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.

scholarly journals THE EFFECTS OF THROMBIN ON PHYTOHEMAGGLUTININ RECEPTOR SITES IN HUMAN PLATELETS

1974 ◽  
Vol 60 (3) ◽  
pp. 541-553 ◽  
Author(s):  
John R. Feagler ◽  
Thomas W. Tillack ◽  
David D. Chaplin ◽  
Philip W. Majerus
Keyword(s):  
Cell Surface ◽  
Affinity Chromatography ◽  
Human Platelet ◽  
Human Platelets ◽  
Cell Surface Receptor ◽  
Platelet Surface ◽  
Release Reaction ◽  
Intramembranous Particles ◽  
Receptor Sites ◽  
Surface Receptor

We have previously demonstrated that lentil phytohemagglutinin (lentil-PHA) binds to human platelet membranes without causing either aggregation or the release reaction. When platelets are treated with thrombin, there is an increase in lentil-PHA binding suggesting the appearance of new receptor sites on the cell surface. We prepared a lentil-PHA-ferritin conjugate using affinity chromatography which was used to saturate cell surface receptor sites. Studies using this conjugate suggest that thrombin causes a complex change in the platelet surface involving a decrease in the number of lentil-PHA receptor sites on the external platelet surface with a marked increase in sites within the center of the canalicular system. These increased sites may result from fusion of granule membranes with the canalicular membranes during the secretion process. There is no obvious relationship between lentil-PHA receptor sites and intramembranous particles.

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 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.

Impaired Platelet Function in Sept8-Deficient Mice In Vitro

2020 ◽  
Author(s):  
Kerstin Jurk ◽  
Katharina Neubauer ◽  
Victoria Petermann ◽  
Elena Kumm ◽  
Barbara Zieger
Keyword(s):  
Platelet Function ◽  
Thrombin Generation ◽  
Human Platelets ◽  
Platelet Surface ◽  
Integrin Αiibβ3 ◽  
Glycoprotein Vi ◽  
Fibrinogen Receptor ◽  
Static Conditions ◽  
The Impact

AbstractSeptins (Septs) are a widely expressed protein family of 13 mammalian members, recognized as a unique component of the cytoskeleton. In human platelets, we previously described that SEPT4 and SEPT8 are localized surrounding α-granules and move to the platelet surface after activation, indicating a possible role in platelet physiology. In this study, we investigated the impact of Sept8 on platelet function in vitro using Sept8-deficient mouse platelets. Deletion of Sept8 in mouse platelets caused a pronounced defect in activation of the fibrinogen receptor integrin αIIbβ3, α-granule exocytosis, and aggregation, especially in response to the glycoprotein VI agonist convulxin. In contrast, δ-granule and lysosome exocytosis of Sept8-deficient platelets was comparable to wild-type platelets. Sept8-deficient platelet binding to immobilized fibrinogen under static conditions was diminished and spreading delayed. The procoagulant activity of Sept8-deficient platelets was reduced in response to convulxin as determined by lactadherin binding. Also thrombin generation was decreased relative to controls. Thus, Sept8 is required for efficient integrin αIIbβ3 activation, α-granule release, platelet aggregation, and contributes to platelet-dependent thrombin generation. These results revealed Sept8 as a modulator of distinct platelet functions involved in primary and secondary hemostatic processes.

Mobilization of Fibrinogen Receptor Sites on Human Platelets Stimulated with ADP is Prevented by Prostacyclin

1979 ◽  
Author(s):  
J. Hawiger ◽  
S. Parkinson ◽  
S. Timmons
Keyword(s):  
Inhibitory Effect ◽  
Human Platelets ◽  
Affinity Constant ◽  
Human Fibrinogen ◽  
Fibrinogen Receptor ◽  
Receptor Sites ◽  
Plasma Factor ◽  
Potent Inhibitory Effect

Fibrinogen is a plasma factor required for aggregation of human platelets by ADP. The mechanism of platelet-ADP-fibrinogen interaction was studied by measuring the equilibrium binding of 125I-fibrinogen to human platelets separated from plasma proteins. Binding of 125I-fibrinogen to platelets not stimulated with ADP was low and unaffected by an excess of unlabel led fibrinogen. However, when platelets were stimulated with 4μM of ADP, there was an eightfold increase In the number of available binding sites for human fibrinogen, with affinity constant of 1.9 x 109M-1. This striking increase in fibrinogen receptor sites on human platelets was specific for ADP as contrasted to ATP, AMP, and adenosine. Prostacyclin (Prostaglandin I2, PGI2), a novel prostaglandin produced by the blood vessel wall, completely blocked this ADP-induced increase in fibrinogen receptor sites on human platelets. The effect of PGI2 was prompt and concentration dependent, reaching maximum at 10-9M. 6-keto PGF2 a stable derivative ot PGI2, did not have such an effect. Thus movement of fibrinogen receptor sites on human platelet membrane stimulated with ADP is prevented by PGI2. This represents a new biologic property of this vascular hormone and contributes to better understanding of its potent inhibitory effect in vitro and in vivo on ADP-induced platelet aggregation requiring mobilization of fibrinogen receptor.

