Rho GTPases in platelet function

Aslan, Joseph E.; McCarty, Owen J. T.

doi:10.1111/jth.12051

Cited by 148 publications

(161 citation statements)

References 174 publications

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“…Additionally, mutETV6 platelets showed defects in functions classically associated with CDC42 (i.e., filopodia formation) and RHOA (i.e., lamellipodia formation and clot retraction). 36 EM also showed platelets of variable sizes and having a more circular instead of discoid shape. RNA sequencing previously performed on mutETV6 transfected cells, patient platelets and leukemia cells did not reveal any modification in Rho GTPase mRNA levels, 1,3 which may be due to variations in the models applied.…”

Section: Discussionmentioning

confidence: 94%

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Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors

et al. 2016

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Variants in ETV6, which encodes a transcription repressor of the E26 transformation-specific family, have recently been reported to be responsible for inherited thrombocytopenia and hematologic malignancy. We sequenced the DNA from cases with unexplained dominant thrombocytopenia and identified six likely pathogenic variants in ETV6, of which five are novel. We observed low repressive activity of all tested ETV6 variants, and variants located in the E26 transformation-specific binding domain (encoding p.A377T, p.Y401N) led to reduced binding to corepressors. We also observed a large expansion of megakaryocyte colony-forming units derived from variant carriers and reduced proplatelet formation with abnormal cytoskeletal organization. The defect in proplatelet formation was also observed in control CD34 + cell-derived megakaryocytes transduced with lentiviral particles encoding mutant ETV6. Reduced expression levels of key regulators of the actin cytoskeleton CDC42 and RHOA were measured. Moreover, changes in the actin structures are typically accompanied by a rounder platelet shape with a highly heterogeneous size, decreased platelet arachidonic response, and spreading and retarded clot retraction in ETV6 deficient platelets. Elevated numbers of circulating CD34 + cells were found in p.P214L and p.Y401N carriers, and two patients from different families suffered from refractory anemia with excess blasts, while one patient from a third family was successfully treated for acute myeloid leukemia. Overall, our study provides novel insights into the role of ETV6 as a driver of cytoskeletal regulatory gene expression during platelet production, and the impact of variants resulting in platelets with altered size, shape and function and potentially also in changes in circulating progenitor levels.

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Section: Discussionmentioning

confidence: 94%

“…36 Therefore, we hypothesize that ETV6 repressive activity is a key regulator of megakaryocyte cytoskeleton remodeling, driven via Rho GTPases in mutETV6 carriers. mutETV6 was associated with a decrease in CDC42 and RHOA expression levels in platelets without affecting RAC1 expression.…”

Section: Discussionmentioning

confidence: 97%

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors

et al. 2016

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“…In platelets, the actin cytoskeleton is essential for filopodia formation and spreading, 28 and is therefore a likely mediator of spatially regulated a-granule secretion. To determine the role of those, first we examined the effects of pharmacologic cytoskeletal inhibitors of actin polymerization (latrunculin A) and microtubule assembly (nocodazole).…”

Section: Resultsmentioning

confidence: 99%

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition

Sakurai

Fitch-Tewfik

Qiu

et al. 2015

Blood

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• The geometric orientation of the underlying matrix regulates platelet a-granule secretion.• On geometrically constrained matrices, platelets selfdeposit additional matrix, providing more cell membrane to extend spreading.Although the biology of platelet adhesion on subendothelial matrix after vascular injury is well characterized, how the matrix biophysical properties affect platelet physiology is unknown. Here we demonstrate that geometric orientation of the matrix itself regulates platelet a-granule secretion, a key component of platelet activation. Using protein microcontact printing, we show that platelets spread beyond the geometric constraints of fibrinogen or collagen micropatterns with <5-mm features. Interestingly, a-granule exocytosis and deposition of the a-granule contents such as fibrinogen and fibronectin were primarily observed in those areas of platelet extension beyond the matrix protein micropatterns. This enables platelets to "self-deposit" additional matrix, provide more cellular membrane to extend spreading, and reinforce platelet-platelet connections. Mechanistically, this phenomenon is mediated by actin polymerization, Rac1 activation, and a IIb b 3 integrin redistribution and activation, and is attenuated in gray platelet syndrome platelets, which lack a-granules, and Wiskott-Aldrich syndrome platelets, which have cytoskeletal defects. Overall, these studies demonstrate how platelets transduce geometric cues of the underlying matrix geometry into intracellular signals to extend spreading, which endows platelets spatial flexibility when spreading onto small sites of exposed subendothelium. (Blood. 2015;126(4):531-538) IntroductionHemostasis begins with platelet adhesion and activation at the site of vascular injury.1 Platelets then secrete their a-and dense granules, leading to further recruitment of other platelets and the formation of a hemostatic plug.2 Extensive research has characterized the underlying biological signaling pathways that govern these processes at the bulk level via in vivo and in vitro techniques.3 However, how platelets interact with their microenvironment at the single-cell level and how these interactions subsequently influence platelet physiology remain poorly understood. For example, although single platelets have long been observed to fill submicron-sized "gaps" in the endothelium, 4 how platelets establish adhesion on such small areas of exposed matrix that is strong enough to withstand the shear forces of the circulation is unknown. Studies of single platelet-matrix interactions may help explain the role of platelets in maintaining vascular integrity and supporting the semipermeable barrier function of the endothelium during homeostatic conditions. 5 Furthermore, because platelets within different regions of the same thrombus exhibit significantly different levels of activation, 6,7 this concept of physical and microenvironmental regulation of platelet physiology at the single-cell level is also important for our overall understanding of clot formation. S...

