Platelet sialic acid and platelet survival after aggregation by ADP

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 876-880
Author(s):  
MA Packham ◽  
MA Guccione ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Sialic Acid ◽  
Human Platelets ◽  
Platelet Surface ◽  
Release Reaction ◽  
Dense Granules ◽  
Total Sialic Acid ◽  
Platelet Survival ◽  
Rabbit Platelets ◽  
Induced Aggregation

Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP- induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.

scholarly journals Platelet sialic acid and platelet survival after aggregation by ADP

Blood ◽  
1980 ◽  
Vol 56 (5) ◽  
pp. 876-880 ◽  
Author(s):  
MA Packham ◽  
MA Guccione ◽  
RL Kinlough-Rathbone ◽  
JF Mustard
Keyword(s):  
Sialic Acid ◽  
Human Platelets ◽  
Platelet Surface ◽  
Release Reaction ◽  
Dense Granules ◽  
Total Sialic Acid ◽  
Platelet Survival ◽  
Rabbit Platelets ◽  
Induced Aggregation

Abstract Some investigators have reported recently that platelet surface sialic acid is decreased during ADP-induced aggregation, whereas others have reported an increase. Since removal of sialic acid from the platelet surface shortens platelet survival, we have determined the survival of platelets that have been aggregatad by ADP. We have also measured the amount of sialic acid in the suspending fluid of platelets after ADP- induced aggregation. ADP-induced aggregation did not cause the loss of sialic acid from rabbit platelets (which do not undergo a release reaction in response to ADP) nor from washed human platelets in a medium containing physiologic concentrations of calcium in which granule contents are not released. In a medium without added calcium, ADP caused the release of 14C-serotonin (42.5% +/- 3%) from human platelets, but less than 4% of the sialic-acid-containing material was released. It seems likely that little of the releasable sialic acid of platelets is in the dense granules or the alpha-granules. Thrombin (5 U/ml) released 90.0% +/- 3.4% of the serotonin from human platelets but only 20.6% +/- 7.4% of the total sialic-acid-containing material. Neuraminidase removed 42.3% of the total sialic acid, presumably from the platelet surface. Rabbit platelets that had been aggregated by ADP and deaggregated survived normally when returned to the circulation. This observation also provides evidence that they had not lost membrane sialic acid during aggregation and deaggregation.

Effects of Inhibitors of ADP-induced Aggregation on Platelet Nucleo-sidediphosphate Kinase (NDK) Activity

1975 ◽  
Author(s):  
M. A. Packham ◽  
J. F. Mustard ◽  
M. A. Guccione ◽  
D. W. Perry
Keyword(s):  
Sulfonic Acid ◽  
Shape Change ◽  
Human Platelets ◽  
Phosphoryl Group ◽  
Platelet Surface ◽  
Rabbit Platelets ◽  
Adenosine Derivatives ◽  
Adp Receptor ◽  
Presence And Absence ◽  
Induced Aggregation

We showed previously (Am. J. Physiol. 227, 1143, 1974) that ADP reacts with NDK on the surface of platelets; when 14C-ADP is added to suspensions of washed rabbit platelets, it is partially converted to 14C-ATP in the suspending fluid. This is more difficult to show with human platelets because of their greater ATPase activity. With both species, -external ATP (10 μM), and other NTP’s at higher concentrations, enhance formation of “C-ATP form “C-ADP. All the NTP’s inhibit ADP-induced aggregation, possibly by acting as external donors of a phosphoryl group which, in their absence, is normally donated to ADP by platelets. Studies of other inhibitors have been done with suspensions -of washed rabbit platelets. The other NDP’s and AMP inhibit, ADP-induced shape change, aggregation, and conversion of 14C-ADP to 14C-ATP, probably by competition for the same site on NDK. AMP inhibits NDK both in the presence and absence of external ATP. Parachloromercuribenzene sulfonic acid has a similar effect. In contrast, prostaglandin E1 and hydroxylamine inhibit ADP-induced shape change and aggregation, but inhibit NDK only in the absence of external ATP, not in its presence. ADP-induced shape change without aggregation occurs in the presence of EGTA or EDTA but only half as much 14C-ADP is converted to 14C-ATP in the first minute. EGTA does not inhibit NDK in the presence of external ATP but EDTA does. Inhibition of NDK by adenosine was observed in some experiments but the adenosine derivatives 2-n-amylthioadenosino, 2-cyclopentylthioadenosine and 2-cyclophexylthioadenosine (Kohjin Co., Tokyo) inhibited strongly both in the presence and absence of external ATP; these compounds also inhibit ADP-induced aggregation. Thus all compounds tested which inhibit NDK also inhibit ADP-induced aggregation, indicating that NDK may be the ADP receptor on the platelet surface. Donation of a phosphoryl group from the platelets to ADP, catalyzed by this enzyme, may be responsible for initiating ADP-induced platelet aggregation.

