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At least 19 recordsLinked to original sources
PubMed1985

Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex.

Several lines of evidence indicate that the platelet membrane glycoprotein IIb-IIIa complex (GP IIb-IIIa) is necessary for the expression of platelet fibrinogen receptors. The purpose of the present study was to determine whether purified GP IIb-IIIa retains the properties of the fibrinogen receptor on platelets. Glycoprotein IIb-IIIa was incorporated by detergent dialysis into phospholipid vesicles composed of 30% phosphatidylcholine and 70% phosphatidylserine. 125I-Fibrinogen binding to the GP IIb-IIIa vesicles, as measured by filtration, had many of the characteristics of 125I-fibrinogen binding to whole platelets or isolated platelet plasma membranes: binding was specific, saturable, reversible, time dependent, and Ca2+ dependent. The apparent dissociation constant for 125I-fibrinogen binding to GP IIb-IIIa vesicles was 15 nM, and the maximal binding capacity was 0.1 mol of 125I-fibrinogen/mol of GP IIb-IIIa. 125I-Fibrinogen binding was inhibited by amino sugars, the GP IIb and/or IIIa monoclonal antibody 10E5, and the decapeptide from the carboxyl terminus of the fibrinogen gamma chain. Furthermore, little or no 125I-fibrinogen bound to phospholipid vesicles lacking protein or containing proteins other than GP IIb-IIIa (i.e. bacteriorhodopsin, apolipoprotein A-I, or glycophorin). Also, other 125I-labeled plasma proteins (transferrin, orosomucoid) did not bind to the GP IIb-IIIa vesicles. These results demonstrate that GP IIb-IIIa contains the platelet fibrinogen receptor.

Amino Acids↗
PubMed1989

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

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.

Adenosine Diphosphate↗
PubMed2004

Interaction between a genetic variant of the platelet fibrinogen receptor and fibrinogen levels in determining the risk of cardiovascular events.

BACKGROUND: The PlA1A2 polymorphism of glycoprotein IIIa (GPIIIa), which affects postoccupancy signaling by the platelet fibrinogen receptor IIbIIIa, has been investigated as a potential genetic risk factor for cardiovascular events in numerous studies, without consistent results. We investigated whether the effect of this genetic variant of the platelet fibrinogen receptor on the risk of cardiovascular events is affected by fibrinogen plasma levels. METHODS: The GPIIIa PlA1A2 polymorphism and fibrinogen levels were determined in 455 men with angiographically documented coronary atherosclerosis. RESULTS: Neither carriership of the rare PlA2 allele nor fibrinogen plasma levels affected the time to cardiovascular event, as assessed in a proportional hazards model. However, there was a significant interaction between PlA2 carriership and fibrinogen plasma levels (P =.002). Carriership of the variant PlA2 allele significantly affected event-free survival only in individuals within the highest fibrinogen quartile (hazard ratio, 2.7; 95% CI, 1.1 to 7.1; P =.03). CONCLUSIONS: We observed a statistically significant interaction between a genetic variant of the platelet fibrinogen receptor and fibrinogen levels in determining the risk of cardiovascular events. This interaction may account for the inconsistent results of genetic association studies investigating this genotype as a genetic risk factor in thrombotic cardiovascular events.

Coronary Artery Disease↗
PubMed2002

Protein kinase C- and calcium-regulated pathways independently synergize with Gi pathways in agonist-induced fibrinogen receptor activation.