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“…Our data also indicate that, further downstream, Rac1 also mediates platelet mechanosensing of substrate stiffness. In platelets, Rac1 acts downstream of integrin α IIb β 3 outside-in signaling to regulate lamellipodia formation, platelet aggregation, and granule secretion (43,44), and can activate Rap1b, which is involved in inside-out integrin α IIb β 3 activation (45). As Rac1 inhibition decreases platelet adhesion and activation on stiffer gels in a dose-dependent manner, it is likely that this integrin-Rac1-Rap1b circuit provides the mechanosensing machinery during platelet adhesion, further α IIb β 3 activation, and integrin clustering when platelets adhered on stiffer substrates, thus increasing adhesion as substrate stiffness increases.…”

Section: Discussionmentioning

confidence: 99%

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation

Qiu

Brown

Myers

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

170

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As platelets aggregate and activate at the site of vascular injury to stem bleeding, they are subjected to a myriad of biochemical and biophysical signals and cues. As clot formation ensues, platelets interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Here, we show for the first time (to our knowledge) that platelets, which are anucleate cellular fragments, sense microenvironmental mechanical properties, such as substrate stiffness, and transduce those cues into differential biological signals. Specifically, as platelets mechanosense the stiffness of the underlying fibrin/fibrinogen substrate, increasing substrate stiffness leads to increased platelet adhesion and spreading. Importantly, adhesion on stiffer substrates also leads to higher levels of platelet activation, as measured by integrin α IIb β 3 activation, α-granule secretion, and procoagulant activity. Mechanistically, we determined that Rac1 and actomyosin activity mediate substrate stiffness-dependent platelet adhesion, spreading, and activation to different degrees. This capability of platelets to mechanosense microenvironmental cues in a growing thrombus or hemostatic plug and then mechanotransduce those cues into differential levels of platelet adhesion, spreading, and activation provides biophysical insight into the underlying mechanisms of platelet aggregation and platelet activation heterogeneity during thrombus formation. mechanotransduction | cell mechanics | platelet cytoskeleton | biomaterials A s the first responders at the site of vascular injury, platelets are subjected to a dynamic microenvironment during the process of hemostasis (1-5). Biochemically, platelets are exposed to diverse and rapidly changing gradients of soluble proteins and agonists such as von Willebrand factor, ADP, and thrombin, all of which drive platelet adhesion and activation (6, 7). During this process, platelet activation may take several forms including activation of platelet α IIb β 3 integrins, secretion of α-and dense granules, and membrane phosphatidylserine (PS) exposure leading to a procoagulant phenotype (8, 9). Biophysically, platelets also activate and aggregate in response to the hemodynamic and shear forces of the circulation (10, 11). As clot formation ensues, platelets then interact with polymerizing fibrin scaffolds, exposing platelets to a large range of mechanical microenvironments. Although the underlying biochemical signaling pathways that govern the fibrinogen-α IIb β 3 -mediated processes have been well characterized, if and how the mechanical cues in the microenvironment affect platelet activation and physiology remain largely unknown. Indeed, as clot structure and mechanics are known to be heterogeneous within the same clot and more recent studies have demonstrated that platelet activation is also vastly heterogeneous within a growing thrombus (12-14), a systematic approach to investigate how platelet activation is affected by the mechanical microenvironment could lead to profoun...

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Rho GTPases in platelet function

Cited by 148 publications

References 174 publications

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation

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Rho GTPases in platelet function

Cited by 148 publications

References 174 publications

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 + progenitors

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 + progenitors

Platelet geometry sensing spatially regulates α-granule secretion to enable matrix self-deposition

Platelet mechanosensing of substrate stiffness during clot formation mediates adhesion, spreading, and activation

Contact Info

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Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors

Germline variants in ETV6 underlie reduced platelet formation, platelet dysfunction and increased levels of circulating CD34 ⁺ progenitors