Platelet ADP Receptors Stimulating Shape Change and Inhibiting Adenylate Cyclase

Physiology ◽  
1992 ◽  
Vol 7 (6) ◽  
pp. 274-278
Author(s):  
RW Colman
Keyword(s):  
Platelet Aggregation ◽  
Membrane Protein ◽  
Adenylate Cyclase ◽  
Shape Change ◽  
Surface Membrane ◽  
Thromboxane A2 ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Adp Receptors

Aggregin, a platelet surface membrane protein required for ADP-induced shape change, aggregation, and fibrinogen binding, is distinct from the receptor coupled to adenylate cyclase. Platelet aggregation by epinephrine, thromboxane A2, and collagen requires ADP binding to aggregin. Thrombin and plasmin are independent of ADP and activate platelet calpain, which cleaves aggregin.

scholarly journals Inhibition of collagen-induced platelet activation by 5'-p- fluorosulfonylbenzoyl adenosine: evidence for an adenosine diphosphate requirement and synergistic influence of prostaglandin endoperoxides

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 565-570 ◽  
Author(s):  
RW Colman ◽  
WR Figures ◽  
LM Scearce ◽  
AM Strimpler ◽  
FX Zhou ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Platelet Surface ◽  
Prostaglandin Endoperoxide ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Absolute Requirement

Abstract The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.

Recognition of platelet-associated fibrinogen by polyclonal antibodies: correlation with platelet aggregation

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2028-2033
Author(s):  
EI Peerschke
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Polyclonal Antibodies ◽  
Surface Membrane ◽  
Adenosine Diphosphate ◽  
Dependent Manner ◽  
Membrane Structures ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Qualitative Changes

Progressive decreases in platelet-bound fibrinogen accessibility to antibody and enzymes were recently reported to occur after adenosine diphosphate (ADP)-induced fibrinogen binding. Because previous studies also indicated that platelets that are activated but not aggregated by ADP in the presence of fibrinogen lose their ability to aggregate in a time-dependent manner despite negligible changes in fibrinogen binding, the present study examined the relationship between platelet aggregation and accessibility of platelet-bound fibrinogen to specific polyclonal antibody F(ab')2 fragments over a 60-minute time course. Although 125I-fibrinogen binding remained virtually unchanged, comparison of antifibrinogen antibody F(ab')2 binding and platelet aggregation 5 minutes and 60 minutes after platelet stimulation with ADP or thrombin showed decreases in F(ab')2 binding of 62% +/- 13% and 73% +/- 7% (mean +/- SD, n = 5), respectively, and decreases of 65% +/- 16% and 60% +/- 10% in platelet aggregation. In contrast, platelets stimulated with A23187 or chymotrypsin retained 87% +/- 16% and 76% +/- 12% of their ability to aggregate over the same time course, and lost only 39% +/- 14% and 36% +/- 12% of their ability to bind antifibrinogen antibody F(ab')2 fragments, respectively. Pretreatment of ADP-stimulated platelets with chymotrypsin largely prevented the progressive loss of platelet aggregability and the accompanying decreased recognition of bound fibrinogen by antifibrinogen F(ab')2 fragments. Preincubation of platelets with cytochalasin D (30 micrograms/mL) also inhibited the decrease in platelet aggregation after exposure of ADP-treated platelets to fibrinogen over a 60-minute time course. This was accompanied by only a 25% +/- 18% decrease in antifibrinogen antibody F(ab')2 binding. Present data support the hypothesis that qualitative changes in platelet-bound fibrinogen correlate with loss of the ability of platelets to aggregate, and implicate both the platelet cytoskeleton and chymotrypsin-sensitive surface membrane structures in modulating qualitative changes in bound fibrinogen on the platelet surface.

