Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists

Abstract The exposure of fibrinogen receptors is an early event in agonist- induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC- PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.

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Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists

Blood ◽

10.1182/blood.v73.1.150.bloodjournal731150 ◽

1989 ◽

Vol 73 (1) ◽

pp. 150-158

Author(s):

SJ Shattil ◽

A Budzynski ◽

MC Scrutton

Keyword(s):

Monoclonal Antibody ◽

Whole Blood ◽

Platelet Activating Factor ◽

Synergistic Interaction ◽

Receptor Expression ◽

Thrombin Inhibitors ◽

Early Event ◽

Fibrinogen Receptor ◽

Fibrinogen Binding ◽

Adrenergic Antagonist

The exposure of fibrinogen receptors is an early event in agonist- induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC- PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.

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Binding of plasma factor XIII to thrombin-receptor activated human platelets

Thrombosis and Haemostasis ◽

10.1160/th09-01-0054 ◽

2009 ◽

Vol 102 (07) ◽

pp. 83-89 ◽

Cited By ~ 16

Author(s):

Béla Nagy ◽

Zsuzsa Simon ◽

Zsuzsa Bagoly ◽

László Muszbek ◽

János Kappelmayer

Keyword(s):

Whole Blood ◽

Factor Xiii ◽

Coagulation Factor ◽

Human Platelets ◽

Thrombin Receptor ◽

Type I ◽

Fibrinogen Receptor ◽

Activated Platelets ◽

Fibrinogen Binding ◽

Plasma Factor

SummaryPlatelet-bound coagulation factor XIII (FXIII) is targeted and concentrated at the site where platelet-rich thrombi are formed. Previous studies were in disagreement about the nature of FXIII binding to platelets. In this study, thrombin-receptor activating peptide (TRAP)-stimulated human whole blood and washed platelets were analysed by flow cytometry for the binding of FXIII using a monoclonal antibody against the A subunit of FXIII (FXIII-A). Here, we demonstrate that FXIII-A positivity significantly increased on activated platelets in whole blood compared to unstimulated sample, but not in washed platelets. GPIIb/IIIa receptor plays an essential role in FXIII binding, as fibrinogen receptor antagonist eptifibatide and fibrinogen binding inhibitor RGDS tetrapeptide significantly prevented the binding of FXIII. Furthermore, stimulated platelets from a patient with severe type I Glanzmann thrombasthenia showed insignificant FXIII-A positivity versus healthy controls. In addition, basal negligible amount of FXIII on washed platelets was only slightly increased when highly purified plasma FXIII (FXIII-A2B2) was added upon platelet activation by TRAP. Similarly, no remarkable FXIII-A positivity was observed when we used FXIII-A2B2 with γA/γA fibrinogen. However, γA/γ' fibrinogen significantly augmented FXIII binding on TRAP-stimulated platelets in the presence of non-activated FXIII-A2B2. We conclude that FXIII-A2B2 of plasma origin binds to thrombin-receptor activated platelets via GPIIb/IIIa receptor-bound fibrinogen with γ’-chain and is not capable of direct platelet binding.

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

Blood ◽

10.1182/blood.v68.6.1224.bloodjournal6861224 ◽

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.

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Fibrin Generation in Heparin-Induced Thrombocytopenia (HIT): Pathomechanistic Background for Novel Therapy and Prophylaxis

Blood ◽

10.1182/blood.v120.21.635.635 ◽

2012 ◽

Vol 120 (21) ◽

pp. 635-635

Author(s):

Valerie Tutwiler ◽

Hyun Sook Ahn ◽

Rudy Fuentes ◽

Vincent M. Hayes ◽

Sergei V. Zaytsev ◽

...

