The microvesicle/CD36 complex triggers a prothrombotic phenotype in patients with non‐valvular atrial fibrillation

The mechanisms responsible for platelet activation, the prothrombotic state, in non‐valvular atrial fibrillation (NVAF) are still obscure. Microvesicles (MVs) can transfer various messages to target cells and may be helpful for exploring the detailed mechanisms. We aimed to investigate the possible mechanisms by which proatherogenic factors of NVAF contribute to platelet activation. Two hundred and ten patients with NVAF were stratified as being at ‘low to moderate risk’ or ‘high risk’ for stroke according to the CHADS2 score. Levels of platelet‐derived MVs (PMVs) and platelet activation were examined. CD36‐positive or CD36‐deficient human platelets were stimulated by MVs isolated from NVAF patients with or without various inhibitors in vitro. Levels of PMVs and platelet activation markers enhanced significantly in high‐risk patients. The MVs isolated from plasma of NVAF patients bound to platelet CD36 and activated platelets by phosphorylating the mitogen‐activated protein kinase 4/Jun N‐terminal kinase 2 (MKK4/JNK2) pathways. However, CD36 deficiency protected against MV‐induced activation of platelets. We reveal a possible mechanism of platelet activation in NVAF and suggest that the platelet CD36 might be an effective target in preventing the prothrombotic state in NVAF.     

Molecular and functional differences induced in thrombospondin-1 by the single nucleotide polymorphism associated with the risk of premature, familial myocardial infarction

A serine (Ser-700) amino acid rather than an asparagine (Asn-700) at residue 700 of thrombospondin-1 has been linked to an increased risk for development of premature, familial heart attacks. We now have identified both functional and structural differences between the Ser-700 and Asn-700 thrombospondin-1 variants. The Ser-700 variant increased the rate and extent of platelet aggregation and showed increased surface expression on platelets compared with the Asn-700 variant. These differences could be ascribed to an enhanced interaction of the Ser-700 variant with fibrinogen on the platelet surface and are consistent with a prothrombotic phenotype in Ser-700 individuals. The Ser-700 variant thrombospondin-1 was conformationally more labile than the Asn-700 variant as demonstrated by increased susceptibility to proteolytic digestion and enhanced susceptibility to unfolding by denaturants. These data suggest a potential molecular and cellular basis for a genetic risk factor associated with early onset myocardial infarction.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

Proteins involved in platelet signaling are differentially regulated in acute coronary syndrome: a proteomic study

Background

Platelets play a fundamental role in pathological events underlying acute coronary syndrome (ACS). Because platelets do not have a nucleus, proteomics constitutes an optimal approach to follow platelet molecular events associated with the onset of the acute episode.

Methodology/Principal Findings

We performed the first high-resolution two-dimensional gel electrophoresis-based proteome analysis of circulating platelets from patients with non-ST segment elevation ACS (NSTE-ACS). Proteins were identified by mass spectrometry and validations were by western blotting. Forty protein features (corresponding to 22 unique genes) were found to be differentially regulated between NSTE-ACS patients and matched controls with chronic ischemic cardiopathy. The number of differences decreased at day 5 (28) and 6 months after the acute event (5). Interestingly, a systems biology approach demonstrated that 16 of the 22 differentially regulated proteins identified are interconnected as part of a common network related to cell assembly and organization and cell morphology, processes very related to platelet activation. Indeed, 14 of those proteins are either signaling or cytoskeletal, and nine of them are known to participate in platelet activation by αIIbβ3 and/or GPVI receptors. Several of the proteins identified participate in platelet activation through post-translational modifications, as shown here for ILK, Src and Talin. Interestingly, the platelet-secreted glycoprotein SPARC was down-regulated in NSTE-ACS patients compared to stable controls, which is consistent with a secretion process from activated platelets.

Conclusions/Significance

The present study provides novel information on platelet proteome changes associated with platelet activation in NSTE-ACS, highlighting the presence of proteins involved in platelet signaling. This investigation paves the way for future studies in the search for novel platelet-related biomarkers and drug targets in ACS.     

Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi‐functional platelets and cross‐species function evolution

Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high‐throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomic technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin‐stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across‐species evolutionary link that the orthologous proteins representing “core proteome”, and the “evolutionary proteome” is actually a relatively static proteome.     

