Identification of a novel, actin-rich structure, the actin nodule, in the early stages of platelet spreading (original) (raw)
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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.
2017
A dynamic, properly organised actin cytoskeleton is critical for the production and haemostatic function of platelets. The Wiskott Aldrich Syndrome protein (WASp) and Actin-Related Proteins 2 & 3 Complex (Arp2/3 complex) are critical mediators of actin polymerisation and organisation in many cell types. In platelets and megakaryocytes, these proteins have been shown to be important for proper platelet production and function. The cortactin family of proteins (Cttn & HS1) are known to regulate WASp-Arp2/3-mediated actin polymerisation in other cell types and so here we address the role of these proteins in platelets using knockout mouse models. We generated mice lacking Cttn and HS1 in the megakaryocyte/platelet lineage. These mice had normal platelet production, with platelet number, size and surface receptor profile comparable to controls. Platelet function was also unaffected by loss of Cttn/HS1 with no differences observed in a range of platelet function assays including aggregat...
Arp2/3 complex is required for actin polymerization during platelet shape change
2010
Platelets undergo a series of actin-dependent morphologic changes when activated by thrombin receptor activating peptide (TRAP) or when spreading on glass. Polymerization of actin results in the sequential formation of filopodia, lamellipodia, and stress fibers, but the molecular mechanisms regulating this polymerization are unknown, The Arp2/3 complex nucleates actin polymerization in vitro and could perform this function inside cells as well. To test whether Arp2/3 regulated platelet actin polymerization, we used recombinant Arp2 protein (rArp2) to generate Arp2-specific antibodies (αArp2). Intact and Fab fragments of αArp2 inhibited TRAPstimulated actin-polymerizing activity in platelet extracts as measured by the pyrene assay. Inhibition was reversed by the addition of rArp2 protein. To test the effect of Arp2/3 inhibition on the formation of specific actin structures, we designed a new method to permeabilize resting platelets while preserving their ability to adhere and to form filopodia and lamellipodia on exposure to glass. Inhibition of Arp2/3 froze platelets at the rounded, early stage of activation, before the formation of filopodia and lamellipodia. By morphometric analysis, the proportion of platelets in the rounded stage rose from 2.85% in untreated to 63% after treatment with αArp2. This effect was also seen with Fab fragments and was reversed by the addition of rArp2 protein. By immunofluorescence of platelets at various stages of spreading, the Arp2/3 complex was found in filopodia and lamellipodia. These results suggest that activation of the Arp2/3 complex at the cortex by TRAP stimulation initiates an explosive polymerization of actin filaments that is required for all subsequent actin-dependent events.
ADF/n-cofilin-dependent actin turnover determines platelet formation and sizing
Blood, 2010
and sizing dependent actin turnover determines platelet formation − ADF/n-cofilin http://bloodjournal.hematologylibrary.org/content/116/10/1767.full.html Updated information and services can be found at: (221 articles) Platelets and Thrombopoiesis Articles on similar topics can be found in the following Blood collections http://bloodjournal.hematologylibrary.org/site/misc/rights.xhtml#repub\_requests
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
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...
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.
Proceedings of the National Academy of Sciences, 2002
We investigated the effect of actin filament barbed end uncapping on Arp23 complex function both in vivo and in vitro. Arp23 complex redistributes rapidly and uniformly to the lamellar edge of activated wild-type platelets and fibroblasts but clusters in marginal actin filament clumps in gelsolin-null cells. Treatment of gelsolin-null platelets with the negative dominant N-WASp C-terminal CA domain has no effect on their residual actin nucleation activity, placing gelsolin actin filament severing, capping, and uncapping function upstream of Arp23 complex nucleation. Actin filaments capped by gelsolin or the gelsolin homolog CapG fail to enhance Arp23 complex nucleation in vitro, but uncapping of the barbed ends of these actin filaments restores their ability to potentiate Arp23 complex nucleation. We conclude that Arp23 complex contribution to actin filament nucleation in platelets and fibroblasts importantly requires free barbed ends generated by severing and uncapping.
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.
Blood, 2003
The platelet receptor for von Willebrand factor (vWF) glycoprotein (GP)Ib-IX-V complex mediates platelet adhesion at sites of vascular injury. The cytoplasmic tail of the GPIb subunit interacts with the actin-binding protein, filamin, anchoring the receptor in the cytoskeleton. In motile cells, the second messenger PtdIns(3,4,5)P 3 induces submembraneous actin remodelling. The inositol polyphosphate 5-phosphatase, SHIP-2, hydrolyzes phosphoinositide 3,4,5 trisphosphate (PtdIns(3,4,5)P 3 ) forming PtdIns(3,4)P 2 , and regulates membrane ruffling via complex formation with filamin (Dyson et al., J.