Effect of the Concentration of Ca2+ in the Suspending Medium on the Responses of Human and Rabbit Platelets to Aggregating Agents

1989 ◽  
Vol 62 (03) ◽  
pp. 968-976 ◽  
Author(s):  
M A Packham ◽  
N L Bryant ◽  
M A Guccione ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Arachidonic Acid ◽  
Close Contact ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Additional Contribution ◽  
Low Concentrations ◽  
Rabbit Platelets

SummaryThe effect of the concentration of Ca2+ in the suspending medium of human and rabbit platelets on aggregation, release of 14C-serotonin, and TXB2 formation in response to ADP, thrombin, l-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (PAF), collagen and arachidonic acid was studied in either platelet-rich plasma anticoagulated with D-phenylalanyl-prolyl-arginyl chloromethylketone (PPACK) or citrate, or suspensions of washed platelets in modified Tyrode-albumin solutions containing 1 mM Mg2+ and concentrations of added Ca2+ ranging from 0 to 5 mM. In response to ADP, thrombin, or PAF, human platelets were stimulated to form TXA2 by close platelet contact in a low- Ca2+ medium; at physiological concentrations of Ca2+, TXB 2formation was much less and declined progressively as the concentration of Ca2+ was raised. When the formation of TXA 2was blocked with aspirin or indomethacin, aggregation and release by human platelets were strongest at physiological concentrations of Ca2+. Rabbit platelet responses differed markedly from those of human platelets because close contact of rabbit platelets in a low-Ca2+ medium did not promote TXA2 formation. Rabbit platelet responses were more strongly inhibited by the lack of added Ca2+ in the medium than the responses of human platelets, possibly because rabbit platelets do not contain releasable Ca2+.In all studies of human platelets in media with low concentrations of Ca2+, the additional contribution to platelet responses of TXA2 formed because of close platelet contact should be considered because TXA2 formation is not usually stimulated in this way at physiological concentrations of Ca2+. When TXA2 formation is blocked, aggregation and release responses to all agonists are greatest at physiological concentrations of Ca2+. Thus, the responses of human platelets in media with low concentrations of Ca2+ (citrated platelet-rich plasma or artificial media to which no Ca2+ has been added) are abnormal in at least two ways, and do not correspond to the responses at physiological concentrations of Ca2+.

Effects of Ethanol on Pathways of Platelet Aggregation In Vitro

1988 ◽  
Vol 59 (03) ◽  
pp. 383-387 ◽  
Author(s):  
Margaret L Rand ◽  
Marian A Packham ◽  
Raelene L Kinlough-Rathbone ◽  
J Fraser Mustard
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Secondary Phase ◽  
Primary Phase ◽  
Rabbit Platelet ◽  
Membrane Phospholipids ◽  
Rabbit Platelets ◽  
Induced Aggregation

SummaryEthanol, at physiologically tolerable concentrations, did not affect the primary phase of ADP-induced aggregation of human or rabbit platelets, which is not associated with the secretion of granule contents. Potentiation by epinephrine of the primary phase of ADP-induced aggregation of rabbit platelets was also not inhibited by ethanol. However, ethanol did inhibit the secondary phase of ADP-induced aggregation which occurs with human platelets in citrated platelet-rich plasma and is dependent on the formation of thromboxane A2. Inhibition by ethanol of thromboxane production by stimulated platelets is likely due to inhibition of the mobilization of arachidonic acid from membrane phospholipids, as ethanol had little or no effect on aggregation and secretion induced by arachidonic acid or the thromboxane mimetic U46619. Rabbit platelet aggregation and secretion in response to low concentrations of collagen, thrombin, or PAF were inhibited by ethanol. Inhibition of the effects of thrombin and PAF was also observed with aspirin-treated platelets. Thus, in addition to inhibiting the mobilization of arachidonate for thromboxane formation that occurs with most agonists, ethanol can also inhibit aggregation and secretion through other effects on platelet responses.

