RESUMO
Binding of coagulation factors to phosphatidylserine (PS)-exposing procoagulant-activated platelets followed by formation of the membrane-dependent enzyme complexes is critical for blood coagulation. Procoagulant platelets formed upon strong platelet stimulation, usually with thrombin plus collagen, are large "balloons" with a small (â¼1 µm radius) "cap"-like convex region that is enriched with adhesive proteins. Spatial distribution of blood coagulation factors on the surface of procoagulant platelets was investigated using confocal microscopy. All of them, including factors IXa (FIXa), FXa/FX, FVa, FVIII, prothrombin, and PS-sensitive marker Annexin V were distributed nonhomogeneously: they were primarily localized in the "cap," where their mean concentration was by at least an order of magnitude, higher than on the "balloon." Assembly of intrinsic tenase on liposomes with various PS densities while keeping the PS content constant demonstrated that such enrichment can accelerate this reaction by 2 orders of magnitude. The mechanisms of such acceleration were investigated using a 3-dimensional computer simulation model of intrinsic tenase based on these data. Transmission electron microscopy and focal ion beam-scanning electron microscopy with Annexin V immunogold-labeling revealed a complex organization of the "caps." In platelet thrombi formed in whole blood on collagen under arterial shear conditions, ubiquitous "caps" with increased Annexin V, FX, and FXa binding were observed, indicating relevance of this mechanism for surface-attached platelets under physiological flow. These results reveal an essential heterogeneity in the surface distribution of major coagulation factors on the surface of procoagulant platelets and suggest its importance in promoting membrane-dependent coagulation reactions.
Assuntos
Fatores de Coagulação Sanguínea/metabolismo , Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Adulto , Anexina A5/metabolismo , Plaquetas/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Simulação por Computador , Humanos , Imageamento Tridimensional , Técnicas In Vitro , Microscopia Confocal , Microscopia Imunoeletrônica , Fosfatidilserinas/sangue , Ativação Plaquetária/fisiologia , Ligação Proteica , Trombina/metabolismo , Trombose/metabolismo , Trombose/patologiaRESUMO
Strongly activated "coated" platelets are characterized by increased phosphatidylserine (PS) surface expression, α-granule protein retention, and lack of active integrin αIIbß3. To study how they are incorporated into thrombi despite a lack of free activated integrin, we investigated the structure, function, and formation of the α-granule protein "coat." Confocal microscopy revealed that fibrin(ogen) and thrombospondin colocalized as "cap," a single patch on the PS-positive platelet surface. In aggregates, the cap was located at the point of attachment of the PS-positive platelets. Without fibrin(ogen) retention, their ability to be incorporated in aggregates was drastically reduced. The surface fibrin(ogen) was strongly decreased in the presence of a fibrin polymerization inhibitor GPRP and also in platelets from a patient with dysfibrinogenemia and a fibrinogen polymerization defect. In contrast, a fibrinogen-clotting protease ancistron increased the amount of fibrin(ogen) and thrombospondin on the surface of the PS-positive platelets stimulated with collagen-related peptide. Transglutaminases are also involved in fibrin(ogen) retention. However, platelets from patients with factor XIII deficiency had normal retention, and a pan-transglutaminase inhibitor T101 had only a modest inhibitory effect. Fibrin(ogen) retention was normal in Bernard-Soulier syndrome and kindlin-3 deficiency, but not in Glanzmann thrombasthenia lacking the platelet pool of fibrinogen and αIIbß3. These data show that the fibrin(ogen)-covered cap, predominantly formed as a result of fibrin polymerization, is a critical mechanism that allows coated (or rather "capped") platelets to become incorporated into thrombi despite their lack of active integrins.