Factors Influencing the Deaggregation of Human and Rabbit Platelets

1983 ◽  
Vol 49 (03) ◽  
pp. 162-167 ◽  
Author(s):  
R L Kinlough-Rathbone ◽  
J F Mustard ◽  
D W Perry ◽  
E Dejana ◽  
J-P Cazenave ◽  
...  
Keyword(s):  
Human Platelets ◽  
Release Reaction ◽  
Factors Influencing ◽  
Platelet Adherence ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe mechanisms involved in platelet deaggregation are unclear. Washed platelets from rabbits or humans aggregated by ADP can be deaggregated by EDTA or PGI2 if the release reaction has not occurred; during deaggregation 125I-fibrinogen dissociates from the platelets. Human platelets suspended in a medium without calcium undergo the release reaction during ADP-induced aggregation; EDTA, PGE, or PGI2 do not deaggregate these platelets although EDTA displaces much of the 125I-fibrinogen that associates with them during aggregation. Rabbit platelets aggregated by low concentrations of releaseinducing stimuli (sodium arachidonate, collagen or thrombin) can be deaggregated by EDTA, PGI2 or PGE1 and 125I-fibrinogen dissociates from them; with high concentrations of collagen or thrombin, deaggregation and dissociation of l25I-fibrinogen is slower. Human platelets that have undergone the release reaction in response to thrombin, collagen or a combination of sodium arachidonate and ADP are not readily deaggregated by EDTA or PGE1. Since aggregation and fibrinogen binding involving the glycoprotein IIb/IIIa complex are readily reversed by EDTA, and since Ca2+ is required for thrombospondin binding to activated platelets, there may be a third type of platelet-platelet adherence that is not disrupted by EDTA; this type of binding plays a greater role with human than with rabbit platelets.

Comparison Of The Major Membrane Glycoproteins Of Human, Rabbit And Rat Platelets

1981 ◽  
Author(s):  
B Toor ◽  
J L McGregor ◽  
K J Clemetson ◽  
L McGregor ◽  
M Dechavanne ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Surface Composition ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Neuraminidase Treatment ◽  
Platelet Surface ◽  
Rabbit Platelets ◽  
Rat Platelets

Rabbit and rat platelets have been extensively investigated under in vitro or in vivo conditions to try to understand the pathology of thrombosis in man. Here, surface-labelling techniques have been used to find out if the platelet surface has a similar composition in these two animals and in man or not. Human, rabbit and rat platelets were isolated, washed and surface-labelled by techniques specific for protein or for sugars (sialic acid or penultimate galactose/N-acetyl galactosamine residues). Labelled platelets were solubilized in sodium dodecyl sulphate and separated under reducing conditions on 7.5 % Laemmli polyacrylamide gels. Dried gels were exposed to film by fluorography or indirect autoradiography. Terminal Gal/Gal NAc residues (no neuraminidase treatment) were strongly labelled with rat and rabbit platelets compared to human platelets which labelled very poorly. Terminal sialic acid labelling with rat and rabbit platelets showed a weak labelling of a glycoprotein (GP) with the same M.Wt. as GPIb which is the most intensely labelled GP in man. However two GP (with rabbits) and one GP (in rats) were intensely labelled at a M.Wt. similar to that of GPIa in man. These GP had a different M.Wt. with terminal Gal/Gal NAc labelling. Bands with a similar M.Wt. to GPIIb and IIIa in man were strongly iodinated with rabbit platelets but with rat platelets only a single band at the position of GPIIb was strongly iodinated. These results strongly indicate that there are considerable differences in surface composition between rabbit, rat and human platelets.

Effect of Amino Sugars on Platelet Aggregation and on Fibrinogen Binding

1984 ◽  
Vol 52 (01) ◽  
pp. 075-080 ◽  
Author(s):  
Raelene L Kinlough-Rathbone ◽  
Marian A Packham ◽  
J Fraser Mustard
Keyword(s):  
Inhibitory Effect ◽  
Human Platelets ◽  
Primary Amines ◽  
Amino Sugars ◽  
Ionophore A23187 ◽  
Release Reaction ◽  
Fibrinogen Binding ◽  
Rabbit Platelets ◽  
High Concentrations ◽  
Induced Aggregation

