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1.
Blood ; 143(6): 548-560, 2024 Feb 08.
Artigo em Inglês | MEDLINE | ID: mdl-37944157

RESUMO

ABSTRACT: Nonmuscle cell contractility is an essential feature underlying diverse cellular processes such as motility, morphogenesis, division and genome replication, intracellular transport, and secretion. Blood clot contraction is a well-studied process driven by contracting platelets. Megakaryocytes (MKs), which are the precursors to platelets, can be found in bone marrow and lungs. Although they express many of the same proteins and structures found in platelets, little is known about their ability to engage with extracellular proteins such as fibrin and contract. Here, we have measured the ability of MKs to compress plasma clots. Megakaryocytes derived from human induced pluripotent stem cells (iPSCs) were suspended in human platelet-free blood plasma and stimulated with thrombin. Using real-time macroscale optical tracking, confocal microscopy, and biomechanical measurements, we found that activated iPSC-derived MKs (iMKs) caused macroscopic volumetric clot shrinkage, as well as densification and stiffening of the fibrin network via fibrin-attached plasma membrane protrusions undergoing extension-retraction cycles that cause shortening and bending of fibrin fibers. Contraction induced by iMKs involved 2 kinetic phases with distinct rates and durations. It was suppressed by inhibitors of nonmuscle myosin IIA, actin polymerization, and integrin αIIbß3-fibrin interactions, indicating that the molecular mechanisms of iMK contractility were similar or identical to those in activated platelets. Our findings provide new insights into MK biomechanics and suggest that iMKs can be used as a model system to study platelet contractility. Physiologically, the ability of MKs to contract plasma clots may play a role in the mechanical remodeling of intravascular blood clots and thrombi.


Assuntos
Células-Tronco Pluripotentes Induzidas , Trombose , Humanos , Megacariócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Plaquetas/metabolismo , Trombose/metabolismo , Fibrina/metabolismo , Plasma
2.
Blood ; 139(12): 1892-1902, 2022 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-34890454

RESUMO

Rebalancing the hemostatic system by targeting endogenous anticoagulant pathways, like the protein C (PC) system, is being tested as a means of improving hemostasis in patients with hemophilia. Recent intravital studies of hemostasis demonstrated that, in some vascular contexts, thrombin activity is sequestered in the extravascular compartment. These findings raise important questions about the context-dependent contribution of activated PC (APC) to the hemostatic response, because PC activation occurs on the surface of endothelial cells. We used a combination of pharmacologic, genetic, imaging, and computational approaches to examine the relationships among thrombin spatial distribution, PC activation, and APC anticoagulant function. We found that inhibition of APC activity, in mice either harboring the factor V Leiden mutation or infused with an APC-blocking antibody, significantly enhanced fibrin formation and platelet activation in a microvascular injury model, consistent with the role of APC as an anticoagulant. In contrast, inhibition of APC activity had no effect on hemostasis after penetrating injury of the mouse jugular vein. Computational studies showed that differences in blood velocity, injury size, and vessel geometry determine the localization of thrombin generation and, consequently, the extent of PC activation. Computational predictions were tested in vivo and showed that when thrombin generation occurred intravascularly, without penetration of the vessel wall, inhibition of APC significantly increased fibrin formation in the jugular vein. Together, these studies show the importance of thrombin spatial distribution in determining PC activation during hemostasis and thrombosis.