Presence of factors that activate platelet aggregation in mitral stenotic patients' plasma

BACKGROUND: Although the association between mitral stenosis (MS) and increased coagulation activity is well recognized, it is unclear whether enhanced coagulation remains localized in the left atrium or whether this represents a systemic problem. To assess systemic coagulation parameters and changes in platelet aggregation, we measured fibrinogen levels and performed in vitro platelet function tests in plasma obtained from mitral stenotic patients' and from healthy control subjects' peripheral venous blood. METHODS: Sixteen newly diagnosed patients with rheumatic MS (Group P) and 16 healthy subjects (Group N) were enrolled in the study. Platelet-equalized plasma samples were evaluated to determine in vitro platelet function, using adenosine diphosphate (ADP), collagen and epinephrine in an automated aggregometer. In vitro platelet function tests in group N were performed twice, with and without plasma obtained from group P. RESULTS: There were no significant differences between the groups with respect to demographic variables. Peripheral venous fibrinogen levels in Group P were not significantly different from those in Group N. Adenosine diphosphate, epinephrine and collagen-induced platelet aggregation ratios were significantly higher in Group P than in Group N. When plasma obtained from Group P was added to Group N subjects' platelets, ADP and collagen-induced, but not epinephrine-induced, aggregation ratios were significantly increased compared to baseline levels in Group N. CONCLUSION: Platelet aggregation is increased in patients with MS, while fibrinogen levels remain similar to controls. We conclude that mitral stenotic patients exhibit increased systemic coagulation activity and that plasma extracted from these patients may contain some transferable factors that activate platelet aggregation.     

JAK2 mutation status, hemostatic risk factors and thrombophilic factors in essential thrombocythemia (ET) patients

The recently discovered JAK2 V617F point mutation, found in 50-60% of ET patients, has been reported to be associated with a higher risk of thrombotic events. In this study, we explored if JAK2 V617F mutation, or coexisting thrombophilic and hemostatic risk factors, contributed to these complications. We examined 32 patients with ET, and looked for pathogenetic JAK2 V617F mutation and prothrombotic genes mutations: factor V Leiden, prothrombin and MTHFR. We also evaluated plasma levels of fibrinogen, factors VIII and XII, AT, protein C, protein S and serum level of homocysteine. Urokinase concentration was assessed in patients' plasma as well as platelet lysates. There was no difference in the number of thrombotic complications between ET patients with and without JAK2 mutation. However, we found a number of thrombophilic and hemostatic risk factors that could contribute to thrombotic complications in ET patients.     

Factor VIII complex in uraemia and effects of haemodialysis.

Levels of the factor VIII complex were found to be raised in patients with chronic renal failure and further raised by regular dialysis. Increased fibrinogen concentrations were also found. These results suggest the existence of a prothrombotic state in uraemia that is exacerbated by haemodialysis. Ristocetin-induced platelet agglutination, however, was depressed in uraemia and worsened by dialysis. This defect may be transferred to normal platelets from dialysed uraemic plasma, suggesting the existence of an inhibitor of the interaction between factor VIII and platelet glycoprotein. These results may help to explain the anomaly of a prolonged bleeding time together with accelerated atherogenesis that is found in patients with uraemia receiving dialysis.     

Fibrinogen distribution on surfaces and in organelles of ADP stimulated human blood platelets

The fibrinogen distribution in platelet organelles after ADP-stimulation was investigated with anti-human fibrinogen using protein A-gold applied to serial sections. Fibrinogen was detected in the so-called alpha-granules of platelets and also in granule protrusions which were observed after ADP-stimulation. The ends of these protrusions were formed as coated membranes and the tips were often in apposition to the surface connected membranes or the plasmalemma. At such places fusion events and hence signs of an exocytosis could be demonstrated by means of cryofixation and cryosubstitution. Examination of serial sections revealed fibrinogen on all these granule profiles. Surface connected membranes, free surfaces and the characteristic structure of the contact zones of aggregated platelets were also labelled by gold particles but less than anticipated. On the platelet surfaces and surface connected membranes fibrinogen was rarely demonstrable with ferritin-labelled anti-human fibrinogen on washed or thrombin-stimulated, almost fibrinogen free platelets. After addition of human fibrinogen to the thrombin stimulated and disaggregated platelets a part of the platelets aggregated spontaneously and formed characteristic contact zones. Anti-human fibrinogen was observed on the free surfaces, in filamentous bridges between the contact spaces and in a tubular surface connected membrane system with involvement of coated membranes at the central ends of these structures. The results indicate the following: all alpha-granules contain fibrinogen; after ADP-stimulation secretion takes place with involvement of coated membranes; during aggregation fibrinogen binds to platelet surfaces and forms contact spaces; fibrinogen is taken up by the surface connected system with involvement of coated membranes.     