Interspecies Comparison of Platelet Aggregation, LIBS Expression and Clot Retraction: Observed Differences in GPIIb-IIIa Functional Activity

1995 ◽  
Vol 74 (06) ◽  
pp. 1551-1556 ◽  
Author(s):  
Lisa K Jennings ◽  
Melanie M White ◽  
Timothy D Mandrell
Keyword(s):  
Platelet Aggregation ◽  
Conformational Changes ◽  
Low Dose ◽  
Species Differences ◽  
Receptor Occupancy ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Clot Retraction ◽  
Fibrinogen Receptor ◽  
Functional Aspects

SummaryWe examined interspecies differences in the function of the platelet fibrinogen receptor, GPIIb-IIIa, by comparing platelet aggregation responses to adenosine diphosphate (ADP) added alone or in combination with a GPIIIa specific monoclonal antibody (mAb), D3. D3 can activate the GPIIb-IIIa receptor in the absence of platelet activation, and it preferentially binds to a region on the GPIIIa subunit after the GPIIb-IIIa complex is occupied by ligand. Using human, monkey, dog, rabbit and pig platelets, we examined whether all species’ platelets bound the D3 mAb similarly, and if the binding of Arg-Gly-Asp-Ser (RGDS) peptides induced the exposure of the anti-LIBS (D3) epitope as previously described for human platelets. We also evaluated how blocking of this neoantigenic region by the D3 mAb affected clot retraction, a process that requires linkage of GPIIb-IIIa with fibrin(ogen) and the platelet cytoskeleton. We found that all species tested bound the D3 mAb. Only in human and monkey platelets did D3 cause aggregation as well as inhibit clot retraction. However, in all species tested, except for pig, D3 prevented disaggregation of platelets typically observed when platelets are treated with low dose ADP. With the exception of pig platelets, there was increased D3 binding to platelets in the presence of RGDS peptides. We propose that this region of GPIIIa is important in the conformational changes that GPIIb-IIIa undergoes during the binding of ligand in most species tested. Our studies suggest 1) there are measurable inter-species differences in GPIIb-IIIa mediated platelet aggregation and clot retraction, 2) LIBS expression due to receptor occupancy is a common but not all-inclusive response and 3) interspecies comparisons may be useful in understanding structural and functional aspects of platelet GPIIb-IIIa.

Junctional Adhesion Molecule-A Is a Novel Csk-Binding Protein Which Recruits Csk to the Integrin aIIbb3 and Suppresses Outside-In Signaling In Platelets

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2120-2120
Author(s):  
Meghna Ulhas Naik ◽  
Ulhas P Naik
Keyword(s):  
Adhesion Molecule ◽  
Conflicts Of Interest ◽  
Human Platelets ◽  
Clot Retraction ◽  
Negative Regulators ◽  
Vessel Occlusion ◽  
Platelet Surface ◽  
Occlusion Time ◽  
Functional Studies ◽  
Signaling Events

Abstract Abstract 2120 Platelet activation is regulated by both positive and negative regulators present within the platelets so that unwanted activation is suppressed, but, when needed, occurs rapidly. A significant amount of effort has been devoted towards understanding the positive regulators of platelet function. However, very little is known about the negative regulators. Dysregulation of the endogenous negative regulators may aid the thrombotic complications seen in various diseases. Upon ligand binding to integrin aIIbb3, a cascade of signaling known as outside-in signaling is induced through the integrin that regulates platelet aggregation and clot retraction. How endogenous negative regulators suppress these events is not well understood. We have previously identified junctional adhesion molecule A (JAM-A), on the platelet surface. We found that Jam-a knockout mice show a prothrombotic phenotype as assessed by significantly (P<0.00001) shortened tail bleeding time, decreased carotid vessel occlusion time and increased pulmonary thromboembolism. Platelet functional studies revealed that Jam-a null platelets were hyperactive to physiological agonists. Surprisingly, inside-out signaling events were not affected in Jam-a null platelets. It is therefore possible that observed hyper-reactivity of Jam-a null platelets could be due to enhanced outside-in signaling. To test this, we performed a clot retraction assay. The clot retraction in wild type (Wt) occurred normally, which began after 1 h and about 50% was completed by 2 h. On the contrary, in Jam-a null platelets, the process of clot retraction was significantly enhanced (P<0.0001). It was initiated before 1h and was completed within 2 h. When analyzed for outside-in signaling events such as b3 tyrosine phosphorylation and c-Src phosphorylation, we found both significantly enhanced in Jam-a null platelets. To assess the mechanism by which JAM-A suppresses outside-in signaling, we analyzed the phosphorylation status of JAM-A. Interestingly, JAM-A was found to be phosphorylated on Y280 in unactivated platelets and rapidly dephosphorylated upon initiation of outside-in signaling. On the other hand, in resting platelets, a minimally phosphorylated S284 residue of JAM-A is rapidly phosphorylated, suggesting that there is a dephosphorylation/phosphorylation switch that may be involved in regulating outside-in signaling. Furthermore, we found that JAM-A associates with integrin aIIbb3 in unactivated human platelets, but this association was disrupted during outside-in signaling as determined by co-immunoprecipitation. We also found Csk, a C-terminal Src kinase, coimmunoprecipitating (IP) with JAM-A from resting, but not activated, platelet lysates, suggesting that JAM-A may be recruiting Csk to unactivated integrins and thus suppressing signaling. To test this, we analyzed association of Csk with integrin in Jam-a null platelets and found that in Wt platelets Csk was abundantly present in the integrin IP, but was completely absent in the integrin IP of the Jam-a null platelet lysates. These results clearly suggest that JAM-A recruits Csk to the integrin and thus suppresses outside-in signaling. Disclosures: No relevant conflicts of interest to declare.