Assuntos
Hemostáticos , Trombose , Animais , Anticoagulantes/farmacologia , Células Endoteliais/metabolismo , Fibrina/metabolismo , Hemostasia , Humanos , Camundongos , Proteína C/farmacologia , Trombina/metabolismo , Trombose/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(39): 24316-24325, 2020 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-32929010

RESUMO

Platelets are best known for their vasoprotective responses to injury and inflammation. Here, we have asked whether they also support vascular integrity when neither injury nor inflammation is present. Changes in vascular barrier function in dermal and meningeal vessels were measured in real time in mouse models using the differential extravasation of fluorescent tracers as a biomarker. Severe thrombocytopenia produced by two distinct methods caused increased extravasation of 40-kDa dextran from capillaries and postcapillary venules but had no effect on extravasation of 70-kDa dextran or albumin. This reduction in barrier function required more than 4 h to emerge after thrombocytopenia was established, reverting to normal as the platelet count recovered. Barrier dysfunction was also observed in mice that lacked platelet-dense granules, dense granule secretion machinery, glycoprotein (GP) VI, or the GPVI signaling effector phospholipase C (PLC) γ2. It did not occur in mice lacking α-granules, C type lectin receptor-2 (CLEC-2), or protease activated receptor 4 (PAR4). Notably, although both meningeal and dermal vessels were affected, intracerebral vessels, which are known for their tighter junctions between endothelial cells, were not. Collectively, these observations 1) highlight a role for platelets in maintaining vascular homeostasis in the absence of injury or inflammation, 2) provide a sensitive biomarker for detecting changes in platelet-dependent barrier function, 3) identify which platelet processes are required, and 4) suggest that the absence of competent platelets causes changes in the vessel wall itself, accounting for the time required for dysfunction to emerge.


Assuntos
Plaquetas/imunologia , Vasos Sanguíneos/imunologia , Hemostasia , Homeostase , Animais , Vasos Sanguíneos/lesões , Vasos Sanguíneos/fisiopatologia , Feminino , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Masculino , Meninges/irrigação sanguínea , Meninges/imunologia , Camundongos , Fosfolipase C gama/genética , Fosfolipase C gama/imunologia , Pele/irrigação sanguínea , Pele/imunologia
4.
Blood ; 136(15): 1773-1782, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-32542378

RESUMO

G protein-coupled receptors are critical mediators of platelet activation whose signaling can be modulated by members of the regulator of G protein signaling (RGS) family. The 2 most abundant RGS proteins in human and mouse platelets are RGS10 and RGS18. While each has been studied individually, critical questions remain about the overall impact of this mode of regulation in platelets. Here, we report that mice missing both proteins show reduced platelet survival and a 40% decrease in platelet count that can be partially reversed with aspirin and a P2Y12 antagonist. Their platelets have increased basal (TREM)-like transcript-1 expression, a leftward shift in the dose/response for a thrombin receptor-activating peptide, an increased maximum response to adenosine 5'-diphosphate and TxA2, and a greatly exaggerated response to penetrating injuries in vivo. Neither of the individual knockouts displays this constellation of findings. RGS10-/- platelets have an enhanced response to agonists in vitro, but platelet count and survival are normal. RGS18-/- mice have a 15% reduction in platelet count that is not affected by antiplatelet agents, nearly normal responses to platelet agonists, and normal platelet survival. Megakaryocyte number and ploidy are normal in all 3 mouse lines, but platelet recovery from severe acute thrombocytopenia is slower in RGS18-/- and RGS10-/-18-/- mice. Collectively, these results show that RGS10 and RGS18 have complementary roles in platelets. Removing both at the same time discloses the extent to which this regulatory mechanism normally controls platelet reactivity in vivo, modulates the hemostatic response to injury, promotes platelet production, and prolongs platelet survival.


Assuntos
Plaquetas/metabolismo , Ativação Plaquetária/genética , Proteínas RGS/genética , Trombopoese/genética , Animais , Plaquetas/efeitos dos fármacos , Sobrevivência Celular/genética , Camundongos , Camundongos Knockout , Fosforilação , Fator de Ativação de Plaquetas/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Contagem de Plaquetas , Proteínas RGS/metabolismo , Trombopoese/efeitos dos fármacos
5.
Proc Natl Acad Sci U S A ; 116(6): 2243-2252, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30674670

