RESUMEN
BACKGROUND: Platelets are central to the process of hemostasis, rapidly aggregating at sites of blood vessel injury and acting as coagulation nidus sites. On interaction with the subendothelial matrix, platelets are transformed into balloonlike structures as part of the hemostatic response. It remains unclear, however, how and why platelets generate these structures. We set out to determine the physiological relevance and cellular and molecular mechanisms underlying platelet membrane ballooning. METHODS AND RESULTS: Using 4-dimensional live-cell imaging and electron microscopy, we show that human platelets adherent to collagen are transformed into phosphatidylserine-exposing balloonlike structures with expansive macro/microvesiculate contact surfaces, by a process that we termed procoagulant spreading. We reveal that ballooning is mechanistically and structurally distinct from membrane blebbing and involves disruption to the platelet microtubule cytoskeleton and inflation through fluid entry. Unlike blebbing, procoagulant ballooning is irreversible and a consequence of Na(+), Cl(-), and water entry. Furthermore, membrane ballooning correlated with microparticle generation. Inhibition of Na(+), Cl(-), or water entry impaired ballooning, procoagulant spreading, and microparticle generation, and it also diminished local thrombin generation. Human Scott syndrome platelets, which lack expression of Ano-6, also showed a marked reduction in membrane ballooning, consistent with a role for chloride entry in the process. Finally, the blockade of water entry by acetazolamide attenuated ballooning in vitro and markedly suppressed thrombus formation in vivo in a mouse model of thrombosis. CONCLUSIONS: Ballooning and procoagulant spreading of platelets are driven by fluid entry into the cells, and are important for the amplification of localized coagulation in thrombosis.
Asunto(s)
Plaquetas/ultraestructura , Acetazolamida/farmacología , Actomiosina/metabolismo , Amidas/farmacología , Animales , Anoctaminas , Trastornos de la Coagulación Sanguínea/sangre , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Trombosis de las Arterias Carótidas/sangre , Trombosis de las Arterias Carótidas/inducido químicamente , Trombosis de las Arterias Carótidas/tratamiento farmacológico , Adhesión Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Forma de la Célula/efectos de los fármacos , Forma de la Célula/fisiología , Tamaño de la Célula/efectos de los fármacos , Micropartículas Derivadas de Células , Cloruros/metabolismo , Colágeno , Citocalasina D/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Ratones , Microtúbulos/efectos de los fármacos , Proteínas de Transferencia de Fosfolípidos/deficiencia , Proteínas de Transferencia de Fosfolípidos/fisiología , Piridinas/farmacología , Sodio/metabolismo , Trombina/biosíntesis , Trombosis/prevención & control , Agua/metabolismoRESUMEN
OBJECTIVE: Platelets are increasingly implicated in processes beyond hemostasis and thrombosis, such as vascular remodeling. Members of the matrix metalloproteinase (MMP) family not only remodel the extracellular matrix but also modulate platelet function. Here, we made a systematic comparison of the roles of MMP family members in acute thrombus formation under flow conditions and assessed platelet-dependent collagenolytic activity over time. APPROACH AND RESULTS: Pharmacological inhibition of MMP-1 or MMP-2 (human) or deficiency in MMP-2 (mouse) suppressed collagen-dependent platelet activation and thrombus formation under flow, whereas MMP-9 inhibition/deficiency stimulated these processes. The absence of MMP-3 was without effect. Interestingly, MMP-14 inhibition led to the formation of larger thrombi, which occurred independently of its capacity to activate MMP-2. Platelet thrombi exerted local collagenolytic activity capable of cleaving immobilized dye-quenched collagen and fibrillar collagen fibers within hours, with loss of the majority of the platelet adhesive properties of collagen as a consequence. This collagenolytic activity was redundantly mediated by platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 but occurred independently of platelet α-granule release (Nbeal2(-/-) mice). The latter was in line with subcellular localization experiments, which indicated a granular distribution of MMP-1 and MMP-2 in platelets, distinct from α-granules. Whereas MMP-9 protein could not be detected inside platelets, activated platelets did bind plasma-derived MMP-9 to their plasma membrane. Overall, platelet MMP activity was predominantly membrane-associated and influenced by platelet activation status. CONCLUSIONS: Platelet-associated MMP-1, MMP-2, MMP-9, and MMP-14 differentially modulate acute thrombus formation and at later time points limit thrombus formation by exerting collagenolytic activity.