Proteolysis of the platelet surface: dissociation of shape change from aggregation

1986 ◽  
Vol 250 (4) ◽  
pp. H550-H557
Author(s):  
E. Kornecki ◽  
Y. H. Ehrlich ◽  
D. H. Hardwick ◽  
R. H. Lenox
Keyword(s):  
Adenylate Cyclase ◽  
Binding Sites ◽  
Prostaglandin E1 ◽  
Direct Evidence ◽  
Shape Change ◽  
Adenylate Cyclase System ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Induced Aggregation ◽  
Platelet Shape

Stimulation of intact platelets by ADP results in a shape change followed by aggregation in the presence of fibrinogen. ADP was found to induce a shape change in chymotrypsin-treated platelets that was similar in extent and initial velocity to that of intact (untreated) platelets. Scanning-electron microscopy verified an ADP-induced shape change in chymotrypsin-treated platelets. This shape change could be completely blocked by stimulators of platelet adenylate cyclase (forskolin, prostaglandin E1, and prostacyclin). On the other hand, the aggregation of chymotrypsin-treated platelets by fibrinogen was not dependent on the presence of ADP and could not be blocked by forskolin, prostaglandin E1, or prostacyclin, even though the levels of cyclic AMP (cAMP) formed in chymotrypsin-treated platelets were comparable to levels that completely inhibited the ADP-induced aggregation of intact platelets. This lack of inhibition of platelet aggregation was not due to degradation of the adenylate cyclase or prostaglandin receptors, since chymotrypsin-treated platelets were found to have a functional adenylate cyclase system that could be stimulated by forskolin, prostaglandin E1, and prostacyclin and inhibited by ADP and epinephrine, similar to that of intact platelets. These results provide direct evidence that cAMP does not interact with fibrinogen binding sites once they have become permanently exposed on the surface of platelets. Pretreatment of platelets with chymotrypsin therefore appears to be a useful tool that allows for the dissociation of platelet shape change from aggregation, without inhibiting either response.

scholarly journals Recognition of platelet-associated fibrinogen by polyclonal antibodies: correlation with platelet aggregation

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2028-2033 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Platelet Aggregation ◽  
Time Course ◽  
Polyclonal Antibodies ◽  
Surface Membrane ◽  
Adenosine Diphosphate ◽  
Dependent Manner ◽  
Membrane Structures ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Qualitative Changes

Abstract Progressive decreases in platelet-bound fibrinogen accessibility to antibody and enzymes were recently reported to occur after adenosine diphosphate (ADP)-induced fibrinogen binding. Because previous studies also indicated that platelets that are activated but not aggregated by ADP in the presence of fibrinogen lose their ability to aggregate in a time-dependent manner despite negligible changes in fibrinogen binding, the present study examined the relationship between platelet aggregation and accessibility of platelet-bound fibrinogen to specific polyclonal antibody F(ab')2 fragments over a 60-minute time course. Although 125I-fibrinogen binding remained virtually unchanged, comparison of antifibrinogen antibody F(ab')2 binding and platelet aggregation 5 minutes and 60 minutes after platelet stimulation with ADP or thrombin showed decreases in F(ab')2 binding of 62% +/- 13% and 73% +/- 7% (mean +/- SD, n = 5), respectively, and decreases of 65% +/- 16% and 60% +/- 10% in platelet aggregation. In contrast, platelets stimulated with A23187 or chymotrypsin retained 87% +/- 16% and 76% +/- 12% of their ability to aggregate over the same time course, and lost only 39% +/- 14% and 36% +/- 12% of their ability to bind antifibrinogen antibody F(ab')2 fragments, respectively. Pretreatment of ADP-stimulated platelets with chymotrypsin largely prevented the progressive loss of platelet aggregability and the accompanying decreased recognition of bound fibrinogen by antifibrinogen F(ab')2 fragments. Preincubation of platelets with cytochalasin D (30 micrograms/mL) also inhibited the decrease in platelet aggregation after exposure of ADP-treated platelets to fibrinogen over a 60-minute time course. This was accompanied by only a 25% +/- 18% decrease in antifibrinogen antibody F(ab')2 binding. Present data support the hypothesis that qualitative changes in platelet-bound fibrinogen correlate with loss of the ability of platelets to aggregate, and implicate both the platelet cytoskeleton and chymotrypsin-sensitive surface membrane structures in modulating qualitative changes in bound fibrinogen on the platelet surface.

