Exposure of a Human Platelet Fibrinogen Receptor by ADP

1979 ◽  
Author(s):  
J. S. Bennett ◽  
G. Vilatre
Keyword(s):  
Platelet Aggregation ◽  
Silicone Oil ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Single Class ◽  
Human Fibrinogen ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Before And After

Fibrinosen is a cofactor for the aggregation of human platelets by ADP but its precise role is not known. In order to clarify the function of fibrinogen in platelet aggregation, we measured the binding of 125I-labeled human fibrinogen to gel-filtered human platelets before and after platelet emulation by ADP. Incubations were performed without stirring to prevent platelet aggregation and secretion. Platelet-bound and free 125I-fibrinogen were separated by centrifugaron of the platelets through silicone oil. Specific fibrinogen binding was that ibrinogen which could be displaced from the platelets by a 10-fold excess unlabeled fibrinogen. Specific fibrinogen binding required platelet stimulation by ADP and either Ca+2 or Mg+2. Specific ending reached equilibrium within 60 sec. Demonstrated saturation kinetics, and did not occur with thrombasthenic platelets. Scatchard analysis demonstrated a single class of ending sites with a Kd of 25 ± 3.9 ug/ml and 39,000 ± 5,000 binding sites per platlet the extent of ADP-induced fibrinogen binding to unstirred platelets was compared to the extent of aggregation of stirred platelets induced by the same concentrations of ADP, correlation 0.96 was seen. This study demonstrates. that a uniform population of fibrinogen receptors is exposed on the platelet surface by ADP. Furthermore, we suggest that the fibrinogen molecules bound to the platelet as a result of ADP stimulation are directly involved in the platelet aggregation response.

scholarly journals Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1224-1231
Author(s):  
SJ Shattil ◽  
HJ Motulsky ◽  
PA Insel ◽  
L Flaherty ◽  
LF Brass
Keyword(s):  
Platelet Aggregation ◽  
Adrenergic Receptors ◽  
Human Platelets ◽  
Receptor Expression ◽  
Platelet Surface ◽  
Prolonged Stimulation ◽  
Fibrinogen Receptor ◽  
Aggregation Response ◽  
Fibrinogen Binding ◽  
Response Expression

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or “desensitization” of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.

Role Of Prostaglandins In Platelet Fibrinogen Receptor Exposure

1981 ◽  
Author(s):  
J S Bennett ◽  
G Vilaire ◽  
J W Burch
Keyword(s):  
Platelet Aggregation ◽  
Blood Donation ◽  
Silicone Oil ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Membrane Receptors ◽  
Scatchard Analysis ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Serotonin Secretion

Fibrinogen binding to membrane receptors exposed by agonists such as ADP and thrombin is a prerequisite for platelet aggregation. However, a role for platelet prostaglandins in this process remains to be clarified. To assess whether platelet prostaglandins can expose fibrinogen receptors, we examined the effects of aspirin (ASA) and indomethacin on fibrinogen binding to ADP and epinephrine- stimulated platelets. Fibrinogen binding was measured by incubating gel-filtered human platelets (GFP) with 125I- labeled fibrinogen, CaCl2 and ADP or epinephrine at 37°C for 3 min without stirring. Free and platelet-bound I-fibrinogen were separated by rapid sedimentation of the platelets through silicone oil. The platelet release reaction, measured as 14C-serotonin secretion, did not occur under these conditions. Fibrinogen binding in response to l-2μM ADP or 10μM epinephrine was inhibited 40-60% by preincubation of platelet-rich plasma with 1 mM ASA for 20 min at 37° C. Similar results were produced by adding 25μM indomethacin to GFP. The inhibitory effect of ASA on ADP-stimulated fibrinogen binding could be overcome by increasing the ADP concentration while the inhibition of epinephrine-stimulated fibrinogen binding could not. However, stimulation of aspirin-treated platelets with both 10μM epinephrine and the prostaglandin endoperoxide PGH2(1μM) restored the extent of fibrinogen binding to that of control platelets stimulated by 10μM epinephrine alone. Scatchard analysis demonstrated that ASA decreased the number but not the affinity of the exposed fibrinogen binding sites. Identical results were obtained if ASA was ingested before blood donation. These studies demonstrate that prostaglandins synthesized in response to platelet stimulation can expose fibrinogen receptors on the platelet surface. Furthermore, these studies support the concept that platelet aggregation can occur through mechanisms independent of platelet ADP secretion.

