CHARACTERIZATION OF THE FIBRINOGEN BINDING SITES USING MONOCLONAL ANTIBODIES TO HOMAN PLATELET MEMBRANE GLYCOPROTEINS IIb/IIIa*

1987 ◽  
Author(s):  
C Ruan ◽  
X Du ◽  
H Wan ◽  
X Hu ◽  
X Xi ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Prolonged Treatment ◽  
Antigenic Determinants ◽  
Membrane Glycoproteins ◽  
Fibrinogen Binding ◽  
Igg1 Subclass ◽  
Academy Of Sciences

Two new murine monoclonal antibodies, SZ-21 and SZ-22, (both IgG1 subclass), were produced by the hybridoma technique using washed human platelets as the immunogen. Both SZ-21 and SZ-22 reanted specifically with normal platelets and megakaryocytes but not with Glanzmann's thrombasthenic platelets which lack the membrane glycoprotein(GP)IIb/IIIa complex. Platelets from 10 normal donors bound 64,500±20,300(x+SD) SZ-21 molecules/platelet with KD4.4±1.5nM and 61,000±19,900 SZ-22 molecules/platelet with KD18.8±6.7nM respectively.Affinity chromatography confirmedthat SZ-21 and SZ-22 reacted with theGPIIb/IIIa complex. SZ-21 inhibited the platelet aggregation and secretioninduced by collagen, arachidonic acidand thrombin,and the second wave of aggregation in response to ADP, adrenaline and ristocetin. The fibrinogen binding to human platelets induced by ADP, arachidonic acid and PAF was also inhibited by SZ-21. But SZ-22 hado effects on platelet aggregation andfibrinogen binding. Western blot analyses indicated that SZ-21reanted with GPIIIa and that SZ-22 bound to GPIIb. When the protein was reduced by 2-mercaptoethand, SZ-22 reacted with the enchain of GPIIb, but SZ-21 lost its ability of binding to GPIIIa, suggested that the antigenic determinants of SZ-21 depended on the integrity ofthe intra αchain disulfide bond of GPIIIa. On chymotrypsin treated platelets,the epitope for SZ-21wasidentified on a 66 KD membrane-bound fragment of GPIIIa. The appearanceofthe 66 KD fragment was related withthechymotrypsin-mediated fibrinogen binding and aggregation. However the prolonged treatment with chymotrypsin reduced the platelet aggregation, which coincided with the appearance of the 66KD fragment, a product of thefurther hydrolysis of the 66KD fragment.These results suggested that the domainbetween the 66KD and 60KD fragments of GPIIIa might be essential for the maintenance of the fibrinogen binding sites.* Supported by the Science fund of the Chinese Academy of Sciences ( project No.82-172 ) and the National Science fund of China ( project No.3860713 ).

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.

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3675-3683 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Toshiaki Aoki ◽  
Masamichi Shiraga ◽  
Yoshiyuki Kurata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Binding Sites ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Fibrinogen Binding ◽  
Group I ◽  
Group Ii ◽  
Inhibition Studies

Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

Association Between Ligand-Induced Conformational Changes of Integrin IIbβ3 and IIbβ3-Mediated Intracellular Ca2+ Signaling

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3675-3683 ◽  
Author(s):  
Shigenori Honda ◽  
Yoshiaki Tomiyama ◽  
Toshiaki Aoki ◽  
Masamichi Shiraga ◽  
Yoshiyuki Kurata ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Conformational Changes ◽  
Binding Sites ◽  
Critical Role ◽  
Thromboxane A2 ◽  
Fibrinogen Binding ◽  
Group I ◽  
Group Ii ◽  
Inhibition Studies

Abstract Platelet IIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of IIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and IIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various IIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as IIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both IIb and β3 subunits. In group II, antagonists can induce LIBS on the IIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on IIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to IIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable IIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate IIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and IIbβ3-mediated intracellular Ca2+ signaling in platelets.

scholarly journals Monoclonal antibodies against porcine platelet membrane glycoproteins Ib and IIb/IIIa

Blood ◽  
1988 ◽  
Vol 72 (5) ◽  
pp. 1740-1747 ◽  
Author(s):  
H Takami ◽  
WL Nichols ◽  
SE Kaese ◽  
RS Miller ◽  
JA Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Human Platelets ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments

Abstract We prepared murine monoclonal antibodies to porcine platelet membrane glycoprotein (GP) Ib and GP IIb/IIIa for further study of the porcine hemostatic mechanism. One monoclonal antibody, designated PP3–4C, blocked Ristocetin-induced platelet agglutination and caused 80% inhibition of Ristocetin-induced 125I-von Willebrand factor (vWF) binding to porcine platelets at a concentration of greater than or equal to 12 micrograms IgG/mL. PP3–4C did not affect adenosine diphosphate (ADP)- or collagen-induced platelet aggregation. Binding of 125I-Fab fragments of PP3–4C to platelets was saturable at 3.7 x 10(4) +/- 0.8 x 10(4) molecules per platelet. Another monoclonal antibody, designated PP3–3A, blocked ADP- or collagen-induced platelet aggregation at 6 micrograms IgG/mL. At a concentration of 10 micrograms IgG/mL, PP3–3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated platelets. PP3–3A did not affect Ristocetin-induced platelet agglutination nor 125I-vWF binding to platelets in the presence of Ristocetin. Binding of 125I-Fab' fragments of PP3–3A to platelets was saturable at 9.8 x 10(4) +/- 1.2 x 10(4) molecules per platelet. PP3–4C antibody (anti-GP Ib) did not bind to human platelets; however, PP3–3A antibody (anti-GP IIb-IIIa) had partial cross-reactivity with human platelets. Immunoaffinity chromatography of solubilized surface-radiolabeled porcine platelets and subsequent sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis demonstrated that PP3–4C recognized a GP with an apparent molecular weight of 160,000 (nonreduced), and 140,000 (reduced). PP3–3A recognized GPs with apparent molecular weights of 130,000 and 80,000 (nonreduced), and 115,000 and 95,000 (reduced). These monoclonal antibodies to porcine platelet membrane GPs, which are structural and functional analogues of human GP Ib and GP IIb/IIIa, will be useful for in vitro and in vivo studies of the mammalian hemostatic mechanism.

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.

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.

FAB FRAGMENTS OF MONOCLONAL ANTIBODIES SPECIFIC FOR PORCINE PLATELET MEMBRANE GLYCOPROTEINS GP IB ANDGP IIB/IIIA

1987 ◽  
Author(s):  
H Takami ◽  
W L Nichols ◽  
S E Kaese ◽  
R S Miller ◽  
J A Katzmann ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Platelet Aggregation ◽  
Platelet Membrane ◽  
In Vivo Studies ◽  
Membrane Glycoproteins ◽  
Fab Fragments ◽  
Igg1 Subclass ◽  
Von Willebrand

For further study of the porcine hemostatic mechanism, we have prepared murine monoclonal antibodies, and F(ab')2 and Fab fragments, specific for porcine platelet membrane glycoproteins GP lb and GP Ilb/IIIa. To avoid production of antibodies to von Willebrand factor (vWF), mice were immunized with platelets obtained from pigs with severe von Willebrand,s disease. One monoclonal antibody (PP3-4C), of IgG1 subclass, caused 85% inhibition of Ristocetin-induced platelet binding of 125I-vWF (porcine) at ≥12 µg IgG/ml. PP3-4C did not affect ADP or collagen-induced platelet aggregation nor inhibit 125I-fibrinogen (porcine) binding. Pepsin and papain digestion, respectively, were used to prepare PP3-4C F(ab')2 and Fab fragments. PP3-4C F(ab')2 at concentrations ≥12 µg/ml caused 80% inhibition of washed platelet agglutination in the presence of vWF and Ristocetin, whereas Fab fragments at concentrations ≥10 µg/ml caused 60% inhibition. Another monoclonal antibody (PP3-3A), of IgG1 subclass, completely inhibited ADP or collagen-induced platelet aggregation at an IgG concentration of 6 µg/ml. At 10 µg IgG/ml PP3-3A completely inhibited binding either of 125I-fibrinogen or of 125I-vWF to ADP-stimulated porcine platelets. PP3-3A did not affect vWF-dependent Ristocetin-induced platelet agglutination, nor 125I-vWF binding to platelets in the presence of Ristocetin. PP3-3A did not bind to platelets which were treated with 10 mM EDTA at 37°C for 60 min. F(ab')2 and Fab fragments were isolated from PP3-3A pepsin or papain digests. Both types of PP3-3A fragments caused 100% inhibition of ADP-induced platelet aggregation, at concentrations ≥6 yg/ml. Immunoprecipitation of surface-radiolabeled porcine platelets and subsequent SDS-PAGE demonstrated that PP3-4C recognized a glycoprotein with molecular weight of 140,000 (under reducing conditions), and 165,000 (non-reduced). PP3-3A recognized glycoproteins with molecular weights of 115,000 and 100,000 (reduced), and 130,000 and 80,000 (non-reduced). Neither monoclonal antibody bound to human platelets. These monoclonal antibodies to porcine platelet membrane glycoproteins which are analogues of human GP lb and GP Ilb/IIIa will be useful for in vitro and in vivo studies to further understanding of mammalian hemostatic mechanisms.