scholarly journals Inhibition of collagen-induced platelet activation by 5'-p- fluorosulfonylbenzoyl adenosine: evidence for an adenosine diphosphate requirement and synergistic influence of prostaglandin endoperoxides

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 565-570
Author(s):  
RW Colman ◽  
WR Figures ◽  
LM Scearce ◽  
AM Strimpler ◽  
FX Zhou ◽  
...  
Keyword(s):  
Platelet Activation ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Adenosine Diphosphate ◽  
Platelet Surface ◽  
Prostaglandin Endoperoxide ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Absolute Requirement

The relative roles of platelet autacoids such as adenosine diphosphate (ADP), prostaglandin endoperoxides, and thromboxane A2 (TXA2) in collagen-induced platelet activation are not fully understood. We reexamined this relationship using the ADP affinity analogue, 5'-p- fluorosulfonylbenzoyl adenosine (FSBA), which covalently modifies a receptor for ADP on the platelet surface, thereby inhibiting ADP- induced platelet activation. Collagen-induced shape change, aggregation, and fibrinogen binding were each fully inhibited under conditions in which FSBA is covalently incorporated and could not be overcome by raising the collagen used to supramaximal concentrations. In contrast, TXA2 synthesis stimulated by collagen under conditions that produced maximum aggregation was only minimally inhibited by FSBA. Since covalent incorporation of FSBA has been previously shown to specifically inhibit ADP-induced activation of platelets, the present study supports the contention that ADP is required for collagen-induced platelet activation. Under similar conditions, indomethacin, an inhibitor of cyclooxygenase, inhibited collagen-induced shape change, indicating that endoperoxides and/or TXA2 also play a role in this response. Shape change induced by low concentrations (10 nmol/L) of the stable prostaglandin endoperoxide, azo-PGH2, was also inhibited by FSBA. These observations indicate a role for ADP in responses elicited by low concentrations of endoperoxides. However, at higher concentrations of azo-PGH2 (100 nmol/L), inhibition by FSBA could be overcome. Thus, the effect of collagen apparently has an absolute requirement for ADP for aggregation and fibrinogen binding and for both ADP and prostaglandins for shape change. Aggregation and fibrinogen binding induced by prostaglandin endoperoxides also required ADP as a mediator, but ADP is not absolutely required at high endoperoxide concentration to induce shape change.

The Effect of the Phospholipase Inhibitor Mepacrine on Platelet Aggregation, the Platelet Release Reaction and Fibrinogen Binding to the Platelet Surface

1981 ◽  
Vol 45 (03) ◽  
pp. 257-262 ◽  
Author(s):  
P D Winocour ◽  
R L Kinlough-Rathbone ◽  
J F Mustard
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Platelet Surface ◽  
Release Reaction ◽  
Fibrinogen Binding ◽  
Platelet Release

SummaryWe have examined whether inhibition by mepacrine of freeing of arachidonic acid from platelet phospholipids inhibits platelet aggregation to collagen, thrombin or ADP, and the release reaction induced by thrombin or collagen. Loss of arachidonic acid was monitored by measuring the amount of 14 C freed from platelets prelabelled with 14 C-arachidonic acid. Mepacrine inhibited 14 C loss by more than 80% but did not inhibit thrombin-induced platelet aggregation and had a small effect on release. ADP-induced platelet aggregation did not cause 14 C loss. Mepacrine inhibited ADP-induced platelet aggregation by inhibiting the association of fibrinogen with platelets during aggregation. The effect of mepacrine on fibrinogen binding could be considerably decreased by washing the platelets but the inhibition of 14 C loss persisted. Platelets pretreated with mepacrine and then washed show restoration of aggregation to collagen. Thus, mepacrine has two effects; 1. it inhibits phospholipases, 2. it inhibits fibrinogen binding. Freeing of arachidonic acid is not necessary for platelet aggregation or the release reaction.