Keyword(s):

Monoclonal Antibody ◽

Platelet Activation ◽

Human Blood ◽

Whole Blood ◽

Platelet Adhesion ◽

Thrombin Inhibitors ◽

Single Chain ◽

Monocyte Activation ◽

Platelet Factor ◽

Fibrin Formation

Abstract Abstract 635 HIT is an immune mediated prothrombotic disorder often associated with life- and limb-threatening thrombosis caused by antibodies to a complex between platelet factor 4 (PF4) and heparin. Platelet activation and clearance are considered key to the pathogenesis. Direct thrombin inhibitors, the most commonly used anticoagulant therapy in the treatment of HIT, provide incomplete prevention against development of new thrombi and little effect on the incidence of loss of limb and life. Thus, there is a need for a better understanding of pathogenesis of HIT and new approaches to therapy. We and others have shown that HIT not only is associated with platelet activation, but also involves activation of monocytes and endothelial cells, which together increase thrombin generation that may affect both the amount and structural properties of the resultant fibrin clot. However, this proposed increase in fibrin formation though suspected, has never been directly investigated. Previously, we have shown in the cremaster muscle laser-injury model of thrombosis induced by the HIT-like murine monoclonal anti-hPF4/heparin antibody KKO that transgenic mice expressing both human PF4 (hPF4+) and hFcγRIIA developed larger, more fibrin-rich occlusive thrombi than in control mice expressing hPF4 or hFcγRIIA alone. To quantify fibrin formation in a more controlled setting, we simulated HIT in a BioFlux microfluidic channel system coated with von Willebrand factor by perfusing whole blood at a venule shear stress of 20 dyne/cm2 at 37°C for 10 min. Platelets were labeled by adding Calcein-AM (3 μM) and fibrin was visualized by adding Alexa 647 labeled fibrinogen (1.5 μg/ml) to the whole blood prior to the perfusion. Using NaCitrate-anticoagulated human blood, we observed that recalcified human blood samples exposed to KKO plus hPF4 formed large platelet thrombi and an extensive fibrin network, with fibers radiating from the platelet aggregates and often organized along the direction of flow. In contrast, control samples exposed to a combination of the non-pathogenic anti-hPF4 monoclonal antibody RTO plus hPF4 showed little fibrin and less organization. Quantitative fluorescence analysis showed nine times more fibrin formed after stimulation with KKO plus PF4 than RTO plus PF4. Inhibition of KKO-mediated platelet activation by blocking FcgRIIA with Fab fragments of monoclonal antibody IV.3 in whole blood suppressed platelet adhesion by > 80%, but decreased fibrin formation by only ∼40%. On the other hand, addition of a selective inhibitor of the Syk tyrosine kinase PRT-060318 (Reilly et al., Blood 2011:117:2241–6; kindly provided by Dr. Uma Sinha, Portola Pharmaceuticals) to whole blood at a concentration of 3 μM suppressed both platelet adhesion and fibrin formation by 80% and 70%, respectively. IV.3 inhibits platelet activation alone, while we have shown that PRT-060318 inhibits both platelet activation and monocyte activation with the subsequent release of tissue factor-rich microparticles. These results provide a mechanistic basis for the use of novel therapies in HIT such as fibrinolytic agents. To do so, we studied a novel chimeric pro-fibrinolytic composed of a C-terminal thrombin-specific activatable low molecular weight urokinase (uPA-T) that has its plasmin-activation site replaced by a thrombin cleavage site and linked at its N-terminus to a single-chain variable region (scFv) that binds with high affinity to human platelet aIIb, designed to deliver the agent to sites of incipient thrombosis. Preliminary results show that uPA-T profoundly suppressed fibrin accumulation in both in vitro and in an in vivo model of HIT. This novel approach to therapy takes advantage of our growing understanding of the pathogenesis of the prothrombotic nature of HIT including monocyte activation and formation of fibrin-rich clots. Such therapeutics may be especially effective as targeted therapy in HIT. Disclosures: Cines: Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

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GPIIb-IIIa Antagonist-induced Reduction in Platelet Surface Factor V/Va Binding and Phosphatidylserine Expression in Whole Blood

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614050 ◽

2000 ◽

Vol 84 (09) ◽

pp. 492-498 ◽

Cited By ~ 25

Author(s):

Lori Krueger ◽

A. L. Frelinger ◽

Marc Barnard ◽

Mary Mascelli ◽

Marian Nakada ◽

...