SummaryThe amino sugars glucosamine, galactosamine and man- nosamine (30 mM) inhibited aggregation of human or rabbit platelets induced by ADP, collagen, thrombin, PAF or high concentrations of sodium arachidonate. 125I-fibrinogen binding during ADP-induced aggregation, and release of amine storage granule contents were also inhibited. Increasing the calcium concentration of the suspending medium to 5 mM did not overcome the inhibitory effect on the release reaction.The amino sugars deaggregated rabbit platelets that had been aggregated by ADP, collagen or thrombin, but deaggregated human platelets readily only when ADP was used as the aggregating agent. Fibrinogen-induced aggregation of chymotrypsin-treated platelets was blocked by the amino sugars. They did not inhibit platelet adherence to a collagen-coated glass surface, nor affect release of granule contents from the adherent platelets. Aggregation and release induced by low concentrations of sodium arachidonate or the divalent cation ionophore A23187 were potentiated, indicating that the effects of the amino sugars on platelets are more complex than simple inhibition of the lectinlike activity that becomes available on the surface of platelets that have undergone the release reaction. One of the effects of the amino sugars, however, is interference with the binding of fibrinogen to platelets. The effects of the amino sugars are shared by other primary amines.

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.

scholarly journals Pathways of Thrombin-Induced Aggregation and Release with Human and Rabbit Platelets

1977 ◽  
Author(s):  
R.L. Kinlough-Rathbone ◽  
D.W. Perry ◽  
M.A. Packham ◽  
J.F. Mustard
Keyword(s):  
Platelet Aggregation ◽  
Direct Effect ◽  
Thromboxane A2 ◽  
Human Platelets ◽  
The Third ◽  
Rabbit Platelets ◽  
Induced Aggregation

There are at least 3 mechanisms involved in thrombin-induced aggregation and release: (1) released ADP, (2) formation of thromboxane A2 and (3) a third mechanism(s). We have examined whether the third pathway is due to formation or release of a substance from platelets which affects other platelets. Washed human platelets were exposed to thrombin (2.5 u/ml) for 15 min at 37°C in the presence of indomethacin to block thromboxane A2 formation. Platelets were removed by centrifugation and the thrombin neutralized with hirudin or DFP. Addition of the superna te to washed human platelets prelabeled with 14C-serotonin caused platelet aggregation but release did not occur. Treatment of the supernate with apyrase, CP/CPK or dialysis abolished aggregation, indicating that the material was ADP. Thus, the mechanism by which thrombin induces aggregation and release with human platelets in the presence of agents which destroy ADP and block the formation of thromboxane A2 is a direct effect of thrombin on platelets and does not involve a substance freed from platelets. In contrast, when washed rabbit platelets were treated with thrombin in the presence of indomethacin and the released ADP was removed, material remained in the supernate which caused aggregation and release from washed rabbit platelets but was without effect on washed human platelets. The activity of this material (MW > 10,000) was not abolished by dialysis or boiling. Therefore rabbit platelets differ from human platelets because they have a mechanism in addition to released ADP, thromboxane A2 and the direct effect of thrombin on platelets that can cause aggregation and release.

EFFECTS OF CELL ADHESION PEPTIDE, Arg-Gly-Asp-Ser (RGDS), ON RESPONSES OF WASHED PLATELETS FROM HUMANS, RABBITS AND RATS

1987 ◽  
Author(s):  
E J Harfenist ◽  
M A Packham ◽  
J F Mustard
Keyword(s):  
Cell Adhesion ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Independent Component ◽  
Tyrode Solution ◽  
Significant Extent ◽  
Aggregation Mechanism ◽  
Rabbit Platelets ◽  
Induced Aggregation ◽  
Rat Platelets

Fibrinogen (Fbg) is a cofactor in the aggregation of human platelets, and washed platelets do not aggregate to a significant extent in response to ADP unless Fbg is added to the suspension; however, exogenous Fbg is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Since, with human platelets, Arg-Gly-Asp-Ser (RGDS) inhibits aggregation and the binding of 125I-Fbg to ADP-stimulated platelets, its effects on the responses of rabbit and rat platelets were studied in an attempt to elucidate the differences in Fbg requirements of platelets from the three species. Aggregation and Fbg binding were studied using washed platelets suspended in Tyrode solution containing albumin, apyrase and 2 mM Ca2+. 50 μM RGDS caused over 80% inhibition of the aggregation of human platelets stimulated with 9 yM ADP in the presence of 0.2 yM Fbg, but only 3-9% inhibition of the ADP-induced aggregation of rabbit or rat platelets regardless of whether exogenous Fbg was added. 50 yM RGDS also inhibited the aggregation of human platelets stimulated with thrombin (0.9 U/mL), but produced no more than 3% inhibition with rabbit or rat platelets. The binding of 125I-Fbg to ADP-stimulated human platelets was inhibited by 80-90% by 30 yM RGDS, but even at 50 μM, RGDS inhibited Fbg binding to rabbit or rat platelets by only 15-27%. The differences were due to the species of platelets, since, with both human and rabbit platelets, human Fbg could be replaced by rabbit Fbg without significantly changing the results. RGDS, added to human platelets that had been aggregated with thrombin, did not cause deaggregation, but did partially inhibit aggregation when added within 1 min; this inhibitory effect was less than when RGDS was added before thrombin, and decreased progressively as the length of time before the addition of RGDS was increased. These observations indicate a difference in aggregation mechanism between human platelets and those from rabbits and rats, and are consistent with a Fbg-independent component to the aggregation of rabbit and rat platelets.