Differential Ability of Agonists to Express Distinct Pools of Fibrinogen (Gpllb/llla) Receptors which Can Mediate the Aggregation of Human Platelets

1989 ◽  
Vol 62 (03) ◽  
pp. 955-961 ◽  
Author(s):  
Ian S Watts ◽  
Rebecca J Keery ◽  
Philip Lumley
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Surface Membrane ◽  
Blood Platelet ◽  
Human Platelets ◽  
Membrane Glycoprotein ◽  
Platelet Surface ◽  
Human Platelet Aggregation ◽  
Differential Ability ◽  
Blood Platelet Aggregation

SummaryWe have investigated the effect of two procedures that modify human platelet surface membrane glycoprotein (Gp) IIb and IIIa complexes upon whole blood platelet aggregation to a range of agonists. (A) Irreversible disruption of complexes by temporary (30 min) Ca2+-deprivation with EGTA at 37° C. (B) Binding of a monoclonal antibody M148 to the complex. EGTA exposure abolished aggregation to ADP, adrenaline and PAF. In contrast, full aggregation curves to collagen and U-46619 could still be established. EGTA exposure reduced M148 binding to platelets by 80%. Excess M148 abolished aggregation to ADP, PAF, collagen and U-46619. However, upon removal of unbound antibody from platelets full aggregation curves to collagen and U-46619 but not to ADP and PAF could be re-established. Thus human platelet aggregation to ADP, PAF and adrenaline appears absolutely dependent upon surface membrane GpIIb/IIIa complexes. In contrast, collagen and U-46619 cause expression of an additional distinct pool of Gp complexes inaccessible to EGTA and M148 in unstimulated platelets which is intimately involved in aggregation to these agonists.

THROMBOSPONDIN SPECIFICALLY INTERACTS WITH AMINO ACID SEQUENCES WITHIN THE A α- AND B β- CHAINS OF FIBRINOGEN

1987 ◽  
Author(s):  
Theresa Bacon-Baguley ◽  
Suzanne Kendra-Franczak ◽  
Daniel Walz
Keyword(s):  
Platelet Aggregation ◽  
Cyanogen Bromide ◽  
Gel Filtration ◽  
Amino Acid Sequences ◽  
Reverse Phase ◽  
Platelet Surface ◽  
Chain Component ◽  
Fibrinogen Binding ◽  
Platelet Aggregate ◽  
Single Peptide

Thrombospondin (TSP) is responsible for the secretion-dependent phase of platelet aggregation. The mechanism of this action is believed to be through the binding of TSP to fibrinogen, resulting in the stabilization of the platelet aggregate. It has been established that the binding of fibrinogen to the platelet surface is dependent upon peptide sequences present, respectively, in the Aa- and y-chains. We have hypothesized that the binding of TSP to fibrinogen is also dependent upon unique fibrinogen peptide sequences. To test this hypothesis we have examined the interaction of TSP and f.ih.r.inogen. using..a.-blat-b.inding assaLy of reduced fibrinogen, the separated fibrinogen chains, selected fibrinogen domains or peptides generated from cyanogen bromide cleaved chains. Iodinated TSP bound specifically to the Aα - and Bβ - chains. Binding to these chains was calcium independent, mutually exclusive and could be blocked either by preincubation of TSP with 9.4 μ M fibrinogen or by preincubation of fibrinogen with 1.1 nM thrombospondin. TSP bound to the D and DD plasmin fragment of fibrinogen; TSP interacted exclusively with the B-chain component of the DD fragment. The cyanogen bromide fragments of the separated Aα - and Bβ -chains were resolved through a combination of gel filtration and reverse-phase chromatography. TSP was found to bind to a single peptide within these fibrinogen chains. These studies demonstrate that thrombospondin interacts with at least two distinct sites on fibrinogen, and these sites differ from those already described for fibrinogen binding to platelets.