Asunto(s)
Plaquetas/enzimología , Colágeno/metabolismo , Colagenasas/sangre , Trombosis/enzimología , Animales , Plaquetas/efectos de los fármacos , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Colagenasas/deficiencia , Colagenasas/genética , Modelos Animales de Enfermedad , Humanos , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteolisis , Trombosis/sangre , Trombosis/genética , Factores de TiempoRESUMEN
Glycogen synthase kinase-3 is a Ser/Thr kinase, tonically active in resting cells but inhibited by phosphorylation of an N-terminal Ser residue (Ser(21) in GSK3α and Ser(9) in GSK3ß) in response to varied external stimuli. Recent work suggests that GSK3 functions as a negative regulator of platelet function, but how GSK3 is regulated in platelets has not been examined in detail. Here, we show that early thrombin-mediated GSK3 phosphorylation (0-30 s) was blocked by PKC inhibitors and largely absent in platelets from PKCα knock-out mice. In contrast, late (2-5 min) GSK3 phosphorylation was dependent on the PI3K/Akt pathway. Similarly, early thrombin-mediated inhibition of GSK3 activity was blocked in PKCα knock-out platelets, whereas the Akt inhibitor MK2206 reduced late thrombin-mediated GSK3 inhibition and largely prevented GSK3 inhibition in PKCα knock-out platelets. More importantly, GSK3 phosphorylation contributes to platelet function as knock-in mice where GSK3α Ser(21) and GSK3ß Ser(9) were mutated to Ala showed a significant reduction in PAR4-mediated platelet aggregation, fibrinogen binding, and P-selectin expression, whereas the GSK3 inhibitor CHIR99021 enhanced these responses. Together, these results demonstrate that PKCα and Akt modulate platelet function by phosphorylating and inhibiting GSK3α/ß, thereby relieving the negative effect of GSK3α/ß on thrombin-mediated platelet activation.
Asunto(s)
Plaquetas/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Agregación Plaquetaria/fisiología , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteína Quinasa C-alfa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Vesículas Secretoras/metabolismo , Trombina/metabolismo , Sustitución de Aminoácidos , Animales , Fibrinógeno/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/genética , Glucógeno Sintasa Quinasa 3 beta , Compuestos Heterocíclicos con 3 Anillos/farmacología , Ratones , Ratones Noqueados , Mutación Missense , Selectina-P/biosíntesis , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/genética , Proteína Quinasa C-alfa/genética , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Piridinas/farmacología , Pirimidinas/farmacología , Vesículas Secretoras/genética , Trombina/farmacologíaRESUMEN
Compelling evidence demonstrates that miR-193a-3p is a tumor suppressor microRNA in many cancer types, and its reduced expression is linked to cancer initiation and progression, metastasis, and therapy resistance. However, its mechanism of action is not consistently described between studies, and often contradicts the pleiotropic role of a microRNA in manipulating several different mRNA targets. We therefore comprehensively investigated miRNA-193a-3p's mode of action in a panel of human cancer cell lines, with a variety of genetic backgrounds, using 1B3, a synthetic microRNA mimic. Interestingly, the exact mechanism through which 1B3 reduced cell proliferation varied between cell lines. 1B3 efficiently reduced target gene expression, leading to reduced cell proliferation/survival, cell cycle arrest, induction of apoptosis, increased cell senescence, DNA damage, and inhibition of migration. SiRNA silencing of 1B3 target mRNAs further highlighted the advantage of the pleiotropic mechanism of 1B3 action, as repression of individual targets did not achieve the same robust effect on cell proliferation in all cell lines. Importantly, a novel lipid nanoparticle-based formulation of 1B3, INT-1B3, demonstrated marked anti-tumor activity as a single agent following systemic administration in tumor-bearing mice. Together, these data strongly support the development of 1B3 as a novel therapeutic agent for treatment of human cancer.
RESUMEN
Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.
RESUMEN
PTEN-induced putative kinase (PINK) 1 is regarded as a master regulator of cellular mitophagy such that loss of function mutations contribute to early onset Parkinson's disease, through aberrant mitochondrial control and function. Mitochondrial function is key to platelet procoagulant activity, controlling the haemostatic response to vessel injury, but can also predispose blood vessels to thrombotic complications. Here, we sought to determine the role of PINK1 in platelet mitochondrial health and function using PINK1 knockout (KO) mice. The data largely show an absence of such a role. Haematological analysis of blood counts from KO mice was comparable to wild type. Quantification of mitochondrial mass by citrate synthase activity assay or expression of mitochondrial markers were comparable, suggesting normal mitophagy in KO platelets. Analysis of mitochondrial permeability transition pore opening, changes in mitochondrial membrane potential and calcium signalling to platelet activation were unaffected by loss of PINK1, whereas subtle enhancements of activation-induced reactive oxygen species were detected. Platelet aggregation, integrin activation, α- and dense granule secretion and phosphatidylserine exposure were unaltered in KO platelets while mouse tail bleeding responses were similar to wild type. Together these results demonstrate that PINK1 does not regulate basal platelet mitophagy and is dispensable for platelet function.
Asunto(s)
Plaquetas/metabolismo , Mitocondrias/genética , Proteínas Quinasas/genética , Animales , Plaquetas/citología , Eliminación de Gen , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , Mitofagia , Activación Plaquetaria , Proteínas Quinasas/metabolismoRESUMEN
BACKGROUND: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. METHODOLOGY/PRINCIPAL FINDINGS: Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/-) platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIb)ß(3) activation, Ca(2+) signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/-) platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. CONCLUSION: Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.