DIFFERENTIAL EFFECT OF TEMPORARY Ca2+ DEPRIVATION UPON ADP-AND COLLAGEN-INDUCED PLATELET AGGREGATION IN HUMAN WHOLE BLOOD

1987 ◽  
Author(s):  
I S Watts ◽  
R J Keery ◽  
P Lumley
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Chelating Agents ◽  
Human Platelets ◽  
Significant Loss ◽  
Response Curves ◽  
Human Whole Blood ◽  
Fibrinogen Receptor ◽  
Receptor Sites ◽  
The Right

In platelets exposed to chelating agents at 37°C and recalcified, aggregation to ADP, adrenaline, collagen and thrombin were either abolished or markedly reduced (1). This phenomenon has been suggested to be caused by disruption, by Ca2+ deprivation, of the fibrinogen receptor (glycoprotein lib Ilia complex) on the surface of the platelet (2). This effect was dependent on the exposure time of Ca2+ chelators and was reduced at lower temperatures. We have investigated the effect of exposure of human platelets to EGTA upon aggregation to a range of agonists at both 37 and 25°C. EGTA (5 mM) was incubated with citrated (12.9 mM) whole blood for either 0, 1, 5, 15 or 30 min. One minute following recalcification (5 mM CaCl2), full aggregation concentration-response curves were constructed to ADP and adrenaline (0.1 to 3 μM), PAF (3 to 100 nM), collagen (0.1 to 4 μg/ml) and U46619 (0.03 to 1 μM), using a platelet counting technique (3). At 25°C, EGTA produced little or no significant loss of sensitivity to any agonist. At 37°C marked rightward shifts of the ADP, adrenaline and PAF aggregation curves occurred which were related to the time of incubation with EGTA (e.g. ADP concentration ratios (CR) of 2.0 (0.8-4.8), 4.7 (2.1-10.7), › 186.6 (135.2-257.6) (95% confidence intervals n=4) obtained at 1, 5 and 15 min respectively). Following 30 min incubation aggregation to all three agonists was abolished (up to ADP 300 pM; adrenaline 100 μM; PAF 10 μM). In contrast, whilst collagen and U46619 concentration-effect curves were displaced to the right following 30 minutes exposure to EGTA (CR =13.6 (6.1-30.4) and 9.0 (4.3-18.7) respectively, n=4) full aggregation curves could still be established. Furthermore, a 60 min incubation with EGTA caused little further effect. Our findings suggest platelet agonists such as collagen and U46619, but not ADP, adrenaline and PAF, can evoke expression of a population of fibrinogen receptor sites involved in platelet aggregation that are inaccessible to EGTA.(1) Zucker, MB & Grant, RA (1978). Blood, 52, 505. (2)Shattil, SJ et al. (1985). Blood, 66, 92. (3) Lumley, P & Humphrey, PPA (1981). J. Pharm. Methods, 6, 153.

Endotoxin-Induced Platelet Activation in Human Whole Blood In Vitro

1988 ◽  
Vol 59 (03) ◽  
pp. 378-382 ◽  
Author(s):  
Gyorgy Csako ◽  
Eva A Suba ◽  
Ronald J Elin
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Atp Release ◽  
Human Platelets ◽  
Lag Phase ◽  
Human Whole Blood ◽  
Dose Dependent

SummaryThe effect of purified bacterial endotoxin was studied on human platelets in vitro. In adding up to 1 μg/mL of a highly purified endotoxin, we found neither aggregation nor ATP release in heparinized or citrated human platelet-rich plasma. On the other hand, endotoxin at concentrations as low as a few ng/mL (as may be found in septic patients) caused platelet aggregation in both heparinized and citrated human whole blood, as monitored by change in impedance, free platelet count, and size. Unlike collagen, the platelet aggregation with endotoxin occurred after a long lag phase, developed slowly, and was rarely coupled with measurable release of ATP. The platelet aggregating effect of endotoxin was dose-dependent and modified by exposure of the endotoxin to ionizing radiation. Thus, the activation of human platelets by “solubilized” endotoxin in plasma requires the presence of other blood cells. We propose that the platelet effect is mediated by monocytes and/or neutrophils stimulated by endotoxin.