THROMBOXANE-A2 MEDIATES THE ACTION OF INOSITOL (1.4.5) TRISPHOSPHATE (IP3) IN SAPONIN-PERMEABILISED PLATELETS

1987 ◽  
Author(s):  
K S Authi ◽  
B J Evenden ◽  
E J Hornby ◽  
N Crawford
Keyword(s):  
Phospholipase A2 ◽  
Shape Change ◽  
Thromboxane A2 ◽  
Thromboxane B2 ◽  
Cyclooxygenase Inhibitors ◽  
Storage Site ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Thromboxane Receptor ◽  
Thromboxane Production

Inositol trisphosphate (IP3) has now been identified as an important intracellular second messenger that can initiate the release of Ca2+ from intracellular stores in a variety of cells, including platelets. We have studied the effects of IP3 on washed platelets permeabilised with saponin (12-14 μg/mi) which allows penetration into the cell of low M.Wt polar molecules. The permeabilised cells show normal responses to the agonists thrombin and collagen. The addition of IP (1-20 μM) after saponin treatment induces shape change, aggregation and secretion of preloaded [14C] 5HT. Concomitant with these responses, thromboxane is produced in a dose related manner. With 20 μM IP3 thromboxane B2 increases from basal levels of 5-4 ± 3-0 ng/ml to 140 ± 23 ng/ml. Both thromboxane production and the platelet responses induced by IP3 are inhibited by pretreatment with the cyclooxygenase inhibitors, indomethacin (EC50 50 μM) and aspirin (EC50 30 μM). Aggregation and secretion responses to IP3 are also inhibited by thromboxane B2 receptor agonists; EPO 92 (R. Jones, Edinburgh) and AH 23848 (Glaxo Ltd.). If Ca2+ EGTA buffers age used with permeabilised platelets to "lock" the cytosolic [Ca2+] at 0.1 μM, thromboxane production is reduced to the basal level. Intact platelets were labelled with Ca2+ (4h incubation) and after washing, resuspension and saponisation, IP3 induced the release of 20% of the cell associated Ca2+. The release was unaffected by pretreatment with antimycin and oligomycin indicating an gndoplasmic reticulum-lige storage site for the sequestered Ca2+. This IP3 -induced Ca2+ release was also not affected by pretreatment with either cyclooxygenase inhibitors or thromboxane receptor antagonists (EPO 92 and AH 23848). We believe these studies indicate that the action of IP3 in sagonised platelets involves release of intracellularly stored Ca2+, activation of phospholipase A2 and cyclooxygenase, and production of thromboxane A2. The release of thromboxane mediates and/or attenuates platelet responses by acting upon platelet surface receptors.

Extracellular fibrinogen binding protein, Efb, from Staphylococcus aureus binds to platelets and inhibits platelet aggregation

2004 ◽  
Vol 91 (04) ◽  
pp. 779-789 ◽  
Author(s):  
Oonagh Shannon ◽  
Jan-Ingmar Flock
Keyword(s):  
Platelet Aggregation ◽  
Potent Inhibitor ◽  
Specific Binding ◽  
Binding Protein ◽  
Dependent Manner ◽  
Platelet Surface ◽  
Putative Receptor ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Dose Dependent

Summary S. aureus produces and secretes a protein, extracellular fibrinogen binding protein (Efb), which contributes to virulence in wound infection. We have shown here that Efb is a potent inhibitor of platelet aggregation. Efb can bind specifically to platelets by two mechanisms; 1) to fibrinogen naturally bound to the surface of activated platelets and 2) also directly to a surface localized component on the platelets. This latter binding of Efb is independent of fibrinogen. The specific binding of Efb to the putative receptor on the platelet surface results in a stimulated, non-functional binding of fibrinogen in a dose dependent manner, distinct from natural binding of fibrinogen to platelets. The natural binding of fibrinogen to GPIIb/IIIa on activated platelets could be blocked by a monoclonal antibody against this integrin, whereas the Efb-mediated fibrinogen binding could not be blocked. The enhanced Efb-dependent fibrinogen binding to platelets is of a nature that does not promote aggregation of the platelets; instead it inhibits aggregation. The anti-thrombotic action of Efb may explain the effect of Efb on wound healing, which is delayed in the presence of Efb.

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