Norcantharidin,a clinical used chemotherapeutic agent,acts as a powerful inhibitor by interfering with fibrinogen–integrin αIIbβ3 binding in human platelets

During platelet activation, fibrinogen binds to its specific platelet receptor, integrin α_IIbβ₃, thus completing the final common pathway for platelet aggregation. Norcantharidin (NCTD) is a promising anticancer agent in China from medicinal insect blister beetle. In this study, we provided the evidence to demonstrate NCTD (0.1–1.0 μM) possesses very powerful antiplatelet activity in human platelets; nevertheless, it had no effects on surface P‐selectin expression and only slight inhibition on ATP‐release reaction in activated platelets. Moreover, NCTD markedly hindered integrin α_IIbβ₃ activation by interfering with the binding of FITC‐labelled PAC‐1. It also markedly reduced the number of adherent platelets and the single platelet spreading area on immobilized fibrinogen as well as clot retraction. Additionally, NCTD attenuated phosphorylation of proteins such as integrin β₃, Src and FAK in platelets spreading on immobilized fibrinogen. These results indicate that NCTD restricts integrin α_IIbβ₃‐mediated outside‐in signalling in human platelets. Besides, NCTD substantially prolonged the closure time in human whole blood and increased the occlusion time of thrombotic platelet plug formation and prolonged the bleeding time in mice. In conclusion, NCTD has dual activities, it can be a chemotherapeutic agent for cancer treatment, and the other side it possesses powerful antiplatelet activity for treating thromboembolic disorders.     

Thrombin-stimulated phosphatidylinositol 3-kinase activity in platelets is associated with activation of PYK2 tyrosine kinase: activation of both enzymes is aggregation independent

In this study, we investigated the activation of a new member of the focal adhesion kinase family of tyrosine kinases, the proline-rich tyrosine kinase, or PYK2, in platelets. We show that PYK2 is tyrosine phosphorylated and its activity is increased during early stages of platelet aggregation. This activation coincided with increased association of phosphatidylinositol (PI) 3-kinase and PYK2, as determined by both anti-PI 3-kinase and anti-PYK2 immunoprecipitates. However, under basal conditions, some association of PYK2 and PI 3-kinase was consistently observed, even though little or no tyrosine phosphorylated PYK2 could be detected. In addition, both increased PI 3-kinase activity and increased PYK2 activity could be detected in immunoprecipitates following thrombin stimulation. All of these events were unaffected by blocking platelet aggregation with arginine-glycine-aspartate-serine (RGDS) peptide, which interferes with binding of the platelet integrin alpha(IIb)beta(3) to fibrinogen. Neither was the activation of the PYK2 kinase activity affected by blocking PI 3-kinase activity. These results support a model in which PYK2 is associated with PI 3-kinase in unstimulated platelets and following activation of platelets, there is an increase in tyrosine phosphorylation of PYK2, increased PYK2 activity, and increased association of PYK2 with PI 3-kinase, which may contribute to the increase in PI 3-kinase activity. All of these were found to be early events independent of subsequent platelet aggregation.     

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.     