Clustering of Integrin αIIb-β3Differently Regulates Tyrosine Phosphorylation of pp72syk, PLCγ2 and pp125FAKin Concanavalin A-stimulated Platelets

1999 ◽  
Vol 81 (01) ◽  
pp. 124-130 ◽  
Author(s):  
Enrico Festetics ◽  
Alessandra Bertoni ◽  
Fabiola Sinigaglia ◽  
Cesare Balduini ◽  
Mauro Torti
Keyword(s):  
Platelet Aggregation ◽  
Tyrosine Phosphorylation ◽  
Concanavalin A ◽  
Tyrosine Kinases ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Natural Ligand ◽  
Fibrinogen Binding

SummaryTyrosine phosphorylation of the non-receptor tyrosine kinases pp72sykand pp125FAKand of the γ2 isoform of phospholipase C (PLCγ2) in human platelets stimulated with the lectin Concanavalin A was investigated. Concanavalin A induced the rapid tyrosine phosphorylation of pp72sykand PLCγ2 with a similar kinetics, while tyrosine phosphorylation of pp125FAKoccurred in a later phase of platelet activation. When compared with other platelet agonists, Concanavalin A revealed to be at least as potent as collagen in inducing tyrosine phosphorylation of PLCγ2 and pp125FAK, while tyrosine phosphorylation of pp72sykinduced by the lectin was much stronger than that induced by thrombin or collagen. Concanavalin A-induced tyrosine phosphorylation of pp72syk, PLCγ2 and pp125FAKwas not dependent on platelet aggregation as it occurred normally even in the absence of sample stirring and when fibrinogen binding to integrin αIIb-β3was inhibited by the peptide RGDS. Tyrosine phosphorylation of pp72syk, PLCγ2 and pp125FAKrequired the binding of the lectin to the platelet surface, but was not observed in platelets treated with succinyl-Concanavalin A, a derivative of the lectin that interacts with the same receptors but does not promote clustering of membrane glycoproteins. Moreover, the aggregation-independent tyrosine phosphorylation of pp125FAKand pp72sykinduced by Concanavalin A required the expression of integrin αIIb-β3on the platelet surface as it was strongly inhibited in platelets from patients affected by Glanzmann thrombasthenia. By contrast, tyrosine phosphorylation of PLCγ2 occurred normally also in thrombasthenic platelets stimulated with Concanavalin A. These results demonstrate that, even in the absence of aggregation, the clustering of integrin αIIb-β3induced by Concanavalin A on the platelet surface directly promotes tyrosine phosphorylation of pp72sykand pp125FAKand provide further evidence that the oligomerization of the fibrinogen receptor promoted by its natural ligand during platelet aggregation may be responsible for the tyrosine phosphorylation of these proteins induced by physiological agonists.

Binding of Recombinant Apolipoprotein(a) to Human Platelets and Effect on Platelet Aggregation

2001 ◽  
Vol 85 (04) ◽  
pp. 686-693 ◽  
Author(s):  
C. Martínez ◽  
J. Rivera ◽  
S. Loyau ◽  
J. Corral ◽  
R. González-Conejero ◽  
...  
Keyword(s):  
Arachidonic Acid ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
High Concentration ◽  
Single Class ◽  
Apo B ◽  
Platelet Surface ◽  
The Individual

SummaryThe interaction of lipoprotein(a) [Lp(a)] with platelets is not well defined, particularly with regards to the individual contribution of the protein components of Lp(a), the apo B-100 and the apolipoprotein apo(a). This study investigated the binding of different recombinant apo(a) [r-apo(a)] isoforms, to human platelets and its effect on platelet aggregation. Scatchard analysis of saturation binding experiments demonstrated that human platelets display a single class of high affinity r-apo(a) binding sites (71 ± 46 molec./platelet, Kd = 5.6 ± 2.0 nmol/L). Platelet activation with strong agonists (thrombin, arachidonic acid) increased 2- to 10-fold the r-apo(a) binding, without affecting the affinity. Competition assays showed that the binding sites are highly specific for r-apo(a) and Lp(a). At high concentration t-PA could also bind to the r-apo(a) binding sites. By contrast, neither fibrinogen nor plasminogen inhibited to the r-apo(a) binding. The lysine analogue EACA inhibits the binding of r-apo(a) to platelets, thus suggesting the involvement of lysine residues in that interaction. Moreover, the r-apo(a) binding to platelets is unlikely mediated by GPIIb/IIIa-attached fibrin since it is not affected by platelet treatment with either LJ-CP8, a monoclonal antibody that specifically blocks fibrinogen binding to GPIIb/IIIa, nor GPRP, an inhibitor of fibrin polymerisation. Finally, we show that the distinct recombinant apo(a) proteins, as well as native Lp(a), promote an aggregation response of platelets to otherwise subaggregant doses of arachidonic acid. This proaggregant effect of r-apo(a) is dependent on its binding to platelets since it requires a minimum incubation time, and it is prevented by EACA at concentration inhibiting the r-apo(a)-platelet interaction.These results suggest that the prothrombotic action of Lp(a) may be in part mediated by modulating the platelet function through the interaction of its apo(a) subunit with a specific receptor at the platelet surface.