Platelet fibrinogen receptor activation is a critical step in platelet plug formation. The fibrinogen receptor (integrin alphaIIbbeta3) is activated by agonist-mediated G(q) stimulation and resultant phospholipase C activation. We investigated the role of downstream signalling events from phospholipase C, namely the activation of protein kinase C (PKC) and rise in intracellular calcium, in agonist-induced fibrinogen receptor activation using Ro 31-8220 (a PKC inhibitor) or dimethyl BAPTA [5,5'-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetra-acetic acid], a high-affinity calcium chelator. All the experiments were performed with human platelets treated with aspirin, to avoid positive feedback from thromboxane A2. In the presence of Ro 31-8220, platelet aggregation caused by U46619 was completely inhibited while no effect or partial inhibition was seen with ADP and the thrombin-receptor-activating peptide SFLLRN, respectively. In the presence of intracellular dimethyl BAPTA, ADP- and U46619-induced aggregation and anti-alphaIIbbeta3 antibody PAC-1 binding were completely abolished. However, similar to the effects of Ro 31-8220, dimethyl BAPTA only partially inhibited SFLLRN-induced aggregation, and was accompanied by diminished dense-granule secretion. When either PKC activation or intracellular calcium release was abrogated, aggregation and fibrinogen receptor activation with U46619 or SFLLRN was partially restored by additional selective activation of the G(i) signalling pathway. In contrast, when both PKC activity and intracellular calcium increase were simultaneously inhibited, the complete inhibition of aggregation that occurred in response to either U46619 or SFLLRN could not be restored with concomitant G(i) signalling. We conclude that, while the PKC- and calcium-regulated signalling pathways are capable of inducing activating fibrinogen receptor independently and that each can synergize with G(i) signalling to cause irreversible fibrinogen receptor activation, both pathways act synergistically to effect irreversible fibrinogen receptor activation.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1981

A role for prostaglandins and thromboxanes in the exposure of platelet fibrinogen receptors.

Exposure of fibrinogen receptors by a variety of agonists is a prerequisite for platelet aggregation. Because the synthesis of prostaglandins and thromboxane A2 also occurs during platelet aggregation we wondered whether these agents participate in the exposure of platelet fibrinogen receptors. Therefore, we measured the binding of human 125I-fibrinogen to gel-filtered normal human platelets after prostaglandin and thromboxane synthesis had been inhibited by aspirin or indomethacin. The fibrinogen binding assay was performed at 37 degrees C but without stirring to prevent the formation of platelet aggregates. Platelet secretion, measured with [14C]serotonin, did not occur during the procedure. Aspirin or indomethacin inhibited fibrinogen binding stimulated by 10 microM epinephrine by 53%, and inhibited fibrinogen binding stimulated by 1-2 microM ADP by 37.1%. However, ADP at concentrations greater than 2 microM returned fibrinogen binding toward control values. Scatchard analysis demonstrated that aspirin decreased the number but not the affinity of the exposed fibrinogen receptors. To determine whether prostaglandins are capable of directly exposing fibrinogen receptors, prostaglandin H2 was used to stimulate platelets in the fibrinogen binding assay. Prostaglandin H2 exposed approximately 54,000 fibrinogen receptors/platelet and corrected the deficit in receptor exposure induced by aspirin. These studies demonstrate that platelet prostaglandins or thromboxane A2 can play a direct role in the exposure of platelet fibrinogen receptors. In addition, they suggest that the synthesis of prostaglandins and thromboxane A2 by stimulated platelets may be all that is required for optimal secondary platelet aggregation.

Adenosine Diphosphate↗
PubMed1995

Non-peptide fibrinogen receptor antagonists. 7. Design and synthesis of a potent, orally active fibrinogen receptor antagonist.

The design, synthesis, and pharmacological evaluation of L-734,217, a potent, low-molecular weight, orally active fibrinogen receptor antagonist, is reported. A strategy for producing low-molecular weight inhibitors from the peptide c-[(Ac)CRGDC] A, previously reported from these laboratories, is outlined. This strategy combines a retrodesign analysis of the conformationally defined cyclic peptide A with stereochemical information present in the arginine-glycine-aspartic acid (RGD) tripeptide sequence, culminating with the discovery of L-734,217. L-734,217 inhibited the aggregation of human, dog, and chimpanzee platelets at concentrations below 100 nM and was found to be > 15000-fold less effective at inhibiting the attachment of human umbilical vein endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting the aggregation of platelets. L-734,217 showed significant ex vivo antiplatelet activity following oral administration in dogs and chimpanzees at doses of 1.0 and 2.0 mg/kg, respectively, and has been selected as a clinical candidate for development as an antithrombotic agent.

Administration, Oral↗
PubMed1987

Induction of the fibrinogen receptor on human platelets by intracellular mediators.

