Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets

Platelets ◽  
1999 ◽  
Vol 10 (2) ◽  
pp. 159-168 ◽  
Author(s):  
James White ◽  
Marlys Krumwiede
Keyword(s):  
Chelating Agents ◽  
Human Platelets

scholarly journals Platelet Aggregation and Secretion: Effects of Anticoagulants and Divalent Cation Chelators

1977 ◽  
Author(s):  
B.A. Lages ◽  
H.J. Weiss
Keyword(s):  
Platelet Aggregation ◽  
Divalent Cation ◽  
Chelating Agents ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Concentration Addition ◽  
Single Wave ◽  
Cation Concentration ◽  
Aggregation Response ◽  
Low Levels

The aggregation and secretion responses of human platelets to ADP and epinephrine have been studied in citrated platelet-rich plasma (cPRP) and in heparinized platelet-rich plasma (hPRP) containing varying concentrations of divalent cation chelating agents. ADP induced a single wave of aggregation in hPRP while E induced biphasic aggregation in both hPRP and cPRP. Secretion of 14C-serotonin (5HT*) occurred in both hPRP and cPRP in response to ADP and E, but generally to a lesser extent in hPRP. The extent of 5HT* secretion in hPRP, relative to that in cPRP, decreased with increasing ADP concentration but remained essentially constant oyer the same range of E concentration. Addition of citrate or EDTA to hPRP produced a clear maximum in ADP-induced 5HT* secretion at levels of 5-20mM citrate or 2mM EDTA and, in some cases, was associated with the appearance of a biphasic aggregation response. With citrate, but not with EDTA, the chelator concentration corresponding to maximum secretion varied directly with the concentration of ADP. These results indicate that ADP-induced secretion is a function of divalent cation concentration but does not occur only at the low levels of divalent cation present in cPRP and that divalent cation may be more directly associated with ADP-platelet interactions than in interactions of platelets and E.

Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets

Platelets ◽  
1999 ◽  
Vol 10 (2-3) ◽  
pp. 159-168 ◽  
Author(s):  
James G. White ◽  
Marlys D. Krumwiede
Keyword(s):  
Chelating Agents ◽  
Human Platelets

Influence of intracellular chelating agents on formation of spike-like pseudopods by human platelets

Platelets ◽  
1999 ◽  
Vol 10 (2) ◽  
pp. 159-168
Author(s):  
James G. White ◽  
Marlys D. Krumwiede
Keyword(s):  
Chelating Agents ◽  
Human Platelets

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.

Defects of Platelet Function Recognized by X-Ray Imaging and Scanning Electron Microscopy

1985 ◽  
Vol 43 ◽  
pp. 610-613
Author(s):  
M.G. Baldini ◽  
S. Morinaga ◽  
D. Minasian ◽  
R. Feder ◽  
D. Sayre ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Cell Biology ◽  
Imaging Technique ◽  
Human Platelets ◽  
X Rays ◽  
X Ray ◽  
X Ray Imaging ◽  
Complex Phenomena ◽  
Scanning Electron

Contact X-ray imaging is presently developing as an important imaging technique in cell biology. Our recent studies on human platelets have demonstrated that the cytoskeleton of these cells contains photondense structures which can preferentially be imaged by soft X-ray imaging. Our present research has dealt with platelet activation, i.e., the complex phenomena which precede platelet appregation and are associated with profound changes in platelet cytoskeleton. Human platelets suspended in plasma were used. Whole cell mounts were fixed and dehydrated, then exposed to a stationary source of soft X-rays as previously described. Developed replicas and respective grids were studied by scanning electron microscopy (SEM).

In Vivo Monitoring of Human Platelets in a Humanised Rag2−/− γ-chain−/− Mouse Model

2015 ◽  
Vol 63 (S 01) ◽  
Author(s):  
C. Heim ◽  
S. Müller ◽  
B. Weigmann ◽  
M. Ramsperger-Gleixner ◽  
N. Koch ◽  
...  
Keyword(s):  
Mouse Model ◽  
Human Platelets ◽  
In Vivo Monitoring

Functional Characterization of PM6/13, a β3-specific (GPIIIa/CD61) Monoclonal Antibody that Shows Preferential Inhibition of Fibrinogen Binding over Fibronectin Binding to Activated Human Platelets

1998 ◽  
Vol 79 (01) ◽  
pp. 177-185 ◽  
Author(s):  
Ashia Siddiqua ◽  
Michael Wilkinson ◽  
Vijay Kakkar ◽  
Yatin Patel ◽  
Salman Rahman ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Platelet Rich Plasma ◽  
Functional Characterization ◽  
Human Platelets ◽  
Western Blots ◽  
Activated Platelets ◽  
Reducing Conditions ◽  
Fibronectin Binding

