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

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 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 Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 887-899
Author(s):  
P Nurden ◽  
M Humbert ◽  
RS Piotrowicz ◽  
C Bihour ◽  
C Poujol ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Gamma Chain ◽  
Rgd Peptides ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Granule Membrane ◽  
Carboxy Terminal

The sequence beta (3)203–228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211–221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding- site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin- stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin- induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.

scholarly journals Distribution of ligand-occupied alpha IIb beta 3 in resting and activated human platelets determined by expression of a novel class of ligand-induced binding site recognized by monoclonal antibody AP6

Blood ◽  
1996 ◽  
Vol 88 (3) ◽  
pp. 887-899 ◽  
Author(s):  
P Nurden ◽  
M Humbert ◽  
RS Piotrowicz ◽  
C Bihour ◽  
C Poujol ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Binding Site ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Gamma Chain ◽  
Rgd Peptides ◽  
Platelet Surface ◽  
Immunogold Staining ◽  
Granule Membrane ◽  
Carboxy Terminal

Abstract The sequence beta (3)203–228 is involved, in a yet undetermined manner, in alpha IIb beta 3 function. We now show that murine monoclonal antibody (MoAb) AP6, specific for beta (3)211–221, binds to alpha IIb beta 3 on adenosine diphosphate (ADP)-activated platelets only when the receptor is occupied by intact fibrinogen. The ligand-induced binding- site reported by AP6 is unique in that it is not expressed following occupancy by either RGD peptides or the gamma-chain carboxy-terminal dodecapeptide. Binding of AP6 to platelets coincides temporally with the binding of the MoAb 9F9, specific for a receptor-induced binding site on fibrinogen. Thus, AP6 reports the binding of fibrinogen to the recognition pocket of alpha IIb beta 3. Its binding to thrombin- stimulated washed platelets correlates with secretion as determined using an MoAb to P-selectin. When ultrathin sections of nonactivated platelets were examined by immunogold staining and electron microscopy, AP6 identified a pool of alpha IIb beta 3 colocalizing with P-selectin and suggesting the presence of alpha IIb beta 3-ligand complexes in the alpha-granule membrane. There was little binding of AP6 to surface alpha IIb beta 3 of unstimulated platelets. After ADP-induced activation, AP6 was abundantly distributed over the entire platelet surface, including pseudopods, but only when fibrinogen was present in the medium. ADP had little effect on AP6 reactivity within platelets. This contrasted with washed platelets and thrombin, where extensive AP6 binding was observed within internal membrane pools as early as 10 to 15 seconds after stimulation. Surface labeling with AP6 followed slower kinetics. Flow cytometry on Triton X-100 permeabilized fixed platelets confirmed AP6 binding to alpha IIb beta 3 within the platelet. Thus, our results provide evidence of (1) a pool of alpha-granule alpha IIb beta 3 occupied by ligand in nonactivated platelets; (2) thrombin- induced activation of alpha IIb beta 3 within the platelet, and (3) thrombin-induced mobilization of ligand-bound alpha IIb beta 3 to the surface.

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.

scholarly journals Synergistic action of two murine monoclonal antibodies that inhibit ADP- induced platelet aggregation without blocking fibrinogen binding

Blood ◽  
1987 ◽  
Vol 69 (2) ◽  
pp. 668-676
Author(s):  
PJ Newman ◽  
RP McEver ◽  
MP Doers ◽  
TJ Kunicki
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Adenosine Diphosphate ◽  
Platelet Surface ◽  
Synergistic Inhibition ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Complete Restoration ◽  
Binding Event ◽  
Murine Monoclonal Antibodies

We have used two murine monoclonal antibodies, each directed against one component of the human platelet membrane glycoprotein (GP) IIb-IIIa complex, to examine further the molecular requirements for fibrinogen binding to the platelet surface and subsequent platelet-platelet cohesion (aggregation). Although neither AP3, which is directed against GPIIIa, nor Tab, which is specific for GPIIb, were individually able to inhibit adenosine diphosphate (ADP)-induced fibrinogen binding, platelet aggregation, or secretion, the combination of AP3 and Tab completely abolished platelet aggregation and the release reaction. Unexpectedly, this synergistic inhibition of platelet-platelet cohesion occurred in the presence of apparently normal fibrinogen binding. Both the number of fibrinogen molecules bound and the dissociation constant for fibrinogen binding remained essentially unchanged in the presence of these two antibodies. Inhibition of aggregation was dependent upon the divalency of both AP3 and Tab because substitution of Fab fragments of either antibody for the intact IgG resulted in a complete restoration of both aggregation and secretion. In contrast to ADP induction, thrombin-activated platelets neither aggregated nor bound fibrinogen in the presence of AP3 plus Tab but were fully capable of secretion, which illustrated the multiple mechanisms by which the platelet surface can respond to different agonists. These data demonstrate that fibrinogen binding to the platelet surface alone is not sufficient to support platelet-platelet cohesion and that an additional post-fibrinogen-binding event(s) that is inhibitable by these two monoclonal antibodies may be required.