scholarly journals Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1224-1231 ◽  
Author(s):  
SJ Shattil ◽  
HJ Motulsky ◽  
PA Insel ◽  
L Flaherty ◽  
LF Brass
Keyword(s):  
Platelet Aggregation ◽  
Adrenergic Receptors ◽  
Human Platelets ◽  
Receptor Expression ◽  
Platelet Surface ◽  
Prolonged Stimulation ◽  
Fibrinogen Receptor ◽  
Aggregation Response ◽  
Fibrinogen Binding ◽  
Response Expression

Abstract Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or “desensitization” of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.

Modulation of Platelet Activation by Native DNA – Initial Description of Platelet Receptor Type, Number and Discrimination for Native DNA

1981 ◽  
Vol 45 (03) ◽  
pp. 263-266 ◽  
Author(s):  
B A Fiedel ◽  
M E Frenzke
Keyword(s):  
Platelet Aggregation ◽  
Human Platelets ◽  
Scatchard Analysis ◽  
Type Number ◽  
Single Class ◽  
Receptor Ligand ◽  
Platelet Receptor ◽  
Platelet Binding ◽  
Ligand Interactions ◽  
Initial Description

SummaryNative DNA (dsDNA) induces the aggregation of isolated human platelets. Using isotopically labeled dsDNA (125I-dsDNA) and Scatchard analysis, a single class of platelet receptor was detected with a KD = 190 pM and numbering ~275/platelet. This receptor was discriminatory in that heat denatured dsDNA, poly A, poly C, poly C · I and poly C · poly I failed to substantially inhibit either the platelet binding of, or platelet aggregation induced by, dsDNA; by themselves, these polynucleotides were ineffective as platelet agonists. However, poly G, poly I and poly G · I effectively and competitively inhibited platelet binding of the radioligand, independently activated the platelet and when used at a sub-activating concentration decreased the extent of dsDNA stimulated platelet aggregation. These data depict a receptor on human platelets for dsDNA and perhaps certain additional polynucleotides and relate receptor-ligand interactions to a physiologic platelet function.

Effect of Calcium and magnesium on Receptor Mediated Binding of Fibrinogen by Platelets

1979 ◽  
Author(s):  
G.A. Marguerie ◽  
E.P. Plow ◽  
T.S. Edgington
Keyword(s):  
Platelet Aggregation ◽  
Shape Change ◽  
Divalent Cations ◽  
Human Platelets ◽  
Optimal Conditions ◽  
Binding Studies ◽  
Single Class ◽  
Fibrinogen Receptor ◽  
Platelet Receptor ◽  
Stimulation Of

When washed human platelets are stimulated by ADP, specific and saturable binding of fibrinogen is rapidly induced, consistent with a discrete cellular receptor. In the absence of divalent cations (<10-6 M), stimulation of plateles by ADP resulted in a well defined shape change but aggregation occurred only when Ca++ or Mg++ were added at mM levels. Deaggergation occurred only in the presence of Ca++ and apyrase. Binding studies demonstrated that 125I-fibrinogen did not interact with platelets in the absence of divalentations. Both Ca++ and Mg++ supported the ADP induced binding, but approximf~ely half as nany 125I-fibrinogen molecules were bound at optimal Mg++ as opposed to optimal Ca++(1 mM). Ca++ and Mg++ did not produce actitive bipping; and affinity of the platelet receptor for 125I-fibrinogen in the presence of either Ca++ or Mg++ was similar suggesting induction by both divalent ions of a single class of receptor. Under optimal conditions of ADP and Ca++ the maximum number of receptors per platelet differed for individuals with a mean of 49,826 ± 22,321 in 12 normal donors with a range of 17,500 to 82,500. 125I-fibrinogen did not dissociate from the platelet unless apyrase and Ca++ were present, paralleling the aggregation studies. The binding or dissociation of fibrinogen and platelet aggregation or deaggregation are consistent with the hypotpesis that induction of the fibrinogen receptor may be implicated in ADP dependent platelet aggregation. Supported by NIH grant HL-16411.

scholarly journals Characterization of the gamma chain platelet binding site on fibrinogen fragment D

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2643-2648 ◽  
Author(s):  
NE Kirschbaum ◽  
MW Mosesson ◽  
DL Amrani
Keyword(s):  
Platelet Aggregation ◽  
Binding Site ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Gamma Chain ◽  
Platelet Surface ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Chain Sequence ◽  
Carboxy Terminal