In Vitro Effects of Ethanol on Rabbit Platelet Aggregation, Secretion of Granule Contents, and Cyclic AMP Levels in the Presence of Prostacyclin

1989 ◽  
Vol 61 (02) ◽  
pp. 254-258 ◽  
Author(s):  
Margaret L Rand ◽  
Peter L Gross ◽  
Donna M Jakowec ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Cyclic Amp ◽  
Inhibitory Effect ◽  
Rabbit Platelet ◽  
Inhibitory Effects ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
In Vitro Effects ◽  
Aggregation Of Platelets

SummaryEthanol, at physiologically tolerable concentrations, inhibits platelet responses to low concentrations of collagen or thrombin, but does not inhibit responses of washed rabbit platelets stimulated with high concentrations of ADP, collagen, or thrombin. However, when platelet responses to high concentrations of collagen or thrombin had been partially inhibited by prostacyclin (PGI2), ethanol had additional inhibitory effects on aggregation and secretion. These effects were also observed with aspirin- treated platelets stimulated with thrombin. Ethanol had no further inhibitory effect on aggregation of platelets stimulated with ADP, or the combination of ADP and epinephrine. Thus, the inhibitory effects of ethanol on platelet responses in the presence of PGI2 were very similar to its inhibitory effects in the absence of PGI2, when platelets were stimulated with lower concentrations of collagen or thrombin. Ethanol did not appear to exert its inhibitory effects by increasing cyclic AMP above basal levels and the additional inhibitory effects of ethanol in the presence of PGI2 did not appear to be brought about by further increases in platelet cyclic AMP levels.

Effects of Bupranolol, a New β-Blocker, on Platelet Functions of Rabbit and Human in Vitro

1976 ◽  
Vol 36 (02) ◽  
pp. 376-387 ◽  
Author(s):  
Teruhiko Umetsu ◽  
Kazuko Sanai ◽  
Tadakatsu Kato
Keyword(s):  
Platelet Aggregation ◽  
Platelet Adhesion ◽  
Human Platelets ◽  
Rabbit Platelet ◽  
Rabbit Platelets ◽  
Β Blocker ◽  
Platelet Aggregates ◽  
Induced Aggregation ◽  
Platelet Functions

SummaryThe effects of bupranolol, a new β-blocker, on platelet functions were investigated in vitro in rabbits and humans as compared with propranolol, a well-known β-blocker. At first, the effect of adrenaline on ADP-induced rabbit platelet aggregation was studied because adrenaline alone induces little or no aggregation of rabbit platelets. Enhancement of ADP-induced rabbit platelet aggregation by adrenaline was confirmed, as previously reported by Sinakos and Caen (1967). In addition the degree of the enhancement was proved to be markedly affected by the concentration of ADP and to increase with decreasing concentration of ADP, although the maximum aggregation (percent) was decreased.Bupranolol and propranolol inhibited the (adrenaline-ADP-)induced aggregation of rabbit platelets, bupranolol being approximately 2.4–3.2 times as effective as propranolol. Bupranolol stimulated the disaggregation of platelet aggregates induced by a combination of adrenaline and ADP, but propranolol did not. Platelet adhesion in rabbit was also inhibited by the β-blockers and bupranolol was more active than propranolol. With human platelets, aggregation induced by adrenaline was inhibited by bupranolol about 2.8–3.3 times as effectively as propranolol.From these findings. We would suggest that bupranolol might be useful for prevention or treatment of thrombosis.

Testosterone Potentiation of Ionophore and ADP Induced Platelet Aggregation : Relationship to Arachidonic Acid Metabolism

1981 ◽  
Vol 46 (02) ◽  
pp. 538-542 ◽  
Author(s):  
R Pilo ◽  
D Aharony ◽  
A Raz
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Unsaturated Fatty Acids ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Arachidonic Acid Metabolism ◽  
Ionophore A23187 ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryThe role of arachidonic acid oxygenated products in human platelet aggregation induced by the ionophore A23187 was investigated. The ionophore produced an increased release of both saturated and unsaturated fatty acids and a concomitant increased formation of TxA2 and other arachidonate products. TxA2 (and possibly other cyclo oxygenase products) appears to have a significant role in ionophore-induced aggregation only when low concentrations (<1 μM) of the ionophore are employed.Testosterone added to rat or human platelet-rich plasma (PRP) was shown previously to potentiate platelet aggregation induced by ADP, adrenaline, collagen and arachidonic acid (1, 2). We show that testosterone also potentiates ionophore induced aggregation in washed platelets and in PRP. This potentiation was dose and time dependent and resulted from increased lipolysis and concomitant generation of TxA2 and other prostaglandin products. The testosterone potentiating effect was abolished by preincubation of the platelets with indomethacin.