Platelet Hyporeactivity in Very Low Birth Weight Neonates

1997 ◽  
Vol 77 (05) ◽  
pp. 1002-1007 ◽  
Author(s):  
Damodara Rajasekhar ◽  
Marc R Barnard ◽  
Francis J Bednarek ◽  
Alan D Michelson
Keyword(s):  
Flow Cytometry ◽  
Binding Site ◽  
Whole Blood ◽  
Very Low Birth Weight ◽  
Thromboxane A2 ◽  
Platelet Surface ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Platelet Binding ◽  
Thromboxane A2 Analogue

SummaryVery few studies have examined platelet function in very low birth weight (VLBW) preterm neonates, because of the relatively large volumes of blood required. In this study, platelet function in clinically stable VLBW neonates was examined by whole blood flow cytometry, which requires only 5 |jl1 of whole blood per assay. The following monoclonal antibodies were used: S12 (P-selectin-specific, reflecting a granule secretion), PAC1 (directed against the fibrinogen binding site exposed on the GPIIb-IIIa complex of activated platelets), F26 (directed against a conformational change in fibrinogen bound to the GPIIb-IIIa complex), and 6D1 (directed against the von Willebrand factor binding site on the GPIb-IX-V complex). VLBW neonates, like normal adults, did not have circulating activated platelets, as determined by the lack of binding of SI2, PAC1, and F26 in the absence of an added agonist. VLBW neonatal platelets were markedly less reactive than adult platelets to thrombin, ADP/epinephrine, and U46619 (a stable thromboxane A2 analogue), as determined by the extent of increase in the platelet binding of SI2, PAC1, and F26, and the extent of decrease in the platelet binding of 6D1. In summary, compared to adults, the platelets of VLBW neonates are markedly hyporeactive to thrombin, ADP/epinephrine and a thromboxane A2 analogue in the physiologic milieu of whole blood, as determined by: 1) the increase in platelet surface P-selectin; 2) the exposure of the fibrinogen binding site on the GPIIb-IIIa complex; 3) fibrinogen binding; and 4) the decrease in platelet surface GPIb. This platelet hyporeactivity may be a factor in the propensity of VLBW neonates to intraventricular hemorrhage. In addition to its previously defined use as a test of platelet hyperreactivity, the present study suggests that whole blood flow cytometry may be useful in the clinical assessment of platelet hyporeactivity.

scholarly journals Immunocytochemical localization of fibrinogen on washed human platelets. Lack of requirement for fibrinogen during adenosine diphosphate-induced responses and enhanced fibrinogen binding in a medium with low calcium levels

Blood ◽  
1988 ◽  
Vol 71 (4) ◽  
pp. 850-860 ◽  
Author(s):  
H Suzuki ◽  
RL Kinlough-Rathbone ◽  
MA Packham ◽  
K Tanoue ◽  
H Yamazaki ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Induced Responses ◽  
Immunocytochemical Localization ◽  
Platelet Surface ◽  
Gold Particles ◽  
Fibrinogen Binding ◽  
Large Numbers ◽  
Lowicryl K4m

Abstract The association of fibrinogen with washed human platelets was examined by immunocytochemistry during aggregation induced by adenosine diphosphate (ADP) and during deaggregation. The platelets were suspended either in a medium containing 2 mmol/L Ca2+ or in a medium containing no added Ca2+ (20 mumol/L Ca2+). Platelets were fixed at several times during aggregation and deaggregation, embedded in Lowicryl K4M, sectioned, incubated with goat antihuman fibrinogen, washed, reacted with gold-labeled antigoat IgG, and prepared for electron microscopy. To determine whether the method detected fibrinogen associated with the platelets, the platelets were pretreated with chymotrypsin (10 U/mL) and aggregated by fibrinogen; gold particles were apparent not only in the alpha granules but on the platelet surface and between adherent platelets as well. In the medium with 2 mmol/L Ca2+, ADP caused extensive aggregation of normal platelets in the presence of fibrinogen (0.4 mg/mL), and gold particles were evident between the adherent platelets and on the platelet surface; when the platelets deaggregated, gold was no longer present on the surface. In a medium without added Ca2+, ADP caused extensive aggregation in the presence of fibrinogen, and large numbers of gold particles were on the platelet surface and even more between adherent platelets. In this medium, the platelets did not deaggregate, and by five minutes, the granules appeared to be swollen or fused. In the absence of external fibrinogen, ADP caused the formation of small aggregates, and fibrinogen was not detected between adherent platelets. Thus, the association of fibrinogen with the platelet surface enhances platelet aggregation but is not essential for the ADP-induced formation of small aggregates. The association of fibrinogen with platelets is greater under conditions in which platelets release their granule contents and do not deaggregate because both endogenous and exogenous fibrinogen take part in aggregation.

