Rap1-Rac1 Circuits Potentiate Platelet Activation (original) (raw)
Related papers
Rac1 Is Essential for Platelet Lamellipodia Formation and Aggregate Stability under Flow
Journal of Biological Chemistry, 2005
The role of Rac family proteins in platelet spreading on matrix proteins under static and flow conditions has been investigated by using Rac-deficient platelets. Murine platelets form filopodia and undergo limited spreading on fibrinogen independent of Rac1 and Rac2. In the presence of thrombin, marked lamellipodia formation is observed on fibrinogen, which is abrogated in the absence of Rac1. However, Rac1 is not required for thrombin-induced aggregation or elevation of F-actin levels. Formation of lamellipodia on collagen and laminin is also Rac1-dependent. Analysis of platelet adhesion dynamics on collagen under flow conditions in vitro revealed that Rac1 is required for platelet aggregate stability at arterial rates of shear, as evidenced by a dramatic increase in platelet embolization. Furthermore, studies employing intravital microscopy demonstrated that Rac1 plays a critical role in the development of stable thrombi at sites of vascular injury in vivo. Thus, our data demonstrated that Rac1 is essential for lamellipodia formation in platelets and indicated that Rac1 is required for aggregate integrity leading to thrombus formation under physiologically relevant levels of shear both in vitro and in vivo. The Rho family of small GTPases, which includes Rac, Rho, and Cdc42 proteins, plays distinct roles in regulating actin assembly and motility. These proteins cycle between an inactive (GDP-bound) and an active (GTP-bound) conformation that can subsequently interact with specific effector proteins. There are three members of the Rac family in mammals, all of which share a common structural arrangement. Rac1 is ubiquitously expressed and is the most extensively studied isoform. Rac2 is specifically expressed in hematopoietic cells, whereas Rac3 is expressed primarily in the brain during development. These three isoforms of Rac share between 89 and 93% identity in their amino acid sequence (1). Initial studies in Swiss 3T3 fibroblasts demonstrated that Rac promotes polymerization of actin at the cell membrane, producing lamellipodia and membrane ruffles (2). This role has since been confirmed in a wide variety of cell types, including platelets, by using constitutively active and dominant negative mutants of Rac (3-5) and through the * This work was supported in part by the Wellcome Trust, British Heart Foundation, and Medical Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains Video 1, which shows the dynamics of thrombin-stimulated wild-type murine platelets spreading on fibrinogen; Video 2, which shows the dynamics of thrombin-stimulated Rac1 Ϫ/Ϫ Rac2 Ϫ/Ϫ murine platelets spreading on fibrinogen; and supplemental Fig. S1.
VAMP-7 links granule exocytosis to actin reorganization during platelet activation
Blood, 2015
Platelet activation results in profound morphologic changes accompanied by release of granule contents. Recent evidence indicates that fusion of granules with the plasma membrane during activation provides auxiliary membrane to cover growing actin structures. Yet little is known about how membrane fusion is coupled with actin reorganization. Vesicle-associated membrane protein-7 (VAMP-7) is found on platelet vesicles and possesses an N-terminal longin domain capable of linking exocytosis to cytoskeletal remodeling. We have evaluated platelets from VAMP-7(-/-) mice to determine whether this VAMP isoform contributes to granule release and platelet spreading. VAMP-7(-/-) platelets demonstrated a partial defect in dense granule exocytosis and impaired aggregation. α-Granule exocytosis from VAMP-7(-/-) platelets was diminished both in vitro and in vivo during thrombus formation. Consistent with a role of VAMP-7 in cytoskeletal remodeling, spreading on matrices was decreased in VAMP-7(-/-...
British Journal of Haematology, 2010
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 reorganization , centralization of secretory granules, and exocytosis of small molecules and proteins from three classes of granules: dense core and a-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. a-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
Journal of Translational Medicine, 2010
Background Platelet activation requires rapid remodeling of the actin cytoskeleton which is regulated by small GTP-binding proteins. By using the Rac1-specific inhibitor NSC23766, we have recently found that Rac1 is a central component of a signaling pathway that regulates dephosphorylation and activation of the actin-dynamising protein cofilin, dense and α-granule secretion, and subsequent aggregation of thrombin-stimulated washed platelets. Objectives To study whether NSC23766 inhibits stimulus-induced platelet secretion and aggregation in blood. Methods Human platelet aggregation and ATP-secretion were measured in hirudin-anticoagulated blood and platelet-rich plasma (PRP) by using multiple electrode aggregometry and the Lumi-aggregometer. Platelet P-selectin expression was quantified by flow cytometry. Results NSC23766 (300 μM) inhibited TRAP-, collagen-, atherosclerotic plaque-, and ADP-induced platelet aggregation in blood by 95.1%, 93.4%, 92.6%, and 70%, respectively. The IC5...