Pretreatment of Human Platelets with Plasmin Inhibits Responses toThrombin, but Potentiates Responses to Low Concentrations of Aggregating Agents, Including the Thrombin Receptor Activating Peptide, SFLLRN

1997 ◽  
Vol 77 (04) ◽  
pp. 741-747 ◽  
Author(s):  
R L Kinlough-Rathbone ◽  
D W Perry ◽  
M L Rand ◽  
M A Packham
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activating Factor ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Fibrinolytic Agents ◽  
Albumin Solution ◽  
Fibrinogen Receptor ◽  
Low Concentrations ◽  
Induced Aggregation

SummaryEffects of plasmin on platelets, that influence subsequent responses to aggregating agents, are relevant to attempts to prevent rethrombosis following administration of fibrinolytic agents. We describe plasmin-induced inhibition of platelet responses to thrombin, but potentiation of responses to other aggregating agents. Washed human platelets were labeled with 14C-serotonin, treated for 30 min at 37° C with 0, 0.1 or 0.2 CU/ml of plasmin, followed by aprotinin, washed and resuspended in a Tyrode-albumin solution with apyrase. Incubation with 0.2 CU/ml of plasmin almost completely inhibited thrombin-induced (0.1 U/ml) aggregation, release of 14C-serotonin, and increase in cytosolic [Ca2+]. In contrast, with plasmin-pretreated platelets, aggregation and release of 14C-serotonin were strongly potentiated in response to low concentrations of the thrombin receptor-activating peptide SFLLRN, ADP, platelet-activating factor, collagen, arachidonic acid, the thromboxane mimetic U46619, and the calcium ionophores A23187 and ionomycin. Aspirin or RGDS partially inhibited potentiation. Plasmin-pretreated platelets resuspended in plasma anticoagulated with FPRCH2C1 (PPACK) also showed enhanced responses to aggregating agents other than thrombin. The contrasting effects on responses to thrombin and SFLLRN are noteworthy. Plasmin cleaves GPIIb/IIIa so that it becomes a competent fibrinogen receptor, and binding of 125I-fibrinogen during ADP-induced aggregation was greatly potentiated within 10 s. Potentiation of aggregation by other agonists may be due to increased binding of released fibrinogen. Thus, platelets freed from a thrombus may have increased responsiveness to low concentrations of aggregating agents other than thrombin. These results provide further support for the use of inhibitors of platelet reactions in conjunction with administration of fibrinolytic agents.

scholarly journals Effects of a Polyoxyethylene Detergent on Platelet Function

1977 ◽  
Author(s):  
P.G. Barton
Keyword(s):  
Lactate Dehydrogenase ◽  
Platelet Rich Plasma ◽  
Secondary Phase ◽  
Human Platelets ◽  
Brij 58 ◽  
Low Concentrations ◽  
High Concentrations ◽  
Glass Surfaces ◽  
Induced Aggregation ◽  
Membrane Destabilization

Low concentrations of a polyoxyethylene detergent, Brij 58, inhibited the secondary phase of platelet aggregation induced by ADP in human citrated platelet rich plasma but had no effect on primary aggregation.Thrombin-induced aggregation of washed human platelets suspended in Tyrode’s buffer was inhibited after incubation of cells with 4.5 × 10-6M detergent. Development of prothrombin-converting activity and efflux of [14C]-serotonin, 45Ca2+ ions and labile endoperoxides were abolished concomitantly. Aggregation of washed platelets by collagen or sodium arachidonate and the attachment of cells to clean glass surfaces were also inhibited by the same concentration of Brij 58 that inhibited thrombin aggregation. This concentration of Brij 58 did not itself produce any release of a cytoplasmic marker, lactate dehydrogenase, from platelets. Higher concentrations of Brij 58, exceeding 10-4 M, lysed the cells liberating all of their serotonin, Ca2+ and lactate dehydrogenase. These results suggest that low concentrations of Brij 58 stabilize a membrane conformation against the action of platelet stimulatory agents while high concentrations produce membrane destabilization and cell lysis. The presence of albumin (BSA) in the suspending fluid increased by tenfold the concentrations of detergent required to “elicit these effects and this could be attributed to competitive binding of the detergent to albumin, demonstrated with [14C]-acetylated Brij 58.