We have used platelets permeabilized with saponin to examine the mechanism by which platelet activation causes the exposure of surface receptors for fibrinogen. Receptor exposure was detected using 125I-fibrinogen and 125I-PAC1, a monoclonal antibody specific for the activated form of the fibrinogen receptor. The potential mediators that were studied included guanyl-5'-yl imidodiphosphate (Gpp(NH)p) and guanosine 5'O-(thiotriphosphate) (GTP gamma S), which cause G protein-dependent phospholipase C activation in platelets; inositol 1,4,5-triphosphate (IP3), which causes Ca2+ release from the platelet dense tubular system; and diacylglycerol and phorbol ester, which activate protein kinase C. Each of these molecules caused fibrinogen and PAC1 binding. The effect of IP3 was mimicked by raising the cytosolic free Ca2+ concentration in the permeabilized platelets. However, IP3 and Ca2+-induced PAC1 binding were abolished by indomethacin or aspirin, which had no effect on PAC1 binding caused by Gpp(NH)p, phorbol ester, or diacylglycerol. This suggests that the response to IP3 and Ca2+ is due to the formation of metabolites of arachidonic acid. One such metabolite, TxA2, is believed to activate platelets by stimulating G protein-dependent phosphoinositide hydrolysis. Indeed, we found that the G protein inhibitor guanyl-5'-yl thiophosphate (GDP beta S) inhibited PAC1 binding caused by a thromboxane A2 analog (U46619), IP3, and Ca2+, but had no effect on diacylglycerol or phorbol ester-induced PAC1 binding. Thrombin-induced PAC1 binding and phosphoinositide hydrolysis were also inhibited by GDP beta S and by pertussis toxin. Increasing the thrombin concentration overcame the inhibition of PAC1 binding caused by GDP beta S but did not overcome the inhibition of phosphoinositide hydrolysis. These observations demonstrate that fibrinogen receptor exposure occurs by at least two routes. One of these, in response to agonists such as thrombin and U46619, is initiated by G protein-dependent phosphoinositide hydrolysis and involves the formation of IP3 and diacylglycerol. IP3 appears to act by stimulating Ca2+-dependent arachidonic acid metabolism which, in turn, triggers further phosphoinositide hydrolysis. Diacylglycerol acts by stimulating protein kinase C. A second route is activated by high concentrations of thrombin and is independent of phosphoinositide hydrolysis.

Antibodies, Monoclonal↗
PubMed1987

Evidence for two functionally different fibrinogen receptors on hemopoietic cells: the glycoprotein IIb-IIIa and the mitogenic fibrinogen receptor.

We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.-P. et al.: Proc. Natl. Acad. Sci. USA 83:6494-6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C-terminal sequence of the fibrinogen gamma chains or by synthetic peptides containing the Arg-Gly-Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which were mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 X 10(-6) M. This value was similar to the KI value of unlabeled fragments D (2.47 X 10(-6) M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb-IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.

Antibodies, Monoclonal↗
PubMed1989

A monoclonal antibody against the platelet fibrinogen receptor contains a sequence that mimics a receptor recognition domain in fibrinogen.

The binding of fibrinogen to its platelet receptor, the glycoprotein IIb-IIIa complex, is mediated, in part, by an Arg-Gly-Asp (RGD) sequence within the fibrinogen A alpha chain. PAC1 is an IgM-kappa murine monoclonal antibody that binds to the platelet fibrinogen receptor, and its binding is inhibited by both fibrinogen and RGD-containing peptides. To identify the regions of PAC1 that interact with the fibrinogen receptor, we determined the mRNA sequences of PAC1 immunoglobulin heavy and light chain variable regions. Five out of the six complementarity-determining regions (CDRs) of PAC1 had entirely germline sequences with no regions of similarity to fibrinogen. However, CDR3 of the PAC1 heavy chain (H-CDR3) was very large and unique due to the insertion of a novel D region segment. H-CDR3 contained a sequence, Arg-Tyr-Asp (RYD), that, if present in the proper conformation, might behave like the RGD sequence in fibrinogen. A 21-residue synthetic peptide encompassing the H-CDR3 region inhibited fibrinogen-dependent platelet aggregation as well as the binding of PAC1 (Ki = 10 microM) and fibrinogen (Ki = 5 microM) to activated platelets. The RYD region of H-CDR3 appeared to be central to its function, because substitution of the tyrosine with glycine increased the inhibitory potency of the peptide by 10-fold, while replacing the tyrosine with D-alanine or inverting the RYD sequence sharply reduced the inhibitory potency. Thus, the linear sequence, RYD, within H-CDR3 of PAC1 appears to mimic the RGD receptor recognition sequence in fibrinogen. This type of immunologic approach could be useful in studying the structural basis of other receptor-ligand interactions.