Assuntos
Plaquetas/metabolismo , Fibrina/metabolismo , Fibrinogênio/metabolismo , Agregação Plaquetária , Trombospondinas/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Western Blotting , Feminino , Citometria de Fluxo , Humanos , Microscopia Confocal , Oligopeptídeos/farmacologia , Fosfatidilserinas/metabolismo , Polimerização/efeitos dos fármacos , Trombastenia/sangue , Trombastenia/metabolismo , Trombose/metabolismo , Transglutaminases/metabolismoRESUMO
Blood platelets are anucleate cell fragments that play a critically important role in hemostasis and thrombosis. Platelets are activated with various agonists that allow them to aggregate, thus forming either hemostatic plugs or pathologic thrombi. Recent studies have revealed that at least two activated platelet subpopulations are formed upon potent stimulation of platelets with collagen and/or thrombin. One of these subpopulations consists of so-called coated platelets that express high levels of phosphatidylserine and retain α-granule proteins, including fibrinogen, on their surface. They also have reduced levels of the main aggregation receptor-activated glycoprotein IIb-IIIa, which might indicate a defect in their proaggregatory ability. In this study, the proaggregatory abilities of coated and noncoated platelets were assessed by means of light transmission aggregometry of suspensions with varying ratios of platelets from one subpopulation to those of a different subpopulation. A mathematical model of platelet aggregation in heterogeneous mixtures was developed to assist in the analysis of experimental data. Flow cytometry was employed to monitor platelet recruitment into aggregates and the ability of platelets to bind external fibrinogen. Finally, confocal microscopy was used to image coated platelets involved into aggregates formed by mechanical shaking. The obtained data revealed to our knowledge a novel mechanism regulating aggregate formation of platelet subpopulations: coated platelets cannot aggregate with each other but can be recruited into aggregates by noncoated platelets.
Assuntos
Plaquetas/citologia , Plaquetas/fisiologia , Agregação Plaquetária/fisiologia , Anticorpos Monoclonais/metabolismo , Plaquetas/efeitos dos fármacos , Fibrinogênio/metabolismo , Fibrinogênio/farmacologia , Humanos , Microscopia Confocal , Modelos Biológicos , Dinâmica não Linear , Agregação Plaquetária/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , SuspensõesRESUMO
Platelets are formed elements of blood. Upon activation, they externalize phosphatidylserine, thus accelerating membrane-dependent reactions of blood coagulation. Activated platelets form two subpopulations, only one of which expresses phosphatidylserine. This study aimed to identify signalling pathways responsible for this segregation. Gel-filtered platelets, intact or loaded with calcium-sensitive dyes, were activated and labelled with annexin V and antibodies, followed by flow cytometric analysis. Calcium Green and Fura Red dyes were compared, and only the latter was able to detect calcium level differences in the platelet subpopulations. Phosphatidylserine-positive platelets produced by thrombin had stably high intracellular calcium level; addition of convulxin increased and stabilized calcium level in the phosphatidylserine-negative subpopulation. PAR1 agonist SFLLRN also induced calcium rise and subpopulation formation, but the resulting platelets were not coated with alpha-granule proteins. Adenylatecyclase activator forskolin inhibited phosphatidylserine-positive platelets formation several-fold, while its inhibitor SQ22536 had no effect. This suggests that adenylatecyclase inactivation is necessary, but not rate-limiting, for subpopulation segregation. Inhibition of mitogen-activated protein kinase kinase (U0126) and glycoprotein IIb-IIIa (Monafram(®)) was without effect, whereas inhibitors of phosphatidylinositol 3-kinase (wortmannin) and Src tyrosine kinase (PP2) decreased the procoagulant subpopulation threefold. These data identify the principal signalling pathways controlling platelet heterogeneity.
Assuntos
Plaquetas/citologia , Plaquetas/metabolismo , Ativação Plaquetária/fisiologia , Transdução de Sinais/fisiologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/metabolismo , Adulto , Anexina A5/metabolismo , Cálcio/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Citometria de Fluxo , Humanos , Fosfatidilserinas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-1/metabolismoRESUMO
Factor XIIIa (fXIIIa) is a transglutaminase that plays a crucial role in fibrin clot stabilization and regulation of fibrinolysis. It is known to bind to procoagulant platelets. In contrast, the zymogen fXIII interaction with platelets is not well characterized. We investigated the interaction of zymogen fXIII with activated platelet subpopulations. Confocal microscopy and flow cytometry using fluorescently labelled factors and antibodies. Phosphatidylserine (PS)-positive activated platelets bound 700 to 800 molecules/cell of fXIII at 100 nM, while both PS-negative activated platelets and resting platelets bound 200 to 400 molecules/cell. The binding was reversible, calcium-independent and linear within the fXIII concentration range of up to 1,000 nM. fXIII predominantly bound to the caps of procoagulant platelets and co-localized with fibrinogen. Exogenous fibrinogen promoted fXIII binding by activated PS-negative platelets; this effect was abolished by the integrin αIIbß3 antagonist monafram. The fXIII binding was 1.5- to 3-fold decreased for platelets from four patients with grey platelet syndrome, and was variable for platelets from six patients with Glanzmann's thrombasthenia. Strong platelet stimulation, fibrinogen and αIIbß3 play essential roles in fXIII binding, without any of them fXIII does not bind to platelets. The preferential binding in the cap-like structures might be important for increasing local fXIII concentration in platelet thrombi.