Inhibition Of Platelet Aggregation, Malonyldialdehyde And Thromboxane Formation By Hydroperoxides Of Arachidonic Acid

1981 ◽  
Author(s):  
D Aharonv ◽  
J B Smith ◽  
M J Silver
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Lipoxygenase Pathway ◽  
Dose Dependent Fashion ◽  
Induced Aggregation ◽  
Layer Chromatography ◽  
Dose Dependent ◽  
Thromboxane Formation

The arachidonate hydroperoxides 12-HPETE and 15-HPETE were biosynthesized from arachidonic acid using partially purified human platelet lipoxygenase or soybean lipoxidase respectively, and isolated by thin layer chromatography. Both compounds inhibited the arachidonic acid- induced aggregation of washed human platelets, suspended in calcium-free Krebs Henseleit solution, in a dose dependent fashion at concentrations between 1 and 50 uM. No inhibition was seen with up to 100 uM of these hydroperoxides when platelet -rich plasma was used. 12-HPETE (in micromolar concentrations) inhibited the formation of both thromboxane B2 (radioimmunoassay) and malonyldialdehyde (spectrophotometrie assay) when washed platelets were incubated with arachidonic acid. The 12-hydroxide, 12-HETE also inhibited platelet aggregation and thromboxane formation, but was less potent than 12-HPETE. We suggest that arachidonate hydroperoxide generated in platelets via the lipoxygenase pathway modulates platelet aggregation induced by arachidonic acid by inhibiting thromboxane formation.

scholarly journals Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium

Blood ◽  
1987 ◽  
Vol 70 (3) ◽  
pp. 647-651 ◽  
Author(s):  
MA Packham ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Calcium Salt ◽  
Close Contact ◽  
Platelet Rich Plasma ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Tyrode Solution ◽  
Low Concentration ◽  
Thromboxane Receptor ◽  
Aggregation Response

Abstract Close platelet-to-platelet contact induced by weak agonists in a medium with a low concentration of Ca2+ leads to thromboxane A2 (TXA2) formation, release of granule contents, and secondary aggregation. These responses do not occur in a medium containing Ca2+ in the physiological range (1 to 2 mmol/L). Experiments were done to determine whether feedback amplification is required to generate amounts of TXA2 that are sufficient to cause secondary aggregation and the reactions associated with it, or whether close platelet-to-platelet contact alone is sufficient to generate enough TXA2 to produce these responses. Platelets were washed and resuspended in a modified Tyrode solution to which no calcium salt was added that contained 0.35% albumin and apyrase. This medium contains 20 mumol/L Ca2+ and 1 mmol/L Mg2+. Platelets were aggregated with adenosine diphosphate (ADP) in the presence of fibrinogen, agglutinated with polylysine, or after pretreatment with chymotrypsin, aggregated with fibrinogen. In the low- Ca2+ medium, all these agonists caused platelets to adhere to each other, followed by secondary aggregation with TXA2 formation and release of granule contents. When Ca2+ (1 to 2 mmol/L), aspirin, or the thromboxane receptor blocker BM 13.177 was present, the secondary responses did not occur; dazoxiben decreased thromboxane formation, but did not prevent secondary aggregation or release. Aspirin-treated platelets were less responsive to ADP, U46619, or TXA2 in the low-Ca2+ medium, which indicated that the secondary responses of untreated platelets were not caused by a generalized increase in sensitivity. The reactions that result from close platelet-to-platelet contact in a low- Ca2+ medium can be caused by a wide variety of weak agonists; the secondary aggregation response and release of granule contents are dependent on TXA2 formation and on feedback amplification by TXA2 or the prostaglandin endoperoxides. The secondary responses caused by weak agonists in citrated platelet-rich plasma (which has a concentration of Ca2+ similar to the low-Ca2+ medium used in the present studies) do not occur at the concentration of Ca2+ in circulating blood and thus may have little biologic relevance.