The Platelet Actin Cytoskeleton Associates with SNAREs and Participates in α-Granule Secretion
Biochemistry, 2010
Following platelet activation, platelets undergo a dramatic shape change mediated by the actin cytoskeleton and accompanied by secretion of granule contents. While the actin cytoskeleton is thought to influence platelet granule secretion, the mechanism for this putative regulation is not known. We found that disruption of the actin cytoskeleton by latrunculin A inhibited α-granule secretion induced by several different platelet agonists without significantly affecting activationinduced platelet aggregation. In a cell-free secretory system, platelet cytosol was required for αgranule secretion. Inhibition of actin polymerization prevented α-granule secretion in this system and purified platelet actin could substitute for platelet cytosol to support α-granule secretion. To determine whether SNAREs physically associate with the actin cytoskeleton, we isolated the Triton X-100 insoluble actin cytoskeleton from platelets. VAMP-8 and syntaxin-2 associated only with actin cytoskeletons of activated platelets. Syntaxin-4 and SNAP-23 associated with cytoskeletons isolated from either resting or activated platelets. When syntaxin-4 and SNAP-23 were tested for actin binding in a purified protein system, only syntaxin-4 associated directly with polymerized platelet actin. These data show that the platelet cytoskeleton interacts with select SNAREs and that actin polymerization facilitates α-granule release. The role of the actin cytoskeleton in granule exocytosis is enigmatic. It has been demonstrated to act both as a physical barrier that limits granule secretion and as a positive regulator of membrane fusion and cargo release. The ability of the resting actin cytoskeleton to serve as a barrier to granule secretion has been demonstrated in neutrophils, neurons, chromaffin cells, melanotrophs, pancreatic beta cells, and acinar cells (1-6). We have previously demonstrated that platelet granules are coated with actin and that the actin cytoskeleton impedes platelet dense granule and α-granule release (7). Partial disruption of this barrier results in augmented and more rapid release of granule contents from platelets. This actin cytoskeletal barrier may help prevent unregulated release of thrombogenic substances into the circulation (7). Yet accumulating evidence indicates that actin polymerization can promote membrane fusion. Actin polymerization contributes to homotypic fusion of yeast vacuoles (8), fusion of phagosomes with endocytotic organelles (9) as well as secretion of granules from neuroendocrine cells (6,10,11), and mast cells (12). In some cells, actomyosin contraction and/ † Supported by NIH grants HL63250 and HL87203 (R.F.) and T32 HL07917 (K.
Arteriosclerosis, thrombosis, and vascular biology, 2013
P latelets serve as the primary cellular mediators of hemostasis and thrombosis. 1,2 These circulating, anucleate fragments of megakaryocytic cells are optimally configured to detect a loss of vascular integrity, adhere to sites of vessel injury, and aggregate to form thrombotic plugs. During the process of platelet activation, platelets undergo a dramatic change in shape from discs to small spheres with filopodial extensions and lamellipodial structures. This process is mediated by changes in the platelet actin cytoskeleton. 3 Accordingly, an array of actin regulatory proteins, including the Wiskott-Aldrich syndrome verprolin-homologous protein SCAR/WAVE, 4 Arp2/3, 5 cortactin, 6 and the Rho GTPases, 7 play roles in regulating platelet morphology through the regulation of actin assembly and lamellipodia dynamics. 3, Through the guanine nucleotide exchange factor-mediated exchange of GDP for GTP, the ≈21-kDa Rho GTPases Rac1 and Cdc42 support the autocatalytic activation of the p21 activated kinases (PAKs). 10 The PAK family of serine/threonine protein kinases represent well-characterized Rho GTPase effectors. 11 As cells adhere to and spread on substrates, PAKs are recruited to focal adhesions and the actin leading edge to orchestrate actin dynamics, lamellipodia formation, and cell motility. A system of PAK substrates, including the LIM domain kinase LIM domain kinase LIMK1, 12 are thought to be major contributors to the PAK-based regulation of actin dynamics. 11 Of the major PAK isoforms in mammalian cells, PAK2 represents the most ubiquitous and highly expressed PAK family member. 11 Although PAK2 was originally described as a kinase activated on platelet stimulation, 13 little is known regarding the role of PAKs in platelet biology. A number of platelet agonists, including glycoprotein receptor VI (GPVI) agonists, have been demonstrated to stimulate PAK autophosphorylation in platelets. 6,14-16 These studies have shown that PAK activation occurs downstream of the Rho GTPases Rac1 and Cdc42 in platelets; however, a specific role for PAK in platelet function has not yet been defined. 