Amino Acid Sequence↗
PubMed1984

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

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.

Blood Platelets↗
PubMed1991

Activation-independent exposure of the GPIIb-IIIa fibrinogen receptor.

Following platelet activation, surface receptors for fibrinogen are exposed. On the activated platelet, glycoprotein IIb-IIIa (GPIIb-IIIa) serves as the receptor for fibrinogen. However, the molecular mechanisms which regulate GPIIb-IIIa fibrinogen receptor exposure are unknown. D3GP3 is an IgG1, kappa monoclonal antibody which is specific for glycoprotein IIIa (GPIIIa). The binding of D3GP3 to GPIIIa, in intact GPIIb-IIIa complexes, induces fibrinogen binding and platelet aggregation. To determine if D3GP3 binding to GPIIIa directly caused the exposure of fibrinogen receptors or, secondarily, due to stimulus response coupling, platelet activation parameters were monitored following the addition of D3GP3 to platelets suspensions. D3GP3 binding did not induce detectable Ca++ mobilization, protein phosphorylation or activation of the pertussis toxin sensitive G-protein subunit alpha-41. Further, D3GP3-induced aggregation was not blocked by PGE1, aspirin, apyrase or the combination of all three reagents. Scanning electron microscopy of D3GP3-induced aggregates demonstrated that the aggregates were composed of discoid platelets. These data suggest that the binding of D3GP3 to GPIIIa induced a conformational change in GPIIb-IIIa such that the fibrinogen receptor was exposed in an activation-independent fashion. This provides evidence that conformational changes in the GPIIb-IIIa complex can result in the transformation of the complex to the high affinity binding competent state.

Antibodies, Monoclonal↗
PubMed2002

A fibrinogen receptor from group B Streptococcus interacts with fibrinogen by repetitive units with novel ligand binding sites.

Group B Streptococcus (GBS) is a frequent cause of bacterial sepsis and meningitis in neonates. During the course of infection, GBS colonizes and invades a number of host compartments, thereby interacting with different host proteins. In the present report, we describe the isolation of the fbsA gene, which encodes a fibrinogen receptor from GBS. The deduced FbsA protein is characterized by repetitive units, each 16 amino acids in length. Sequencing of the fbsA gene from five different GBS strains revealed significant variation in the number of repeat-encoding units. The deletion of the fbsA gene in the genome of GBS 6313 completely abolished fibrinogen binding, suggesting that FbsA is the major fibrinogen receptor in this strain. Growth of the fbsA deletion mutant in human blood was significantly impaired, indicating that FbsA protects GBS from opsonophagocytosis. In Western blot experiments with truncated FbsA -proteins, the repeat region of FbsA was identified as mediating fibrinogen binding. Using synthetic peptides, even a single repeat unit of FbsA was demonstrated to bind to fibrinogen. Spot membrane analysis and competitive binding experiments with peptides carrying single amino acid substitutions allowed the prediction of a fibrinogen-binding motif with the consensus sequence G-N/S/T-V-L-A/E/M/Q-R-R-X-K/R/W-A/D/E/N/Q-A/F/I/L/V/Y-X-X-K/R-X-X.

Amino Acid Sequence↗
PubMed1983

Exposure of fibrinogen receptors on fresh and stored platelets by ADP and epinephrine as single agents and as a pair.