Contrasting effects of rabbit and human platelets on chikungunya virus infectivity

1977 ◽  
Vol 23 (9) ◽  
pp. 1237-1244 ◽  
Author(s):  
M. A. Chernesky ◽  
R. P. B. Larke
Keyword(s):  
Electron Microscopy ◽  
Chikungunya Virus ◽  
Human Platelets ◽  
Heat Inactivation ◽  
Rabbit Platelet ◽  
Virus Infectivity ◽  
Stabilizing Effect ◽  
Rabbit Platelets ◽  
Section Electron ◽  
Platelet Aggregates

Chikungunya virus infectivity was markedly stabilized in the presence of washed suspensions of human platelets but rapidly disappeared in similar preparations of rabbit platelets. Supernatant fluids collected from human platelets had some stabilizing effect on chikungunya virus over a 6-day incubation period at 37 °C. Rabbit platelet supernatant fluid had no virus-stabilizing effect, nor did it demonstrate any capacity to inactivate virus as compared to whole rabbit platelet preparations. Thin-section electron microscopy demonstrated that chikungunya virus formed an association with human platelets by becoming entrapped in platelet aggregates; during this process some of the platelets appeared to have undergone degranulation and lysis. Rabbit platelets exposed to chikungunya virus for 24 h demonstrated a considerable amount of platelet degranulation and lysis but virus was not visualized eitherin association with platelet membranes or within phagocytic vacuoles in the platelet cytoplasm. Human platelets, which appear to be more stable under these incubation conditions, may protect chikungunya virus infectivity from heat inactivation by surrounding viruses with large platelet aggregates whereas rabbit platelets, which appear to be more fragile, do not afford this type of protection. Thus, chikungunya virus in the presence of rabbit platelets may become inactivated by heat or may become bound irreversibly to membranes in such a fashion that infectivity assay and electron microscopy techniques may prove to be too insensitive for detection of virus.

The role of PAR4 in thrombin-induced thromboxane production in human platelets

2003 ◽  
Vol 90 (08) ◽  
pp. 299-316 ◽  
Author(s):  
Chin-Chung Wu ◽  
Tsong-Long Hwang ◽  
Chang-Hui Liao ◽  
Sheng-Chu Kuo ◽  
Fang-Yu Lee ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Calcium Influx ◽  
Selective Inhibition ◽  
Therapeutic Benefit ◽  
Human Platelets ◽  
Agonist Peptide ◽  
Low Concentrations ◽  
Thromboxane Production ◽  
Thromboxane Formation

SummaryThere are two protease-activated receptors (PARs), PAR1 and PAR4, in human platelets. It has been suggested that PAR1 mediates platelet responses to low concentrations of thrombin, whereas PAR4 mediates signaling only at high concentrations. In the present study, we used a selective PAR4 blocker, YD-3, to investigate the role of PAR4 in thrombin-induced thromboxane formation in human platelets. YD-3 completely prevented thromboxane production by either a low concentration of thrombin (0.1 U/ml) or the PAR4 agonist peptide GYPGKF. In contrast, YD-3 did not affect thromboxane production caused by the PAR1 agonist peptide SFLLRN, collagen or arachidonic acid. YD-3 also decreased [3H]arachidonic acid release from thrombin-stimulated platelets. Moreover, desensitization of platelets with GYPGKF prevented low thrombin-induced thromboxane formation. The decreased thromboxane production by YD-3 is linked to inhibition of calcium influx in thrombin-stimulated platelets. These results suggest that PAR4 plays an important role in the regulation of thromboxane formation in platelets responding to thrombin through prolonged elevation of [Ca2+]i and activation of phospholipase A2. These data also indicate that PAR4 can be activated by relatively low concentrations of thrombin in human platelets. The selective inhibition of thrombin-induced thromboxane production by YD-3 may be of therapeutic benefit for thrombotic diseases.

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