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Upon activation with physiological stimuli, human platelets undergo morphological changes, centralizing their organelles and secreting effector molecules at the site of vascular injury. Previous studies have indicated that the actin filaments and microtubules of suspension-activated platelets play a critical role in granule movement and exocytosis; however, the participation of these cytoskeleton elements in adhered platelets remains unexplored. α- and β-dystrobrevin members of the dystrophin-associated protein complex in muscle and non-muscle cells have been described as motor protein receptors that might participate in the transport of cellular components in neurons. Recently, we characterized the expression of dystrobrevins in platelets; however, their functional diversity within this cellular model had not been elucidated. The present study examined the contribution of actin filaments and microtubules in granule trafficking during the platelet adhesion process using cytoskeleton-disrupting drugs, quantification of soluble P-selectin, fluorescence resonance transfer energy analysis and immunoprecipitation assays. Likewise, we assessed the interaction of α-dystrobrevins with the ubiquitous kinesin heavy chain. Our results strongly suggest that microtubules and actin filaments participate in the transport of alpha and dense granules in the platelet adhesion process, during which α-dystrobrevins play the role of regulatory and adaptor proteins that govern trafficking events.
The critical role of the platelet is to sense vascular damage and respond by secreting components that promote primary haemostasis and clot formation. Activated platelets initiate signalling cascades that lead to cytoskeletal re-organization, centralization of secretory granules, and exocytosis of small molecules and proteins from three classes of granules: dense core and α-granules and secretory lysosomes (Rendu & Brohard-Bohn, 2001). Platelet granules are the most prominent structural features, and upon activation, they coalesce in the centre of the platelet and fuse with the open canalicular system (OCS). The OCS represents a membrane reservoir that is evaginated onto the platelet surface during interaction with surfaces (Stenberg et al, 1984; Escolar et al, 1989), fusing with the plasma membrane (Ginsberg et al, 1980). The release of the granule contents into the OCS and their diffusion into the extracellular environment exert a paracrine role to activate other platelets in the immediate area that are critical to the formation of the haemostatic thrombus (Escolar & White, 1991; White & Escolar, 1991).
Dense core granules mainly contain small molecules, such as adenosine diphosphate (ADP), serotonin and calcium, which are critical for further platelet activation and vasoconstriction. α-Granules represent the storage sites for a diverse set of proteins, such as platelet factor 4, von Willebrand factor, platelet-derived growth factor and P-selectin, which play roles in clot formation and initiating wound healing. Platelets also release lysosomal enzymes, such as cathepsins and hexosaminidase, which may play a role in clot remodelling or in further platelet activation (Anitua et al, 2004). To date, more than 300 proteins and small molecules have been shown to be secreted from activated platelets (Coppinger et al, 2004).
Studies performed in suspended platelets have shown contradictory arguments for the participation of microtubules or microfilaments. One study demonstrated that microtubules are intimately involved in the movement of intracellular granules (Sneddon, 1971); other studies that induced the stabilization of microtubules with taxol did not inhibit the secretion process (White & Rao, 1982, 1983). It has also been demonstrated that actin polymerization, granule centralization and membrane fusion act synergistically in the promotion of granule secretion (Painter & Ginsberg, 1984), while other studies suggest that cytochalasins do not block agonist-mediated granule secretion (Kirkpatrick et al, 1980; Cox, 1988). Recent results indicate that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates α-granule and dense granule secretion (Flaumenhaft et al, 2005).
We recently demonstrated that the diversity of expression of the Dystrophin associated protein complex (DAPC) plays a key role in the platelet haemostatic functions, participating as a membrane scaffold as well as having a signalling role (Cerecedo et al, 2005, 2006a). Dystrobrevins are cytoplasmic components of the DAPC that link the actin cytoskeleton to the extracellular matrix in skeletal muscle (Blake et al, 2002); they are the product of two different genes coding for two highly similar proteins, i.e. α- and β-dystrobrevin (Ambrose et al, 1997; Peters et al, 1997; Blake et al, 1998). Dystrobrevins have been characterized in association with dystrophin isoforms and utrophin in several non-muscle tissues including platelets (Cerecedo et al, 2005). Functional diversity of α- and β-dystrobrevin within the same cell type has been demonstrated in recent studies in which these proteins bind to the cargo-binding domain of kinesin family member 5A, thus participating in the transport of cellular components (Torreri et al, 2005).