Identification of the phosphotyrosine proteome from thrombin activated platelets

Signalling cascades are regulated both positively and negatively by tyrosine phosphorylation. Integrin mediated platelet adhesion triggers signal transduction cascades involving translocation of proteins and tyrosine phosphorylation events, ultimately causing large signalling complexes to be assembled. In resting platelets, a small number of phosphorylated proteins are evident with molecular mass of 50-62 kDa and 120-130 kDa. In thrombin activated human platelets, however, there is a large increase in the number of tyrosine phosphorylated signalling proteins detected. These proteins include pCas (130 kDa), FAK (125 kDa), PI(3)k (85 kDa) and src (85 kDa). However, it is unlikely that this list of proteins represents all the dynamic changes that occur after platelet activation and it is understood that more proteins remain unidentified. In this study, we propose a method for the isolation of the phosphotyrosine proteome from both resting and thrombin activated human platelets. All the dynamic phosphotyrosine events that occur in the platelet after thrombin activation were isolated by immunoprecipitation, using the monoclonal antibody 4G10, allowing us to obtain higher concentrations of relatively low abundant proteins. The resulting phosphotyrosine proteomes were separated by two-dimensional gel electrophoresis. Sixty-seven proteins were reproducibly found to be unique in the thrombin activated platelet proteome when compared to resting platelets. We have positively identified ten of these proteins by Western blotting and matrix-assisted laser desorption/ionisation-time of flight mass spectrometry and these include FAK, Syk, ALK-4, P2X6 and MAPK kinase kinase. This proteomics approach to understanding the signalling events following platelet activation may elucidate potential drug targets for the treatment of coronary thrombosis.     

Characteristics of collagen-induced fibrinogen binding to human platelets

Polymerized type I calf skin collagen induced a time-dependent specific binding of 125I-fibrinogen to washed human platelets. Binding occurred more rapidly in a shaken rather than in an unstirred system. It was linear in the range 0.05-0.3 microM added fibrinogen and was saturated at higher fibrinogen concentrations (more than 0.8 microM). Scatchard analysis showed a single population of binding sites (16530 +/- 5410 per platelet) with a Kd = 0.53 +/- 0.23 microM. Collagen-induced 125I-fibrinogen binding to platelets was completely inhibited by ADP antagonists such as creatine phosphate/creatine phosphokinase and AMP, and partially inhibited by pretreatment of the platelets with aspirin. With both normal and aspirin-treated platelets a close correlation was observed between the amount of 125I-fibrinogen bound and the extent of dense granule secretion. Our results confirm that fibrinogen becomes bound to platelet surface receptors during collagen-induced platelet aggregation and suggest that secreted ADP is an essential cofactor in this process.     

Ligand inhibition of the platelet glycoprotein IIb-IIIa complex function as a calcium channel in liposomes

Platelet glycoproteins IIb and IIIa function as a fibrinogen receptor on the activated platelet. We have shown that these glycoproteins can be incorporated onto the surface of phosphatidylcholine vesicles with retention of fibrinogen and antibody binding properties and can permit Ca2+ transit across the phospholipid bilayer. In the current study we demonstrate that this apparent Ca2+ channel function is specifically inhibited by the synthetic analogue of the fibrinogen gamma COOH-terminal peptide, His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (His-12-Val), but not by the adhesive protein sequence Arg-Gly-Asp-Ser (RGDS). Prior incubation of IIb-IIIa liposomes with RGDS prevented Ca2+ transit inhibition by 25 microM His-12-Val, analogous to RGDS inhibition of His-12-Val binding to platelets. His-12-Val inhibited a minor component of transmembrane Ca2+ influx into ADP and thrombin-activated human platelets but had no effect on steady-state platelet 45Ca flux. These data indicate that ligand binding may exert a regulatory influence on transmembrane Ca2+ influx into activated platelets. The difference in inhibitory potency of the peptides studied may be related to differences in conformational changes in the glycoprotein IIb-IIIa complex induced by His-12-Val and RGDS, steric considerations, or differences in interactions with glycoprotein IIb Ca2+ binding domains.     

Proteomic analysis of resting and thrombin‐stimulated platelets reveals the translocation and functional relevance of HIP‐55 in platelets

The platelet surface is a dynamic interface that changes rapidly in response to stimuli to co‐ordinate the formation of thrombi at sites of vascular injury. Tight control is essential as loss of organisation may result in the inappropriate formation of thrombi (thrombosis) or excessive bleeding. In this paper we describe the comparative analysis of resting and thrombin‐stimulated platelet membrane proteomes and associated proteins to identify proteins important to platelet function. Surface proteins were labelled using a biotin tag and isolated by NeurtrAvidin affinity chromatography. Liquid phase IEF and SDS‐PAGE were used to separate proteins, and bands of increased intensity in the stimulated platelet fractions were digested and identified by FT‐ICR mass spectrometry. Novel proteins were identified along with proteins known to be translocated to the platelet surface. Furthermore, many platelet proteins revealed changes in location associated with function, including G6B and Hip‐55. HIP‐55 is an SH3‐binding protein important in T‐cell receptor signalling. Further analysis of HIP‐55 revealed that this adaptor protein becomes increasingly associated with both Syk and integrin β3 upon platelet activation. Analysis of HIP‐55 deficient platelets revealed reduced fibrinogen binding upon thrombin stimulation, suggesting HIP‐55 to be an important regulator of platelet function.     

