scholarly journals Thrombospondin promotes platelet aggregation

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 109-115 ◽  
Author(s):  
GP Tuszynski ◽  
VL Rothman ◽  
A Murphy ◽  
K Siegler ◽  
KA Knudsen
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Receptor System ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Surface Receptor ◽  
Lines Of Evidence

Abstract Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.

scholarly journals Thrombospondin promotes platelet aggregation

Blood ◽  
1988 ◽  
Vol 72 (1) ◽  
pp. 109-115
Author(s):  
GP Tuszynski ◽  
VL Rothman ◽  
A Murphy ◽  
K Siegler ◽  
KA Knudsen
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Receptor System ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Surface Receptor ◽  
Lines Of Evidence

Thrombospondin (TSP), isolated from human platelets, promotes aggregation of both nonstimulated platelets and platelets stimulated with thrombin or ADP. The TSP-promoted aggregation is specific since a monoclonal antibody against TSP inhibits the effect of exogenously added TSP and inhibits thrombin-induced platelet aggregation in the absence of added TSP. Several lines of evidence suggest that TSP mediates its effect on aggregation of nonstimulated and stimulated platelets through different platelet-surface receptor systems. The TSP- promoted aggregation of nonstimulated platelets was inhibited by a monoclonal antibody to platelet glycoprotein IV (GPIV), but not by a monoclonal antibody to the fibrinogen receptor, GPIIb-IIIa. In contrast, the antibody to GPIIb-IIIa totally inhibited the TSP- potentiated aggregation of thrombin-stimulated platelets, whereas the antibody to GPIV has no effect. Thus, these studies suggest that TSP promotes platelet aggregation by at least two mechanisms--one dependent on and one independent of the platelet fibrinogen receptor system.

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.

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.

scholarly journals Platelet-melanoma cell interaction is mediated by the glycoprotein IIb- IIIa complex

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 658-663
Author(s):  
H Boukerche ◽  
O Berthier-Vergnes ◽  
E Tabone ◽  
JF Dore ◽  
LL Leung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Cell Interaction ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Human Malignant Melanoma

A human malignant melanoma cell line (M3Dau) was observed by electron microscopy to interact directly with human platelets and induced platelet aggregation. Fab fragments of a monoclonal antibody MoAb (LYP18), directed against the platelet glycoprotein (GP) IIb-IIIa complex, inhibited platelet-melanoma interactions and platelet-platelet aggregation. M3Dau melanoma cells bind LYP 18 and synthesize IIb-IIIa- like GPs. When the melanoma cells were preincubated with LYP 18, tumor- platelet interaction did not occur, suggesting that the interaction may be mediated by the IIb-IIIa-like GPs present on the melanoma cell surface. Glanzmann's thrombasthenic platelets, lacking GPIIb and IIIa, did not interact with melanoma cells, indicating that the platelet GPIIb-IIIa complex is also necessary for the platelet-melanoma cell interaction. This work demonstrates the importance of the IIb-IIIa-like GPs, present on M3Dau melanoma cells, in mediating tumor-platelet interactions.

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.

scholarly journals Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation

Blood ◽  
1992 ◽  
Vol 80 (1) ◽  
pp. 113-120 ◽  
Author(s):  
TW Chow ◽  
JD Hellums ◽  
JL Moake ◽  
MH Kroll
Keyword(s):  
Monoclonal Antibody ◽  
Shear Stress ◽  
Platelet Aggregation ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Platelet Glycoprotein ◽  
Platelet Surface ◽  
Von Willebrand ◽  
Induced Aggregation ◽  
Willebrand Factor

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.

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 Redistribution of the fibrinogen receptor of human platelets after surface activation.

1984 ◽  
Vol 99 (3) ◽  
pp. 822-829 ◽  
Author(s):  
J C Loftus ◽  
R M Albrecht
Keyword(s):  
Human Platelets ◽  
Surface Activation ◽  
Clot Retraction ◽  
Platelet Surface ◽  
Surface Receptors ◽  
Fibrinogen Receptor ◽  
Normal Platelet ◽  
Receptor Sites ◽  
Surface Receptor ◽  
Peripheral Areas

We investigated the whole cell distribution of the platelet membrane receptor for fibrinogen in surface-activated human platelets. Fibrinogen-labeled colloidal gold was used in conjunction with platelet whole mount preparations to visualize directly the fibrinogen receptor. Unstimulated platelets fail to bind fibrinogen, and binding was minimal in the stages of activation immediately following adhesion. The amount of fibrinogen bound per platelet increased rapidly during the shape changes associated with surface activation until 7,600 +/- 500 labels were present at saturation. Maximal binding of fibrinogen was followed by receptor redistribution. During the early stages of spreading, fibrinogen labels were uniformly distributed over the entire platelet surface, including pseudopodia, but the labels become progressively centralized as the spreading process continued. In well spread platelets, labels were found over the central regions, whereas peripheral areas were cleared of receptors. Receptor redistribution during spreading was accompanied by cytoskeletal reorganization such that a direct correlation was seen between the development of specific ultrastructural zones and the distribution of surface receptor sites suggesting a link between the surface receptors and the cytoskeleton. The association of fibrinogen receptors with contractile elements of the cytoskeleton, which permits coordinated receptor centralization, is important to the understanding of the role of fibrinogen in normal platelet aggregation and clot retraction.

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.

scholarly journals Platelet-melanoma cell interaction is mediated by the glycoprotein IIb- IIIa complex

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 658-663 ◽  
Author(s):  
H Boukerche ◽  
O Berthier-Vergnes ◽  
E Tabone ◽  
JF Dore ◽  
LL Leung ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Melanoma Cell ◽  
Melanoma Cell Line ◽  
Cell Interaction ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Human Malignant Melanoma

Abstract A human malignant melanoma cell line (M3Dau) was observed by electron microscopy to interact directly with human platelets and induced platelet aggregation. Fab fragments of a monoclonal antibody MoAb (LYP18), directed against the platelet glycoprotein (GP) IIb-IIIa complex, inhibited platelet-melanoma interactions and platelet-platelet aggregation. M3Dau melanoma cells bind LYP 18 and synthesize IIb-IIIa- like GPs. When the melanoma cells were preincubated with LYP 18, tumor- platelet interaction did not occur, suggesting that the interaction may be mediated by the IIb-IIIa-like GPs present on the melanoma cell surface. Glanzmann's thrombasthenic platelets, lacking GPIIb and IIIa, did not interact with melanoma cells, indicating that the platelet GPIIb-IIIa complex is also necessary for the platelet-melanoma cell interaction. This work demonstrates the importance of the IIb-IIIa-like GPs, present on M3Dau melanoma cells, in mediating tumor-platelet interactions.

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