NEW INSIGHTS INTO THE ANTIPLATELET ACTIVITY OF FORSKOLIN: ROLE OF PLASMA ADENOSINE AND SPECIES DIFFERENCES

1987 ◽  
Author(s):  
Kailash C Agarwal
Keyword(s):  
Platelet Aggregation ◽  
Adenylate Cyclase ◽  
Whole Blood ◽  
Inhibitory Action ◽  
Rat Plasma ◽  
Human Platelets ◽  
Nucleoside Transport ◽  
Antiplatelet Activity ◽  
Human Whole Blood ◽  
Plasma Adenosine

Forskolin stimulates adenylate cyclase by interacting with the catalytic subunit and inhibits platelet aggregation. This inhibition is greatly potentiated by adenosine (Ado) which stimulates adenylate cyclase through membrane-bound Ado receptors. Forskolin is 2-4 fold more potent as an inhibitor of collagen-induced rat platelet aggregation as compared to human platelets (IC50 values, in rat PRP, 0.5-0.8 μM; in human PRP, 1.5-2 μM). However, if the blood is pretreated with adenosine deaminase (ADA), an enzyme that degrades Ado to inosine, the inhibitory action of forskolin is greatly reduced producing similar effects both in human and rat PRPs (IC50, 2-3 μM) and whole blood (IC50, 4.6 μM). Both 5’-methylthioadenosine (MTA, 50-100 μM), an antagonist of Ado receptors, and 2’,5’-dideoxyadenosine (DDA, 100 μM), an inhibitor of adenylate cyclase, reverse the inhibition of platelet aggregation in rat PRP, whereas, no reversal is seen in human PRP. When Ado in the rat plasma is degraded by ADA pretreatment, DDA or MTA shows no reversal as seen in human PRP. The inhibitory action of forskolin (1-2 μM), which is only weakly inhibitory alone (<20%) in human whole blood, can be greatly potentiated (100% inhibition) by the inhibitors of nucleoside transport, dipyridamole (10 μM) or dilazep (2 μM). Only slight potentiation is seen in rat whole blood suggesting that rat plasma Ado levels are not affected significantly perhaps due to weakly active erythrocytic nucleoside transport system. Sato and Ui (In: Physiology and Pharmacology of Adenosine, Daly et al, Eds. Raven Press, 1983, 1-11), have shown that rat plasma contains much higher Ado levels (7.55 ± 0.51 pM) than human plasma (0.29 ± 0.08 μM). These studies demonstrate that plasma adenosine plays an important role in the forskolin antiplatelet activity which can be greatly potentiated in human whole blood by the clinically used drugs, dipyridamole and dilazep. (Supported by US PHS Grant CA 07340).

Mobilization of Fibrinogen Receptor Sites on Human Platelets Stimulated with ADP is Prevented by Prostacyclin

1979 ◽  
Author(s):  
J. Hawiger ◽  
S. Parkinson ◽  
S. Timmons
Keyword(s):  
Inhibitory Effect ◽  
Human Platelets ◽  
Affinity Constant ◽  
Human Fibrinogen ◽  
Fibrinogen Receptor ◽  
Receptor Sites ◽  
Plasma Factor ◽  
Potent Inhibitory Effect

Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from plasma proteins. Binding of 125I-fibrinogen to platelets not stimulated with ADP was low and unaffected by an excess of unlabel led fibrinogen. However, when platelets were stimulated with 4μM of ADP, there was an eightfold increase In the number of available binding sites for human fibrinogen, with affinity constant of 1.9 x 109M-1. This striking increase in fibrinogen receptor sites on human platelets was specific for ADP as contrasted to ATP, AMP, and adenosine. Prostacyclin (Prostaglandin I2, PGI2), a novel prostaglandin produced by the blood vessel wall, completely blocked this ADP-induced increase in fibrinogen receptor sites on human platelets. The effect of PGI2 was prompt and concentration dependent, reaching maximum at 10-9M. 6-keto PGF2 a stable derivative ot PGI2, did not have such an effect. Thus movement of fibrinogen receptor sites on human platelet membrane stimulated with ADP is prevented by PGI2. This represents a new biologic property of this vascular hormone and contributes to better understanding of its potent inhibitory effect in vitro and in vivo on ADP-induced platelet aggregation requiring mobilization of fibrinogen receptor.