IL-17A increases ADP-induced platelet aggregation

The increased risk of thromboembolism and higher incidence of cardiovascular disorders are among the most common causes of morbidity in patients suffering from autoimmune diseases. In this study we tested the hypothesis that IL-17A, a key pro-inflammatory cytokine involved in the development of autoimmune diseases, exerts pro-aggregant effects on both human and mouse platelets. Human or murine platelets were incubated with IL-17A for 2 min at 37 °C prior the addition of the stimuli. Aggregation was monitored in a light transmission aggregometer measuring changes in turbidity with continuous observation over a 5-min interval after the addition of the stimuli. IL-17RA, CD42b and CD62P expression as well as fibrinogen bindings were measured by FACS while Erk-2 phosphorylation was analyzed by western blot using phospho-specific antibodies. Pre-incubation with IL-17A increased ADP-, but not collagen-induced platelet aggregation and accelerated CD62P expression and exposure of fibrinogen binding sites. These effects were associated with a faster kinetic of ADP-induced Erk-2 phosphorylation and were lost in platelets deficient in the IL-17 receptor. Together these results unveil a novel aspect of the inflammatory nature of IL-17A suggesting, at the same time, that therapeutic strategies targeting this cytokine might provide further benefit for the treatment of autoimmune diseases by reducing the risk of cardiovascular-related pathologies.     

The number and affinity of fibrinogen receptors in blood platelets of patients with ischemic stroke]

Parameters of fibrinogen binding with blood platelets (number of receptors and their affinity) have been studied in patients with ischemic stroke. Due to the increased platelet ability to aggregate in the ischemic diseases such studies seem helpful. The studies involved 13 patients with ischemic stroke. Blood platelets collected from younger patients (under 50 years) possessed significantly higher number of receptors binding fibrinogen than blood platelets of healthy individuals (p less than 0.02). These receptors significantly more strongly bound ligand than those in the control group (p less than 0.05), and in the group of older patients with stroke (less than 0.05). Fibrinogen binding to blood platelets in patients over 50 years of age did not differ significantly from that in the control group. These results may indicate, that the increased platelet aggregation might be a significant pathogenic factor of the stroke in younger patients.     

Model for the regulation of platelet volume and responsiveness by the trans-membrane Na+/K(+)-pump.

The correspondence between K+ uptake in platelets to their responsiveness was studied using 86Rb+ as an analogue of K+. An average 86Rb+ uptake rate of 0.73 (+/- 0.140) x 10(-15) mole Rb+/min-plt (n = 20) was observed. By the use of K(+)-influx inhibitors, we were able to distinguish three distinct 86Rb+ uptake pathways: an ouabain-sensitive (61% +/- 2% inhibitable) pump and two equivalent channels, only one of which is sensitive to furosemide. Other platelet parameters were also examined in conjunction with K(+)-uptake. Platelets incubated with ouabain exhibited an overall rise in their cell volume (MPV) with incubation time (delta MPV = 7.4 x 10(-17) L/min-1 plt-1). Concomitantly, over 24 hours, a steady decrease in platelet number was recorded by blood cell coulter, which correlated inversely with the counts of particles, which by their size resemble white blood cells (r = 0.89). On a cellular level, incubation with ouabain induced greater expression of surface fibrinogen-receptor (GPIIb), increased binding of FITC-labelled fibrinogen, and increased responsiveness to ADP. Our observations suggest the following sequence of events: Ouabain turns off the Na+/K(+)-ATPase pump, which leads to water accumulation in platelets and concomitant increased MPV. Greater expression of fibrinogen receptors on the distended platelet surface corresponds to spontaneous microaggregate formation as well as greater responsiveness to agonists. Our model links volume regulation, the expression of fibrinogen receptors, and the sensitivity of platelets to agonists to the activity of the Na+/K(+)-ATPase pump.     

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.