Platelet concentrates stored at 22 degrees C have a marked decrease in their aggregation response to adenosine diphosphate (ADP) or epinephrine but a normal response to these agents when used as a pair. Since platelet stimulation involves exposure of receptors for fibrinogen, we studied fibrinogen binding to platelets from fresh and stored concentrates. Following stimulation with 10 microM ADP or 20 microM epinephrine, platelet suspensions from fresh concentrates bound 125I-fibrinogen in a reaction that reached completion within 30 min. Significantly less binding occurred in suspensions from platelet concentrates that had been stored for 5 days at 22 degrees C. When stimulated by ADP and epinephrine as a pair (2 microM each), binding of fibrinogen to platelets was complete within 10-15 min and was not significantly decreased in suspensions from stored concentrates. We also investigated the effect of storage on the glycoprotein IIb-IIa complex, thought to be a specific receptor for fibrinogen on the platelet surface. Binding of a monoclonal antibody specific for this complex (B59.2) to platelet suspensions was unaffected by 5 days of storage. Furthermore, B59.2 inhibited aggregation, secretion, and fibrinogen binding of fresh and stored platelets stimulated with the pair of agents just as it did with single agents. We conclude that storage for 5 days at 22 degrees C impairs the exposure of fibrinogen receptors on platelets in response to ADP or epinephrine when used as single agents, without affecting the glycoprotein IIb-IIIa complex quantitatively. The function of the receptor is normal in response to the pair of agents.

Adenosine Diphosphate↗
PubMed1995

On the structure and function of platelet integrin alpha IIb beta 3, the fibrinogen receptor.

Platelet membrane glycoprotein (GP) IIb/IIIa (alpha IIb beta 3), a Ca(2+)-dependent heterodimer, serves as an inducible receptor for fibrinogen and other adhesive plasma proteins, and is the most thoroughly studied integrin receptor. Intensive research during the past several years has elucidated the major features of its biosynthetic pathway, covalent structure, domain organization, and topography, and we are beginning to get an insight into the cellular mechanisms controlling integrin function. The emerging picture indicates that platelet-specific elements initiate at the cytoplasmic domains of GPIIb/IIIa a signal that leads to conformational changes within the integrin's extracellular domains and expression of the fibrinogen receptor. The simultaneous occupancy on adjacent platelets of receptors with dimeric fibrinogen molecules leads to platelet aggregation. Further structural alterations promote clustering of occupied GPIIb/IIIa complexes and their attachment to the remodelling cytoskeletal network. This interaction provides the physical link for clot retraction to occur and appears to regulate the compartmentalization, and local activation, of a multienzymatic complex which translates the ligand-binding information into time-dependent irreversibility of the fibrinogen-GPIIb/IIIa interaction. Platelet GPIIb/IIIa plays, thus, a central role in thrombus formation both in health and disease: abnormalities in the platelet adhesive mechanisms responsible for the formation of the hemostatic plug, lead to major pathophysiologic disorders, ranging from severe bleeding to thrombosis. It is, therefore, not surprising that GPIIb/IIIa has been the subject of intensive research during the last decades, since a detailed knowledge of the molecular biology and the mechanism underlying the platelet activation and aggregation processes may aid in the rational design of both an effective gene replacement therapy, and of potent and specific anti-thrombotic drugs. The aim of this minireview is to summarize many functional and structural data from different laboratories in the perspective of an emerging model that may help us to understand structure-function relationships of GPIIb/IIIa and of other members of the integrin family.

Amino Acid Sequence↗
PubMed1991

A comparison of the fibrinogen receptor distribution on adherent platelets using both soluble fibrinogen and fibrinogen immobilized on gold beads.