SummaryWe report the characterization of a monoclonal antibody (MAb) PM6/13 which recognises glycoprotein IIIa (GPIIIa) on platelet membranes and in functional studies inhibits platelet aggregation induced by all agonists examined. In platelet-rich plasma, inhibition of aggregation induced by ADP or low concentrations of collagen was accompanied by inhibition of 5-hydroxytryptamine secretion. EC50 values were 10 and 9 [H9262]g/ml antibody against ADP and collagen induced responses respectively. In washed platelets treated with the cyclooxygenase inhibitor, indomethacin, PM6/13 inhibited platelet aggregation induced by thrombin (0.2 U/ml), collagen (10 [H9262]g/ml) and U46619 (3 [H9262]M) with EC50 = 4, 8 and 4 [H9262]g/ml respectively, without affecting [14C]5-hydroxytryptamine secretion or [3H]arachidonate release in appropriately labelled cells. Studies in Fura 2-labelled platelets revealed that elevation of intracellular calcium by ADP, thrombin or U46619 was unaffected by PM6/13 suggesting that the epitope recognised by the antibody did not influence Ca2+ regulation. In agreement with the results from the platelet aggregation studies, PM6/13 was found to potently inhibit binding of 125I-fibrinogen to ADP activated platelets. Binding of this ligand was also inhibited by two other MAbs tested, namely SZ-21 (also to GPIIIa) and PM6/248 (to the GPIIb-IIIa complex). However when tested against binding of 125I-fibronectin to thrombin stimulated platelets, PM6/13 was ineffective in contrast with SZ-21 and PM6/248, that were both potent inhibitors. This suggested that the epitopes recognised by PM6/13 and SZ-21 on GPIIIa were distinct. Studies employing proteolytic dissection of 125I-labelled GPIIIa by trypsin followed by immunoprecipitation with PM6/13 and analysis by SDS-PAGE, revealed the presence of four fragments at 70, 55, 30 and 28 kDa. PM6/13 did not recognize any protein bands on Western blots performed under reducing conditions. However Western blotting analysis with PM6/13 under non-reducing conditions revealed strong detection of the parent GP IIIa molecule, of trypsin treated samples revealed recognition of an 80 kDa fragment at 1 min, faint recognition of a 60 kDa fragment at 60 min and no recognition of any product at 18 h treatment. Under similar conditions, SZ-21 recognized fragments at 80, 75 and 55 kDa with the 55kDa species persisting even after 18 h trypsin treatment. These studies confirm the epitopes recognised by PM6/13 and SZ-21 to be distinct and that PM6/13 represents a useful tool to differentiate the characteristics of fibrinogen and fibronectin binding to the GPIIb-IIIa complex on activated platelets.

Potentiation by Adrenaline of Ca2+ Influx and Mobilization in Stimulated Human Platelets: Dissociation from Thromboxane Generation and Aggregation

1988 ◽  
Vol 59 (02) ◽  
pp. 212-215 ◽  
Author(s):  
M J Powling ◽  
R M Hardisty
Keyword(s):  
Human Platelets ◽  
The Other ◽  
Fibrinogen Binding ◽  
Thromboxane Production

SummaryIn a medium containing 1 mM extracellular Ca2+ (Ca2+o), the prior addition of 0.5 pM adrenaline to quin 2-loaded human platelets increased both the rate and amplitude of the rise in cytosolic free Ca2+ (Ca2+i) in response to sub-threshold concentrations of thrombin and PAF and these effects were not prevented by blocking either fibrinogen binding and aggregation or cyclo-oxygenase. In the presence of 2 mM EGTA ([Ca2+o] >100 nM), the rate, but not the extent of rise of [Ca2+i] was enhanced by adrenaline, and this was also unaffected by blockade of cyclo-oxygenase. Addition of adrenaline 1 min after the other agonist in the presence of 1 mM Ca2+o resulted in aggregation without further elevation of [Ca2+i]. Adrenaline thus enhances both influx and intracellular mobilization of Ca2+ by a mechanism independent of both fibrinogen binding and thromboxane production, but these effects do not fully explain its potentiation of aggregation by other agonists

Ticlopidine Facilitates the Deaggregation of Human Platelets Aggregated by Thrombin

1994 ◽  
Vol 71 (01) ◽  
pp. 091-094 ◽  
Author(s):  
M Cattaneo ◽  
B Akkawat ◽  
R L Kinlough-Rathbone ◽  
M A Packham ◽  
C Cimminiello ◽  
...  
Keyword(s):  
Cardiovascular Disease ◽  
Platelet Aggregation ◽  
Prostaglandin E1 ◽  
Human Platelet ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Before And After ◽  
Normal Human ◽  
Platelet Aggregates ◽  
Induced Aggregation

SummaryNormal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80–90% 14C-serotinin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5× the activity of thrombin) and PGE1 (10 μmol/1) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 μmol/1) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.

Monoamine Oxidase and Other Mitochondrial Enzymes in Density Subpopulations of Human Platelets

1988 ◽  
Vol 59 (01) ◽  
pp. 029-033 ◽  
Author(s):  
K G Chamberlain ◽  
D G Penington
Keyword(s):  
Monoamine Oxidase ◽  
Protein Concentration ◽  
Close Correlation ◽  
Human Platelets ◽  
Mitochondrial Enzymes ◽  
Density Fractions ◽  
Percoll Gradients ◽  
Normal Human ◽  
The Mean ◽  
Very High

SummaryNormal human platelets have been separated according to density on continuous Percoll gradients and the platelet distribution divided into five fractions containing approximately equal numbers of platelets. The mean volumes and protein contents of the platelets in each fraction were found to correlate positively with density while the protein concentration did not differ significantly between the fractions. Four mitochondrial enzymes (monoamine oxidase, glutamate dehydrogenase, cytochrome oxidase and NADP-dependent isocitrate dehydrogenase) were assayed and their activities per unit volume were found to increase in a very similar monotonie fashion with platelet density. When MAO and GDH were assayed on the same set of density fractions the correlation between the two activities was very high (r = 0.94–1.00, p <0.001) and a similar close correlation was found between MAO and ICDH. The results support the hypothesis that high density platelets either have a higher concentration of mitochondria or have larger mitochondria than low density platelets.

Sign in / Sign up

Export Citation Format

Share Document