Effect of prenylated flavonoids and chalcones isolated from Artocarpus species on platelet aggregation in human whole blood

2010 ◽  
Vol 64 (3) ◽  
pp. 365-369 ◽  
Author(s):  
Ibrahim Jantan ◽  
Yusyila H. Mohd Yasin ◽  
Shajarahtunnur Jamil ◽  
Hasnah Sirat ◽  
Norazah Basar
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Human Whole Blood ◽  
Prenylated Flavonoids

Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity

1995 ◽  
Vol 74 (06) ◽  
pp. 1551-1556 ◽  
Author(s):  
Lisa K Jennings ◽  
Melanie M White ◽  
Timothy D Mandrell
Keyword(s):  
Platelet Aggregation ◽  
Conformational Changes ◽  
Low Dose ◽  
Species Differences ◽  
Receptor Occupancy ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Functional Aspects

SummaryWe examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

Binding of plasma factor XIII to thrombin-receptor activated human platelets

2009 ◽  
Vol 102 (07) ◽  
pp. 83-89 ◽  
Author(s):  
Béla Nagy ◽  
Zsuzsa Simon ◽  
Zsuzsa Bagoly ◽  
László Muszbek ◽  
János Kappelmayer
Keyword(s):  
Whole Blood ◽  
Factor Xiii ◽  
Coagulation Factor ◽  
Human Platelets ◽  
Thrombin Receptor ◽  
Type I ◽  
Fibrinogen Receptor ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Plasma Factor

SummaryPlatelet-bound coagulation factor XIII (FXIII) is targeted and concentrated at the site where platelet-rich thrombi are formed. Previous studies were in disagreement about the nature of FXIII binding to platelets. In this study, thrombin-receptor activating peptide (TRAP)-stimulated human whole blood and washed platelets were analysed by flow cytometry for the binding of FXIII using a monoclonal antibody against the A subunit of FXIII (FXIII-A). Here, we demonstrate that FXIII-A positivity significantly increased on activated platelets in whole blood compared to unstimulated sample, but not in washed platelets. GPIIb/IIIa receptor plays an essential role in FXIII binding, as fibrinogen receptor antagonist eptifibatide and fibrinogen binding inhibitor RGDS tetrapeptide significantly prevented the binding of FXIII. Furthermore, stimulated platelets from a patient with severe type I Glanzmann thrombasthenia showed insignificant FXIII-A positivity versus healthy controls. In addition, basal negligible amount of FXIII on washed platelets was only slightly increased when highly purified plasma FXIII (FXIII-A2B2) was added upon platelet activation by TRAP. Similarly, no remarkable FXIII-A positivity was observed when we used FXIII-A2B2 with γA/γA fibrinogen. However, γA/γ' fibrinogen significantly augmented FXIII binding on TRAP-stimulated platelets in the presence of non-activated FXIII-A2B2. We conclude that FXIII-A2B2 of plasma origin binds to thrombin-receptor activated platelets via GPIIb/IIIa receptor-bound fibrinogen with γ’-chain and is not capable of direct platelet binding.

Platelet aggregation in human whole blood after chronic administration of aspirin

1987 ◽  
Vol 46 (1) ◽  
pp. 133-140 ◽  
Author(s):  
J.P. De La Cruz ◽  
S. Camara ◽  
I. Bellido ◽  
T. Carrasco ◽  
F.Sanchez De La Cuesta
Keyword(s):  
Platelet Aggregation ◽  
Whole Blood ◽  
Chronic Administration ◽  
Human Whole Blood
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