The distribution of fibrinogen receptors was determined on the surface of adherent platelets using both direct labeling with the ligand fibrinogen which was immobilized on gold particles (Fg-Au) and indirect immunogold (Ig-Au) labeling of bound soluble fibrinogen identified with a rabbit polyclonal anti-fibrinogen antibody. Two distinctly different patterns of labeling were obtained and appeared to depend on whether solid phase fibrinogen (Fg-Au) or soluble phase released fibrinogen were bound to the membrane receptor. The membrane-bound Fg-Au reorganized in patterns that closely mimicked the organization of the underlying cytoskeleton. In approximately 18% of the adherent platelets, Fg-Au was seen in channels or vesicle-like structures lying deep to the platelet surface suggesting internalization into the open canalicular system and/or endocytosis. The labeling pattern obtained when identifying the location of membrane-bound soluble released fibrinogen by Ig-Au was diffuse and lacked the organizational patterns characteristic of Fg-Au. Unlike the Fg-Au probe, early dendritic platelets were heavily labeled by the soluble phase fibrinogen using the Ig-Au technique. Although the label covered the entire exposed platelet membrane in fully spread platelets, labeling over the peripheral web was more dense than that over the intermediate or granulomere zone. The diffuse organization and heavier peripheral distributional pattern of the glycoprotein IIb-IIIa (GP IIb-IIIa) receptor in fixed, adherent platelets, was also seen with the GP IIb-IIIa receptor-specific antibody AP-2. The binding of both the Fg-Au and Ig-Au were inhibited using the tetrapeptide Arg-Gly-Asp-Ser (RGDS) (93% and 98% inhibition, respectively), AP-2 (98% and 97%, respectively) and platelets from patients with Glanzmann's thrombasthenia (GT) (99% and 98%, respectively). The data presented provides the first report that receptor reorganization, following binding of fibrinogen, appears to be related to the state of the ligand. Substrate bound fibrinogen (i.e., Fg-Au or fibrinogen bound to another platelet) induces receptor translocation toward the platelet granulomere in a capping-like phenomenon. On the other hand, the binding of soluble released fibrinogen results in formation of microclusters and short linear arrays in a diffuse distribution but does not induce central movement of receptors. Furthermore, double labeling studies clarify that Fg-Au does not identify all available fibrinogen receptors as many are occupied by soluble released fibrinogen. The data presented provides an interesting new perspective on what constitutes an appropriate ligand-receptor stimulus sufficient to induce receptor reorganization.

Amino Acid Sequence↗
PubMed1982

Evidence that changes in platelet cyclic AMP levels regulate the fibrinogen receptor on human platelets.

Fibrinogen binds to human platelets after specific receptor sites are exposed by thrombin, ADP, epinephrine, and other stimuli. Since prostaglandin I2 (PGI2), a potent activator of platelet adenylate cyclase, prevents mobilization of the fibrinogen receptor by aggregating agents, we investigated the relationship between platelet cAMP levels and fibrinogen receptor status in thrombin-stimulated human platelets. A dose-dependent rise in platelet cAMP in response to two adenylate cyclase agonists, PGI2 and forskolin, correlated with progressive inhibition of fibrinogen binding. Moreover, the receptor inhibition produced by either agonist was sustained up to 2 h and was associated with a persistent increase in cAMP levels. The phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine, in the presence of a subthreshold concentration of PGI2 also raised cAMP and inhibited fibrinogen binding. In contrast, the effects of PGI2 on both cAMP and fibrinogen binding were markedly attenuated by 9-(tetrahydro-2-furyl) adenine, an adenylate cyclase inhibitor. These results indicate that the inhibition of fibrinogen binding by PgI2 is linked to its effect on cAMP levels and suggest that elevation of platelet cAMP levels from any cause prevents exposure of the fibrinogen receptor.

Blood Platelets↗
PubMed1990

The down-regulation of the mitogenic fibrinogen receptor (MFR) in serum-containing medium does not occur in defined medium.

Normal human hemopoietic cells such as early bone marrow progenitors, or lymphoma-derived cell lines such as Raji or JM cells, possess a low-affinity receptor specific for fibrinogen. This receptor triggers a mitogenic effect. It differs from the glycoprotein IIb-IIIa which is involved in fibrinogen-induced platelet aggregation. We demonstrate here that this mitogenic fibrinogen receptor (MFR) can be internalized or reexpressed, depending on culture conditions. Internalization was temperature-dependent. At 37 degrees C in the presence of cycloheximide or actinomycin D, the half-life of cell surface MFRs was 2 h, independent of receptor occupancy. Binding of fibrinogen to the MFR resulted in a down-regulation which was fibrinogen dose-dependent. This occurred in serum-supplemented medium but not in defined medium supplemented with fatty acids. Reexpression of MFRs could be induced in 28 to 42 h by serum removal. The down-regulation of mitogenic receptors in plasma or serum could explain why normal cells do not proliferate in the peripheral blood.

Blood↗