Studies on the Binding of 3H-SR121566, an Inhibitor of Gp IIb-IIIa Activation

2001 ◽  
Vol 85 (04) ◽  
pp. 702-709 ◽  
Author(s):  
P. Savi ◽  
G. Zamboni ◽  
O. Rescanières ◽  
J. M. Herbert
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Gamma Chain ◽  
Activated Platelets ◽  
Fibrinogen Binding ◽  
Direct Competition ◽  
Stimulation Of

SummarySR121566 is a new synthetic agent which inhibits the binding of fibrinogen to activated platelets, and platelet aggregation. 3H-SR121566 bound with nanomolar affinity (KD ranging from 45 to 72 nM) to Gp IIb-IIIa expressing cells only. On activated human platelets, this ligand allowed the detection of a maximal number of 100-140,000 binding sites. The binding of SR121566 to platelets, was displaced by several agents including RGD-containing peptides and synthetic RGD mimetics, but not by ReoPro®, a humanised monoclonal antibody which inhibits the binding of fibrinogen to the Gp IIb-IIIa complex. Neither the fibrinogen dodecapeptide nor fibrinogen itself were able to compete with SR121566 whether platelets were activated or not.Flow cytometry studies indicated that SR121566 which did not activate Gp IIb-IIIa by itself, dose-dependently prevented the detection of activation-induced binding sites on TRAP-stimulated platelets in the presence or absence of exogenous fibrinogen, indicating a direct effect on the activation state of the Gp IIb-IIIa complex. Moreover, SR121566 was able to reverse the activation of Gp IIb-IIIa and to displace the binding of fibrinogen when added up to 5 min after TRAP stimulation of platelets. When added at later times (15 to 30 min), SR121566 failed to displace fibrinogen binding, even if SR121566 binding sites were still accessible and the Gp IIb-IIIa complex not activated.In conclusion, our study is in accordance with the finding that fibrinogen is recognised by the activated Gp IIb-IIIa complex through the dodecapeptide sequence present on its gamma chain, and that this interaction is inhibited by SR121566 by preventing and reversing the activated conformation of Gp IIb-IIIa and not by direct competition with fibrinogen.

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.

The FcR γ Chain Potentiates Outside-In Integrin Signaling.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 215-215 ◽  
Author(s):  
Brian Boylan ◽  
Debra K. Newman ◽  
Peter J. Newman
Keyword(s):  
Platelet Aggregation ◽  
Platelet Activation ◽  
Second Messengers ◽  
Chain Complex ◽  
Adenosine Diphosphate ◽  
Adaptor Proteins ◽  
Immunoblot Analysis ◽  
Human Platelets ◽  
Rgd Peptides ◽  
Fibrinogen Binding

Abstract The FcRγ chain is a 12 kDa homodimer that exists in the platelet plasma membrane non-covalently associated with platelet membrane glycoprotein (GP)VI. Interaction of laminin or collagen fibers in the vessel wall with GPVI results in rapid transmission of activation signals into the cell - a process that is dependent on Src family kinase-mediated phosphorylation of Immunoreceptor tyrosine-based activation motifs (ITAMs) within the cytoplasmic domain of FcRγ. Tyrosine phosphorylated FcRγ, in turn, recruits the tyrosine kinase Syk, which then initiates a signal amplification pathway involving assembly of a series of adaptor proteins, activation of phospholipase Cγ2, and generation of second messengers that facilitate granule secretion, activation of the platelet integrins, and platelet aggregation. We and others have recently shown that loss of the GPVI/FcRγ chain complex from the surface of either human or murine platelets renders them unresponsive to collagen and laminin. Unexpectedly, GPVI/FcRγ-chain-depleted platelets also exhibited moderately reduced reactivity to adenosine diphosphate (ADP) and thrombin, which activate platelets by binding to the G protein coupled receptors (GPCRs) P2Y1, P2Y12, PAR1, and PAR4, respectively. Because GPCR-mediated platelet activation has not previously been thought to involve signals emanating from the GPVI/FcRγ-chain complex, we sought to determine whether the FcRγ chain might function to amplify platelet activation responses following GPCR-mediated signaling. Human platelets were exposed to ADP, thrombin or the TXA2-mimetic, U46619, allowed to aggregate, and detergent lysed. Phosphotyrosine immunoblot analysis revealed that the FcRγ chain became rapidly phosphorylated on its ITAMs during platelet aggregation induced by either thrombin or U46619. Interestingly, FcRγ chain phosphorylation was completely inhibited when platelets were activated in the presence of RGD peptides, which allow platelet activation and granule release, but prevent fibrinogen binding to the major platelet integrin, GPIIb-IIIa. The observation that the FcRγ chain becomes tyrosine phosphorylated following GPCR signaling in an RGD-inhibitable manner suggests that integrin-associated kinases might be capable of phosphorylating FcRγ, either directly or indirectly, downstream of ligand binding, though evidence for this remains to be determined. Taken together, these dataprovide support for the notion that the FcRγ chain functions as a general amplifier of platelet activation,helps to explain why GPVI-depleted platelets exhibit blunted responses to agonists specific for GPCRs, andmay lead to important new insights into the mechanism of action of contemplated GPVI-based therapeutics.

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