Granulocyte-platelet interactions and platelet fibrinogen receptor exposure

1988 ◽  
Vol 255 (3) ◽  
pp. H651-H658 ◽  
Author(s):  
E. Kornecki ◽  
Y. H. Ehrlich ◽  
R. Egbring ◽  
M. Gramse ◽  
R. Seitz ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Human Platelets ◽  
Granulocyte Elastase ◽  
Fibrinogen Receptor ◽  
Fibrinogen Binding ◽  
Low Concentrations ◽  
Number Of Binding Sites ◽  
Proteolytic Action ◽  
Aggregation Of Platelets

We have examined the interaction of human granulocyte elastase with human platelets. Incubation of human platelets with human granulocyte elastase exposed active fibrinogen-binding sites as evidenced by 125I-labeled fibrinogen binding and spontaneous fibrinogen-induced platelet aggregation. The aggregation of platelets by fibrinogen occurred at low concentrations of human granulocyte elastase (0.5–1 microgram/ml). Platelets pretreated with human granulocyte elastase exposed an average of 10,500 fibrinogen binding sites per platelet, i.e., about one-third the number of binding sites exposed by optimal concentrations of ADP. With the use of a polyclonal antiplatelet membrane antibody, the glycoproteins IIb (GPIIb), IIIa (GPIIIa), and a 60,000-Da (60 kDa) protein (66 kDa in a reduced system) derived from GPIIIa were immunoprecipitated from the surface of detergent extracts of human 125I-radiolabeled platelets pretreated with increasing concentrations of human granulocyte elastase. Experiments performed by immunoblotting with use of polyclonal and monoclonal antibodies directed to GPIIIa showed that pretreatment of human platelets with granulocyte elastase resulted in the appearance of an additional proteolytic derivative of GPIIIa migrating with an apparent molecular mass of 120 kDa in a nonreduced system. GPIIIa appears to be the preferred substrate of elastase, since GPIIb was not degraded by human granulocyte elastase. We conclude that 1) the proteolytic action of human granulocyte elastase on platelet GPIIIa results in the formation of two major hydrolytic products, and 2) human granulocyte elastase exposes active fibrinogen-binding sites associated with the GPIIb/GPIIIa complex, resulting in direct platelet aggregation by fibrinogen.

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

Abstract 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.

Detection Of High Affinity Fibrinogen Binding Sites On Human Platelets Stimulated By ADP: Comparison Of Two Methods Of Platelet Separation

1981 ◽  
Author(s):  
Stefan Niewiarowski ◽  
Thomas A Morinelli ◽  
Elizabeth Kornecki
Keyword(s):  
Platelet Aggregation ◽  
Binding Sites ◽  
Gel Filtration ◽  
Human Platelets ◽  
Binding Studies ◽  
Test Analysis ◽  
High Affinity ◽  
Fibrinogen Binding ◽  
Binding Data ◽  
Specific Receptors

Binding of fibrinogen to specific receptors on human platelets exposed by ADP results in platelet aggregation. There are controversial data regarding classes and number of fibrinogen receptors, the values range from one to two classes and 1,000-80,000 receptors per platelet as reported in the literature. We have studied the interaction of fibrinogen with a) platelets washed by differential centrifugation according to Mustard and colleagues (washed platelets - WP) and with b) gel-filtered platelets (GFP). Platelet aggregation was studied with 100 μM ADP and with various concentration of fibrinogen. Maximal velocities of aggregation for WP and GFP were 81 and 47 units per min, respectively, and the Km values for fibrinogen calculated from the rate of aggregation were 0.9 × 10-7M for WP and 5.8 × 10-7M for GFP. The level of platelet fibrinogen released into the suspension from WP and GFP amounted to 2.4 μg and 15.0 μg per 10 9 platelets/ml, respectively, as measured by the staphylococcal clumping test. Analysis of 125I-fibrinogen binding data by the method of Scatchard and Feldman revealed 1,300 high affinity receptors (KD 3.2 × 10-8M) and 80,000 low affinity receptors (KD 5.6 × 10-5M) for WP. The binding of 125I-fibrinogen to GFP was greatly diminished. The number of fibrinogen receptors exposed by ADP on GFP and their binding affinity are under investigation in our laboratory. In conclusion, GFP were less sensitive to fibrinogen than were WP as shown in the aggregation and 125I-fibrinogen binding studies. It appears that the method of platelet separation is critical for the assessment of fibrinogen binding. Platelet activation and release of intact platelet fibrinogen during gel-filtration may interfere with the detection of high affinity fibrinogen binding sites.

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