RESUMO

Extensive studies have detailed the molecular regulation of individual components of the hemostatic system, including platelets, coagulation factors, and regulatory proteins. Questions remain, however, about how these elements are integrated at the systems level within a rapidly changing physical environment. To answer some of these questions, we developed a puncture injury model in mouse jugular veins that combines high-resolution, multimodal imaging with functional readouts in vivo. The results reveal striking spatial regulation of platelet activation and fibrin formation that could not be inferred from studies performed ex vivo. As in the microcirculation, where previous studies have been performed, gradients of platelet activation are readily apparent, as is an asymmetrical distribution of fibrin deposition and thrombin activity. Both are oriented from the outer to the inner surface of the damaged vessel wall, with a greater extent of platelet activation and fibrin accumulation on the outside than the inside. Further, we show that the importance of P2Y12 signaling in establishing a competent hemostatic plug is related to the size of the injury, thus limiting its contribution to hemostasis to specific physiologic contexts. Taken together, these studies offer insights into the organization of hemostatic plugs, provide a detailed understanding of the adverse bleeding associated with a widely prescribed class of antiplatelet agents, and highlight differences between hemostasis and thrombosis that may suggest alternative therapeutic approaches.


Assuntos
Coagulação Sanguínea , Hemostasia , Ferimentos e Lesões/sangue , Animais , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Modelos Animais de Doenças , Fibrina/metabolismo , Masculino , Camundongos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Trombose/metabolismo , Trombose/patologia , Veias/lesões , Ferimentos e Lesões/etiologia
6.
Small ; 17(15): e2004889, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33150735

RESUMO

Hemostasis is an innate protective mechanism that plays a central role in maintaining the homeostasis of the vascular system during vascular injury. Studying this essential physiological process is often challenged by the difficulty of modeling and probing the complex dynamics of hemostatic responses in the native context of human blood vessels. To address this major challenge, this paper describes a microengineering approach for in vitro modeling of hemostasis. This microphysiological model replicates the living endothelium, multilayered microarchitecture, and procoagulant activity of human blood vessels, and is also equipped with a microneedle that is actuated with spatial precision to simulate penetrating vascular injuries. The system recapitulates key features of the hemostatic response to acute vascular injury as observed in vivo, including i) thrombin-driven accumulation of platelets and fibrin, ii) formation of a platelet- and fibrin-rich hemostatic plug that halts blood loss, and iii) matrix deformation driven by platelet contraction for wound closure. Moreover, the potential use of this model for drug testing applications is demonstrated by evaluating the effects of anticoagulants and antiplatelet agents that are in current clinical use. The vascular injury-on-a-chip may serve as an enabling platform for preclinical investigation of hematological disorders and emerging therapeutic approaches against them.


Assuntos
Trombose , Lesões do Sistema Vascular , Fibrina , Hemostasia , Humanos , Dispositivos Lab-On-A-Chip
7.
Blood ; 127(12): 1598-605, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-26738537

RESUMO

Previous studies have shown that hemostatic thrombi formed in response to penetrating injuries have a core of densely packed, fibrin-associated platelets overlaid by a shell of less-activated, loosely packed platelets. Here we asked, first, how the diverse elements of this structure combine to stem the loss of plasma-borne molecules and, second, whether antiplatelet agents and anticoagulants that perturb thrombus structure affect the re-establishment of a tight vascular seal. The studies combined high-resolution intravital microscopy with a photo-activatable fluorescent albumin marker to simultaneously track thrombus formation and protein transport following injuries to mouse cremaster muscle venules. The results show that protein loss persists after red cell loss has ceased. Blocking platelet deposition with an αIIbß3antagonist delays vessel sealing and increases extravascular protein accumulation, as does either inhibiting adenosine 5'-diphosphate (ADP) P2Y12receptors or reducing integrin-dependent signaling and retraction. In contrast, sealing was unaffected by introducing hirudin to block fibrin accumulation or a Gi2α gain-of-function mutation to expand the thrombus shell. Collectively, these observations describe a novel approach for studying vessel sealing after injury in real time in vivo and show that (1) the core/shell architecture previously observed in arterioles also occurs in venules, (2) plasma leakage persists well beyond red cell escape and mature thrombus formation, (3) the most critical events for limiting plasma extravasation are the stable accumulation of platelets, ADP-dependent signaling, and the emergence of a densely packed core, not the accumulation of fibrin, and (4) drugs that affect platelet accumulation and packing can delay vessel sealing, permitting protein escape to continue.


