and its modification by methylprednisolone

Endotoxin given intraperitoneally to rats induced leucopenia and pulmonary oedema over 28 hr. The output of cyclo-oxygenase products PGE2, PGF2 alpha, TxB2, and 6-oxo-PGF1 alpha, from the perfused lungs isolated from these rats was measured using as substrate either exogenous arachidonate or endogenous arachidonate stimulated by the calcium ionophore A23187. From exogenous or endogenous arachidonate, the major effects of endotoxin were increased TxB2 and PGF2 alpha output and decreased 6-oxo-PGF1 alpha output. Treatment with methylprednisolone 30 min after endotoxin prevented the pulmonary oedema and leucopenia. The output of TxB2 and PGF2 alpha was unaltered, whereas that of 6-oxo-PGF1 alpha was increased. Our results do not support the suggestion that the beneficial effects of methylprednisolone in this model of acute lung injury are due to inhibition of prostanoid synthesis. Benefit may be better correlated with increased PGI2 formation.     

The H1-histamine antagonist dithiaden inhibits human platelet function in vitro

The H-histamine receptor antagonist Dithaden inhibited in a dose-dependent manner, human platelet 1 aggregation in vitro that was induced with stimuli in the following rank order of potency: thrombin > A23187 > adrenaline > ADP. The aggregation of platelets in plasma induced with adrenaline was inhibited by Dithiaden (DIT) (both the first and the second phase) and the onset of the second phase was prolonged significantly. In a concentration-dependent manner DIT inhibited thrombin- and calcium ionophore A23187-induced {3H}arachidonic acid liberation from, and peroxidation (measured as malondialdehyde formation) of membrane phospholipids. The same effect of DIT was found for the inhibition of thromboxane B suggested that DIT does not inhibit stimulated platelets at specific histamine receptors sites but rather at the phospholipase A and thromboxane synthase level. Results from in vitro experiments should be verified in 2 vivo . generation. It is 2.     

Evidence for a role for Galphai1 in mediating weak agonist-induced platelet aggregation in human platelets: reduced Galphai1 expression and defective Gi signaling in the platelets of a patient with a chronic bleeding disorder

We have examined platelet functional responses and characterized a novel signaling defect in the platelets of a patient suffering from a chronic bleeding disorder. Platelet aggregation responses stimulated by weak agonists such as adenosine diphosphate (ADP) and adrenaline were severely impaired. In comparison, both aggregation and dense granule secretion were normal following activation with high doses of collagen, thrombin, or phorbol-12 myristate-13 acetate (PMA). ADP, thrombin, or thromboxane A2 (TxA2) signaling through their respective Gq-coupled receptors was normal as assessed by measuring either mobilization of intracellular calcium, diacylglycerol (DAG) generation, or pleckstrin phosphorylation. In comparison, Gi-mediated signaling induced by either thrombin, ADP, or adrenaline, examined by suppression of forskolin-stimulated rise in cyclic AMP (cAMP) was impaired, indicating dysfunctional Galphai signaling. Immunoblot analysis of platelet membranes with specific antiserum against different Galpha subunits indicated normal levels of Galphai2,Galphai3,Galphaz, and Galphaq in patient platelets. However, the Galphai1level was reduced to 25% of that found in normal platelets. Analysis of platelet cDNA and gDNA revealed no abnormality in either the Galphai1 or Galphai2 gene sequences. Our studies implicate the minor expressed Galphai subtype Galphai1 as having an important role in regulating signaling pathways associated with the activation of alphaIIbbeta3 and subsequent platelet aggregation by weak agonists.     

Hermansky-Pudlak platelets: further studies on release reaction and protein phosphorylations

Platelets from a patient with the Hermansky-Pudlak syndrome were studied. These platelets had decreased amounts of serotonin and adenine nucleotides, and a decreased number of mepacrine-labeled dense bodies. beta-Thromboglobulin and acid hydrolases contained in alpha-granules and lysosomes respectively were present in normal amount. Platelets in platelet-rich plasma did not respond to collagen, but arachidonic acid and ionophore A 23187 induced normal aggregation and normal thromboxane (TX) synthesis. Alpha-granule release was found impaired and remained subnormal even with high doses of inducers. In response to thrombin aggregation, release and TX synthesis of isolated metrizamide gradient platelets were found at lower than normal levels. Phosphorylation of P20 and P43 proteins was normal. Only a combination of ADP plus thrombin could restore a normal aggregation, with normal alpha-granule and lysosome release and normal TX synthesis. These results indicated that in the absence of dense bodies: the release of other granules is impaired; the TX synthesis is delayed except when induced by arachidonic acid and A 23187 ionophore; the absence of dense bodies could be compensated for by the addition of ADP which restores the impaired release reaction and TX formation; and P20 and P43 polypeptides were phosphorylated as rapidly as those in normal platelets.     

Platelet aggregation and dense granule secretion in a colony of dogs with spontaneous hypertension.

AIM: Since canine hereditary essential hypertension has been previously reported in a colony of Siberian husky dogs, we tested the suitability of this model for use in studies on the platelet defect identified in humans with essential hypertension. METHODS: Platelet aggregation and dense granule ATP secretion were measured in dogs with essential hypertension and normotensive control dogs. RESULTS: The platelets from the hypertensive group showed significantly increased aggregation and secretion in response to stimulation with ADP. There was no significant increase in aggregation or secretion when platelets from hypertensive dogs were stimulated by platelet-activating factor, thrombin, calcium ionophore A23187 or phorbol myristate acetate. CONCLUSION: The increased aggregation and secretion responses in platelets from the hypertensive dogs suggest that a defect similar to that reported in humans with essential hypertension exists and that further investigation is warranted.     

In vitro and ex vivo effects of indobufen on human platelet aggregation, the release reaction and thromboxane B2 production

We have done a comprehensive study in normal volunteers of the in vitro and ex vivo effects of the antiplatelet agent indobufen on platelet aggregation, the release reaction and thromboxane B2 (TxB2) production as induced by different concentrations of aggregating agents. At low concentrations (10 microM), indobufen completely inhibited secondary platelet aggregation, the release reaction and TxB2 production stimulated by ADP, epinephrine and low concentrations of platelet-activating factor (PAF acether). Higher concentrations of indobufen (100 microM) completely inhibited TxB2 production, platelet aggregation and ATP release induced by arachidonic acid (1 mM) or collagen (2 micrograms/ml). The inhibitory effect was partially overcome by higher concentrations of arachidonic acid (2 mM). Data obtained ex vivo 2 h after the oral administration of 200 mg indobufen to 8 normal volunteers were in keeping with those of the in vitro study. We conclude that indobufen inhibits platelet aggregation and the release reaction by inhibiting the platelet arachidonate pathway.