Absence of Myosin on Platelet Membranes Supported by Membrane Iodination, Immuno-Fluorescence and Functional Studies

1979 ◽  
Author(s):  
J.C. Mattson ◽  
W.J. Esselman ◽  
M.L. Schwarz ◽  
C.A. Zuiches
Keyword(s):  
Membrane Proteins ◽  
Protein Complex ◽  
Human Platelet ◽  
Human Platelets ◽  
Platelet Membrane ◽  
Clot Retraction ◽  
Platelet Surface ◽  
Functional Studies ◽  
Sds Page ◽  
Formalin Fixed

Immunofluorescence and functional studies were performed utilizing the immunoglobulin fraction of antisera raised against chromatographically purified human platelet myosin. When non-permeable, formalin fixed platelets were used, no FITC staining of the platelet membrane occurred indicating the myosin is not present on the platelet surface. When similar studies were performed on formalin fixed platelets using antibodies raised against the contractile protein complex, thrombosthenin, membrane fluorescence occurred. Autoradiographs of SDS-PAGE gels of the immune precipitate produced by reacting 125I labeled human platelet thrombosthenin with antithrombosthenin demonstrated that anti-thrombosthenin antisera was capable of reacting with at least three iodinated proteins In addition to myosin. 125I lactoperoxidase catalyzed labeling of the external membrane proteins of resting and ADP stimulated platelets indicated no external labeling of myosin although actin appeared to be labeled. In functional studies, incubation of human platelets with antimyosin did not produce aggregation nor did it Inhibit subsequent aggregation by ADP, collagen or epinephrine. Similarly, treatment of intact platelets with antimyosin did not cause Inhibition of clot retraction. These studies support the thesis that myosin is not localized on the external platelet membrane.

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.

Fibrinogen receptor movement on the ventral surface of platelets

1990 ◽  
Vol 48 (3) ◽  
pp. 22-23
Author(s):  
S.L. Goodman ◽  
Q.J. Lai ◽  
K. Park ◽  
R.M. Albrecht
Keyword(s):  
Thrombus Formation ◽  
Blood Platelets ◽  
Blood Platelet ◽  
Ventral Surface ◽  
Clot Retraction ◽  
Ligand Complex ◽  
Platelet Surface ◽  
Fibrinogen Receptor ◽  
Straight Lines

The fibrinogen receptor (glycoprotein Ilb/IIIa) on the blood platelet surface is important in platelet adhesion, cohesion, and thrombus formation. Previously, we showed that the fibrinogen receptors of surface adherent platelets become motile once ligand is bound. Subsequent to the binding of fibrinogen to GP Ilb/IIIa, either as soluble fibrinogen or conjugated to colloidal gold, the receptor-ligand complex moves from areas overlying the peripheral regions of the platelet to regions overlying more central areas. This movement occurs in relatively straight lines and requires the intact actin and myosin cytoskeleton. This receptor’s motility may be involved in clot retraction and thrombus formation. In vivo, blood platelets contact surfaces coated with fibrinogen and other proteins. Since GP Ilb/IIIa is an adhesion receptor we wished to determine if platelet contact with a fibrinogen coated surface would initiate movement of receptors on the surface-adherent or ventral face of the platelets, and if platelets move or redistribute surface adsorbed fibrinogen.

Sign in / Sign up

Export Citation Format

Share Document