The goal of this study was to identify the contribution of actin filaments and microtubules to granule trafficking in adhered platelets. Immunoprecipitation assays and fluorescence resonance energy transfer (FRET) analysis utilizing disrupting cytoskeleton drugs were performed to corroborate that actin filaments and microtubules contribute to α- and dense granule mobilization in adhered platelets, and enabled the identification of the α-dystrobrevins as part of the platelet transport machinery in close association with ubiquitous kinesin heavy chain (UKHC).
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- Materials and methods
Dramatic platelet shape change upon surface activation involves the fragmentation of the microtubule ring, as well as the formation of actin-based structures, such as the contractile ring (Bearer, 1995), which provokes concurrent centralization of granules preceding granule-content release. However, the influence of the cytoskeleton on granule secretion has been a controversial subject. Inhibition of tubulin using monoclonal antibodies inhibited platelet granule secretion; in contrast, the microtubule stabilizing agent paclitaxel suggested that microtubule reorganization does not influence granule secretion (White & Rao, 1982, 1983). F-actin disassembly might be required for normal granule secretion, because studies utilizing actin polymerization inhibitors did not block agonist-mediated granule secretion (Kirkpatrick et al, 1980; Cox, 1988; Lefebvre et al, 1993). Additionally, it has been demonstrated that the actin cytoskeleton interferes with platelet exocytosis and differentially regulates α-granule and dense granule secretion (Flaumenhaft et al, 2005). However, it is important to note that similar studies have not been described in adhered platelets.
Our morphological analysis confirmed that initially, platelet granules were centralized to the granulomere; however, as the process continued, they were translocated near the plasma membrane. We also observed that granule dispersion can proceed in the absence of microtubules or microfilaments, although with different distribution patterns.
Recently, using the microtubule-inhibitor Col and the actin filament-inhibitor CD, we found that microtubules and actin filaments were highly dependent upon each other, and that removing either component dramatically changed the organization of the other (Cerecedo et al, 2008). It is evident that platelet cell spreading requires extensive cellular remodelling, which was inhibited by treatment with CD or Col, as well as with jasplakinolide or taxol. The present study showed that the reduced sP-selectin levels observed following independent treatment with depolymerizing or stabilizing drugs was not statistically significant, except for jasplakinolide, as well as for simultaneous treatment with both cytoskeleton inhibitors and both cytoskeleton stabilizers. In all cases, granule-content extrusion was diminished, but not prevented, as reflected in sP-selectin levels.
In addition, it is very probable that the low quantities of sP-selectin observed with simultaneous use of stabilizer or inhibitor drugs was due to the feasible fusion of the membrane granule with the membranous system, which is a critical event for the release of platelet granule contents into the extracellular environment (Furie et al, 2001; Zhou & Freed, 2009). It is probable that the interaction between granules and cytoplasm membranes or OCS was too close, thus, it was not affected by the use of cytoskeleton inhibitors or stabilizers. It is also feasible that jasplakinolide interrupted granule centralization and that amplification of the OCS in a greater extension disturbed the re-distribution of microtubules, thus the occurrence of granule translocation to the plasma membrane.
According to our results, it is pertinent to propose that microtubules, probably via kinesin motors, counteract the confining effect of the actin-based contractile ring by increasing short-range mobility of granules at the plasma membrane. Thus, actin and microtubules may play opposing and complementary roles in order to fine-tune mobility and to direct granules at target sites on the plasma membrane. At these sites, the fusing membranes are orchestrated by the universal machinery denominated Soluble N-ethylmaleimide (NEM)-sensitive attachment protein receptors (SNARE) (Lemons et al, 1997; Flaumenhaft et al, 1999).
In neurons, motor proteins, such as kinesin, transport cargoes along microtubules and are involved in the targeting and localization of specific proteins within distinct molecular and functional domains. Recent studies have suggested a role for dystrobrevin as a motor protein receptor binding to the cargo-binding domain of the kinesin heavy chain (Ceccarini et al, 2005).
The present study found that co-distribution of α-dystrobrevins with kinesin and microtubules in the granulomere zone strongly suggest that α-dystrobrevins might play a role in granule transport as an adaptor molecule upon surface adhesion.
In conclusion, we speculate that granule dynamics during the adhesion process is strictly regulated by actin filaments and microtubules. This begins with the centralization of granules at the granulomere zone by the actin-based contractile ring; however, as the process continues, microtubules are re-organized from the granulomere, translocating the granules to the plasma membrane by kinesin motors. During this process, α-dystrobrevins play a modulator active role in mediating the recruitment of motor proteins to granules.