Keyword(s):

Unfractionated Heparin ◽

Whole Blood ◽

Venous Blood ◽

Annexin V ◽

Procoagulant Activity ◽

Thrombin Inhibitors ◽

Thrombin Inhibitor ◽

Factor V ◽

Platelet Surface ◽

Fibrinogen Binding

SummaryIn addition to inhibition of platelet aggregation, GPIIb-IIIa antagonists may reduce thrombotic events via other mechanisms. In a novel whole blood flow cytometric system, we investigated the effects of GPIIb-IIIa antagonists, in the presence or absence of thrombin inhibitors, on platelet surface-bound factor V/Va and platelet surface phospholipids. Diluted venous blood was incubated with either buffer or a GPIIb-IIIa antagonist (abciximab, tirofiban, or eptifibatide). Some samples were pre-incubated with clinically relevant concentrations of unfractionated heparin (UFH), a low molecular weight heparin, a direct thrombin inhibitor, or buffer only. Platelets were then activated and labeled with mAb V237 (factor V/Va-specific) or annexin V (binds phosphatidylserine), fixed, and analyzed by flow cytometry. In the absence of thrombin inhibitors, GPIIb-IIIa antagonists (especially abciximab) significantly reduced agonist-induced platelet procoagulant activity, as determined by reduced binding of V237 and annexin V. At high pharmacologic concentrations, unfractionated heparin and enoxaparin, but not hirudin, further reduced factor V/Va binding to the surface of activated platelets in the presence of GPIIb-IIIa antagonists. Agonist-induced platelet procoagulant activity was reduced in a patient with Glanzmann’s thrombasthenia. We conclude that GPIIb-IIIa antagonists reduce platelet procoagulant activity in whole blood and heparin and enoxaparin augment this reduction. Fibrinogen binding to GPIIb-IIIa is important in the generation of platelet procoagulant activity.

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

Blood ◽

10.1182/blood.v64.1.59.bloodjournal64159 ◽

1984 ◽

Vol 64 (1) ◽

pp. 59-63 ◽

Cited By ~ 1

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.

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

Blood ◽

10.1182/blood.v64.1.59.59 ◽

1984 ◽

Vol 64 (1) ◽

pp. 59-63 ◽

Cited By ~ 19

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.

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miR-204-5p and platelet function regulation: insight into a mechanism mediated by CDC42 and GPIIbIIIa

Thrombosis and Haemostasis ◽

10.1055/a-1497-9649 ◽

2021 ◽

Author(s):

Alix Garcia ◽

Sylvie Dunoyer-Geindre ◽

Séverine Nolli ◽

Catherine Strassel ◽

Jean-Luc Reny ◽

...

Keyword(s):