6, Here, we demonstrate that PAK activity downstream of the platelet collagen receptor, GPVI, is necessary for platelet aggregation, secretion, and lamellipodia formation as well as platelet aggregate stability under physiological conditions of shear. Platelet PAK activation occurs rapidly on stimulation with the GPVI agonist, collagen-related peptide (CRP). Through inhibition of platelet PAK, we show that PAK effectors LIMK1 and MAPK/ERK kinase (MEK) are Objective-Rho GTPase proteins play a central role in regulating the dynamics of the platelet actin cytoskeleton. Yet, little is known regarding how Rho GTPase activation coordinates platelet activation and function. In this study, we aimed to characterize the role of the Rho GTPase effector, p21 activated kinase (PAK), in platelet activation, lamellipodia formation, and aggregate formation under shear. Approach and Results-Stimulation of platelets with the glycoprotein receptor VI agonist, collagen-related peptide, rapidly activated PAK in a time course preceding phosphorylation of PAK substrates, LIM domain kinase LIMK1 and the MAPK/ERK kinase MEK, and the subsequent activation of MAPKs and Akt. Pharmacological inhibitors of PAK blocked signaling events downstream of PAK and prevented platelet secretion as well as platelet aggregation in response to collagen-related peptide. PAK inhibitors also prevented PAK activation and platelet spreading on collagen surfaces. PAK was also required for the formation of platelet aggregates and to maintain aggregate stability under physiological shear flow conditions.
To cite this article: Calaminus SDJ, Thomas S, McCarty OJT, Machesky LM, Watson SP. Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading. J Thromb Haemost 2008; 6: 1944-52.
Blood, 2013
Platelet granule secretion is important not only for hemostasis and thrombosis, but also for a variety of physiological processes including inflammation, angiogenesis and malignancy. Vesicle Associated Membrane Proteins (VAMPs) are a group of v-SNARE proteins resident on the platelet granule surface that participate in granule secretion. Platelets contain several VAMP isoforms including VAMP-2, VAMP-3, VAMP-7, and VAMP-8. VAMP-7 is unique in that it contains an N-terminal profilin-like longin domain. Previous work by our group demonstrated spatial segregation of granules expressing different VAMPs during platelet spreading. Granules expressing VAMP-3 and VAMP-8 localized to the granulomere of spreading platelets, while those expressing VAMP-7 moved towards the periphery. Based on this observation, we proposed that VAMP-7+ granules move to the periphery of the spreading platelet to add membrane to growing actin structures. To assess this hypothesis, platelets from VAMP-7 null mice we...
FEBS Letters, 1995
The integrin etnb/33-mediated redistribution of the tyrosine kinases pp125 rAg and pp60 src and the small GTP-binding proteins CDC42Hs and RaplB from the membrane skeleton to the cytoskeleton was found to be reversible: upon prolonged platelet aggregation (up to 15 rain) induced by the thrombin-receptor activating peptide (TRAP) these signalling proteins dissociated from the cytoskeleton and reappeared in the membrane skeleton. Addition of the extracellular Ca 2+ chelator EGTA and the intracellular Ca 2+ chelator BAPTA/AM 30 s after TRAP allowed platelet aggregation and the association of pp125 rAK, pp60 s'c, CDC42Hs and RaplB with the cytoskeleton, but prevented their dissociation from the cytoskeleton. The results indicate that the prolonged elevation of cytosolic Ca 2+ in stimulated platelets leads to the dissociation of signalling proteins from the eytoskeleton.
The human blood platelet circulates in the blood as a non-adherent disk. Upon receiving signals of blood vessel damage, the platelet reorganizes its actin cytoskeleton which transforms it into a spiky dynamic adherent glue. This transformation involves a temporal sequence of four morphologically distinct steps that can be reproducible in vitro. The actin dynamics that underlie these shape changes depend on a large number of actin-binding proteins. Maintenance of the discoid shape requires actin-binding proteins that inhibit these reorganizations, whereas transformation involves other proteins, some to disassemble old filaments and others to polymerize new ones. F-actin-affinity chromatography identified a large set of actin-binding proteins including VASP, Arp2 and 2E4/kaptin. Recent discoveries show that VASP inhibits filament disassembly and Arp2/3 is required to polymerize new filaments. Morphological analysis of the distribution of these actin-binding proteins in spread platelets together with biochemical measurements of their interactions with actin lead to a model of interactions with actin that mediate shape change.