MECHANISMS OF PLATELET AGGREGATION BY ACIDIC MUCOPOLYSACCHARIDE EXTRACTED FROM STICHOPUS JAPONICUS SELENKA IN HUMANS AND RABBITS

1987 ◽  
Author(s):  
J Z Li ◽  
E C-Y Lian
Keyword(s):  
Platelet Aggregation ◽  
Ammonium Sulfate ◽  
Human Platelets ◽  
Platelet Inhibitors ◽  
Rabbit Plasma ◽  
Human Fibrinogen ◽  
Stichopus Japonicus ◽  
Saturated Ammonium Sulfate ◽  
Rabbit Platelets ◽  
Induced Aggregation

It has been reported that acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus selenka) (SJAMP) induced the aggregation of human and animal platelets by an unknown mechanism, using platelet-rich plasma (prp) and washed human and rabbit platelets we studied the effects of storage, platelet inhibitors, and various plasmas and their fractions on SJAMP-induced platelet aggregation. we found that the lowest concentrations of SJAMP required for aggregation of human and rabbit platelets were 0.4 and 2 ug/ml respectively. The reactivity of human platelets to SJAMP decreased with time after drawing of blood; rabbit platelets did not show this phenomenon. Platelet inhibitors such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose, and EDTA inhibited by 50 to 100% the aggregation of human platelets induced by SJAMP; but these inhibitors had no effect on SJAMP-induced aggregation of rabbit platelets. Washed human and rabbit platelets were not aggregated by SJAMP. The aggregation of washed human platelets by SJAMP was restored completely by human or rabbit plasma, by human fibrinogen, or by 0 to 30% saturated ammonium sulfate fraction but not by serum. The aggregation of rabbit platelets by SJAMP could only be restored by rabbit plasma or serum, or by 50 to 60% saturated ammonium sulfate fraction. The data indicate that the mechanisms of aggregation of human and rabbit platelets by SJAMP are different. THe SJAMP-induced human platelet aggregation is dependent upon metabolism, release of ADP and the cyclooxygenase pathway requiring fibrinogen and Ca++. The aggregation of rabbit platelets induced by SJAMP is independent of metabolism, release of ADP and cyclooxygenase pathway, and does not require fibrinogen and Ca++, but needs certain protein(s) in the 50 to 60% saturated ammonium sulfate fraction of rabbit plasma.

Protease-activated receptor 1 (PAR1) signalling desensitization is counteracted via PAR4 signalling in human platelets

2011 ◽  
Vol 436 (2) ◽  
pp. 469-480 ◽  
Author(s):  
Knut Fälker ◽  
Linda Haglund ◽  
Peter Gunnarsson ◽  
Martina Nylander ◽  
Tomas L. Lindahl ◽  
...  
Keyword(s):  
Receptor Activation ◽  
Human Platelets ◽  
Dense Granule ◽  
Receptor Internalization ◽  
Functional Responses ◽  
Protease Activated Receptors ◽  
Dense Granules ◽  
Dense Granule Secretion ◽  
Granule Secretion ◽  
Induced Aggregation

PARs (protease-activated receptors) 1 and 4 belong to the family of G-protein-coupled receptors which induce both Gα12/13 and Gαq signalling. By applying the specific PAR1- and PAR4-activating hexapeptides, SFLLRN and AYPGKF respectively, we found that aggregation of isolated human platelets mediated via PAR1, but not via PAR4, is abolished upon homologous receptor activation in a concentration- and time-dependent fashion. This effect was not due to receptor internalization, but to a decrease in Ca2+ mobilization, PKC (protein kinase C) signalling and α-granule secretion, as well as to a complete lack of dense granule secretion. Interestingly, subthreshold PAR4 activation rapidly abrogated PAR1 signalling desensitization by differentially reconstituting these affected signalling events and functional responses, which was sufficient to re-establish aggregation. The lack of ADP release and P2Y12 receptor-induced Gαi signalling accounted for the loss of the aggregation response, as mimicking Gαi/z signalling with 2-MeS-ADP (2-methylthioadenosine-5′-O-diphosphate) or epinephrine (adrenaline) could substitute for intermediate PAR4 activation. Finally, we found that the re-sensitization of PAR1 signalling-induced aggregation via PAR4 relied on PKC-mediated release of both ADP from dense granules and fibrinogen from α-granules. The present study elucidates further differences in human platelet PAR signalling regulation and provides evidence for a cross-talk in which PAR4 signalling counteracts mechanisms involved in PAR1 signalling down-regulation.

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