Binding Assay ◽

Platelet Reactivity ◽

Receptor Expression ◽

Hematopoietic Stem ◽

Fibrinogen Receptor ◽

Fibrinogen Binding ◽

Cardiovascular Patients ◽

Functional Mechanisms ◽

The Impact ◽

Insight Into

Background: Several platelet-derived miRNAs are associated with platelet reactivity (PR) and clinical outcome in cardiovascular patients. We previously showed an association between miR-204-5p and PR in stable cardiovascular patients, but data on functional mechanisms are lacking. Aims: To validate miR-204-5p as a regulator of PR in platelet-like structures (PLS) derived from human megakaryocytes and to address mechanistic issues. Methods: Human hematopoietic stem cells were differentiated into megakaryocytes, enabling the transfection of miR-204-5p and the recovery of subsequent PLS. The morphology of transfected megakaryocytes and PLS was characterized using flow cytometry and microscopy. The functional impact of miR-204-5p was assessed using a flow assay, the quantification of the activated form of the GPIIbIIIa receptor and a fibrinogen-binding assay. qPCR and western blot were used to evaluate the impact of miR-204-5p on a validated target, CDC42. The impact of CDC42 modulation was investigated using a silencing strategy. Results: miR-204-5p transfection induced cytoskeletal changes in megakaryocytes associated with the retracted protrusion of proPLS, but it had no impact on the number of PLS released. Functional assays showed that the PLS produced by megakaryocytes transfected with miR-204-5p were more reactive than controls. This phenotype is mediated by the regulation of GPIIbIIIa expression, a key contributor in platelet–fibrinogen interaction. Similar results were obtained after CDC42 silencing, suggesting that miR-204-5p regulates PR, at least in part, via CDC42 downregulation. Conclusions: We functionally validated miR-204-5p as a regulator of the PR that occurs through CDC42 downregulation and regulation of fibrinogen receptor expression.

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NORMAL EXPRESSION OF THROMBOSPONDIN RECEPTOR SITES ON PLATELETS OF THREE GLANZMANN THROMBASTHENIC PATIENTS

10.1055/s-0038-1644740 ◽

1987 ◽

Author(s):

J L McGregor ◽

H Boukerche

Keyword(s):

Monoclonal Antibody ◽

Platelet Aggregation ◽

Whole Blood ◽

Binding Sites ◽

Competitive Binding ◽

Fibrinogen Binding ◽

Receptor Sites ◽

Low Concentrations ◽

Normal Expression ◽

Stimulation Of

A well characterised anti-thrombospondin (TSP) monoclonal antibody (Mab) LYP8 was used to investigate the presence of TSP receptors on Glanzmann thrombasthenic (G.T.) platelets. LYP8 inhibited platelet aggregation induced by low concentrations of thrombin (0.05 U/ml) or collagen (0.5 ug/ml). The presence of LYP8 did not affect the number of sites and Kd of 125I-fibrinogen binding to thrombin-stimulated normal platelets. Binding of LYP8 to normal platelets was minimal in whole blood (300 IgG molecules/ olatelet), increased in citrated PRP (1187 ± 209 IgG molecules/ platelet) and washed platelets (2967 ± 1278 IgG molecules/platelet) . Thrombin stimulation of platelets, washed in the presence of 2 mM calcium, increased the number of LYP8 binding sites (14917 ± 42n IgG molecules/platelet). Addition of EDTA (5mM) to thrombin-stimulated platelets did not reduce the number of LYP8 binding sites. The number of LYP8 binding sites on thrombin-stimulated platelets of three Glanzmann thrombasthenic patients (showing an absence of the glycoprotein (GP) lib and IIIa)was similar to normals in the presence of 2 mM calcium or 5 mM EDTA. In competitive binding, Mab LYP18 directed against the GPIIb-IIIa complex did not inhibit the binding of labelled monoclonal antibody LYP8. These results strongly suggest that TSP binds to a membrane receptor different from the GPIIb-IIIa complex in the presence of calcium or EDTA. This unidentified receptor may be GPIV also known as GPIIIb (Asch, A. et al. Clin. Res. 1986, 34:450A).

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

Blood ◽

10.1182/blood.v68.6.1224.1224 ◽

1986 ◽

Vol 68 (6) ◽

pp. 1224-1231 ◽

Cited By ~ 36

Author(s):

SJ Shattil ◽

HJ Motulsky ◽

PA Insel ◽

L Flaherty ◽

LF Brass

Keyword(s):

Platelet Aggregation ◽

Adrenergic Receptors ◽

Human Platelets ◽

Receptor Expression ◽

Platelet Surface ◽

Prolonged Stimulation ◽

Fibrinogen Receptor ◽

Aggregation Response ◽

Fibrinogen Binding ◽

Response Expression

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

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