Abstract Glycoprotein (GP) IIb/IIIa on adenosine diphosphate (ADP)-activated human platelets interacts with specific sites on the fibrinogen molecule leading to aggregation. We characterized the platelet-binding site on the gamma chains of fibrinogen using plasmic fragments D gamma A and D gamma'. Fragment D gamma A, which contains the carboxy terminal gamma A400–411 platelet-binding sequence (HHLGGAKQAGDV), was 70-fold more active than the synthetic gamma A400–411 peptide in inhibiting ADP- induced platelet aggregation. Fragment D gamma A inhibited fibrinogen binding and also bound directly to ADP-activated platelets. The Kd values determined for fibrinogen and fragment D gamma A binding were 0.55 mumol/L and 1.2 mumol/L, respectively. In contrast, fragment D gamma', which differs from fragment D gamma A with respect to its gamma chain sequence from position 408 to the COOH-terminus at position 427, did not inhibit platelet aggregation or fibrinogen binding, and did not bind directly to the platelet surface. Denaturation of fragment D gamma A with guanidine-HCl caused a loss of inhibitory activity in platelet aggregation assays. These data indicate that the native conformation of the gamma chain platelet-binding site on fibrinogen is important for optimal binding to GPIIb/IIIa.

scholarly journals A murine monoclonal antibody that blocks fibrinogen binding to normal platelets also inhibits fibrinogen interactions with chymotrypsin- treated platelets

Blood ◽  
1984 ◽  
Vol 64 (1) ◽  
pp. 59-63 ◽  
Author(s):  
EI Peerschke ◽  
BS Coller
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Membrane Glycoproteins ◽  
Molecular Weights ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Unstimulated Control ◽  
Lines Of Evidence

We recently described a monoclonal antibody, 10E5 , that completely blocks adenosine diphosphate (ADP) induced fibrinogen binding to platelets and aggregation induced by ADP, epinephrine, and thrombin. Multiple lines of evidence indicate that 10E5 binds to platelet membrane glycoproteins IIb and/or IIIa. Because it has been reported that platelets treated with chymotrypsin aggregate when fibrinogen is added, we tested the effect of 10E5 antibody on chymotrypsin-induced fibrinogen binding and platelet aggregation. Aspirin-treated human platelets were washed in modified Tyrode's buffer (pH 7.5), incubated for 5 minutes at 22 degrees C with 300 micrograms/mL chymotrypsin, and washed again. The amount of 10E5 antibody bound to these platelets (37,232 +/- 2,928 molecules/platelet; mean +/- SEM, N=9) was similar to that bound to unstimulated control platelets (36,910 +/- 2,669) and did not differ significantly from the amount of antibody bound to ADP- treated platelets (P less than .01, N = 5). The amount of 10E5 bound to chymotrypsin-treated platelets correlated directly with the amount of fibrinogen bound to separate aliquots of the same platelet samples (r = .876, P less than .001). The 10E5 antibody caused virtually complete inhibition of both the binding of fibrinogen to chymotrypsin-treated platelets and the aggregation induced by exogenous fibrinogen. Immunoprecipitation studies of 125I-labeled chymotrypsin-treated platelets revealed that the 10E5 antibody bound proteins with molecular weights characteristic of glycoproteins IIb and IIIa. These data suggest that the fibrinogen receptor on chymotrypsin-treated platelets is identical to that on ADP-treated platelets and that this receptor is either near to, or on, the glycoprotein IIb/IIIa complex.

A New Method for the Assay of Exposed Platelet Fibrinogen Receptor Using a Chemiluminescent Label

1995 ◽  
Vol 74 (06) ◽  
pp. 1546-1550 ◽  
Author(s):  
Makoto Katoh ◽  
Susumu Chishima ◽  
Nobukazu Kiuchi ◽  
Tomihiro Ikeo ◽  
yasuhiko Sasaki
Keyword(s):  
Binding Sites ◽  
High Selectivity ◽  
Binding Assay ◽  
Specific Binding ◽  
Human Platelets ◽  
Assay System ◽  
Human Fibrinogen ◽  
Fibrinogen Receptor ◽  
Plot Analysis ◽  
Fibrinogen Binding

SummaryAssay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative I-labeled fibrinogen (acridinium-fibrinogen).Human fibrinogen in saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa was assayed by measuring chemiluminescence emitted on addition of 0.1 N NaOH containing 0.06% H202 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with a Kd of 326 nM and Bmax of 7.8 pmol/108 platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen γ-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 μM and 31.3μM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotop.

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