Assuntos
Proteínas Sanguíneas/metabolismo , Hemostasia , Microvasos/lesões , Microvasos/patologia , Trombose/patologia , Difosfato de Adenosina/metabolismo , Animais , Proteínas Sanguíneas/análise , Fibrina/análise , Fibrina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/metabolismo , Ativação Plaquetária , Contagem de Plaquetas , Trombose/sangue , Trombose/metabolismo
9.
Blood ; 126(24): 2611-20, 2015 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-26407691

RESUMO

Most platelet agonists activate platelets by binding to G-protein-coupled receptors. We have shown previously that a critical node in the G-protein signaling network in platelets is formed by a scaffold protein, spinophilin (SPL), the tyrosine phosphatase, Src homology region 2 domain-containing phosphatase-1 (SHP-1), and the regulator of G-protein signaling family member, RGS18. Here, we asked whether SPL and other RGS18 binding proteins such as 14-3-3γ regulate platelet reactivity by sequestering RGS18 and, if so, how this is accomplished. The results show that, in resting platelets, free RGS18 levels are relatively low, increasing when platelets are activated by thrombin. Free RGS18 levels also rise when platelets are rendered resistant to activation by exposure to prostaglandin I2 (PGI2) or forskolin, both of which increase platelet cyclic adenosine monophosphate (cAMP) levels. However, the mechanism for raising free RGS18 is different in these 2 settings. Whereas thrombin activates SHP-1 and causes dephosphorylation of SPL tyrosine residues, PGI2 and forskolin cause phosphorylation of SPL Ser94 without reducing tyrosine phosphorylation. Substituting alanine for Ser94 blocks cAMP-induced dissociation of the SPL/RGS/SHP-1 complex. Replacing Ser94 with aspartate prevents formation of the complex and produces a loss-of-function phenotype when expressed in mouse platelets. Together with the defect in platelet function we previously observed in SPL(-/-) mice, these data show that (1) regulated sequestration and release of RGS18 by intracellular binding proteins provides a mechanism for coordinating activating and inhibitory signaling networks in platelets, and (2) differential phosphorylation of SPL tyrosine and serine residues provides a key to understanding both.


Assuntos
Ativação Plaquetária/fisiologia , Proteínas RGS/fisiologia , Animais , Plaquetas/efeitos dos fármacos , Células CHO , Colforsina/farmacologia , Cricetinae , Cricetulus , AMP Cíclico/fisiologia , Epoprostenol/farmacologia , Transplante de Tecido Fetal , Fígado/embriologia , Transplante de Fígado , Camundongos , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/fisiologia , Fosforilação , Fosfosserina/metabolismo , Fosfotirosina/metabolismo , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteína Tirosina Fosfatase não Receptora Tipo 6/fisiologia , Quimera por Radiação , Receptores de Trombina/agonistas , Sistemas do Segundo Mensageiro/fisiologia , Transdução de Sinais/fisiologia , Trombina/farmacologia
10.
Blood ; 125(10): 1623-32, 2015 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-25477496

RESUMO

Hermansky-Pudlak syndrome (HPS) is characterized by oculocutaneous albinism, bleeding diathesis, and other variable symptoms. The bleeding diathesis has been attributed to δ storage pool deficiency, reflecting the malformation of platelet dense granules. Here, we analyzed agonist-stimulated secretion from other storage granules in platelets from mouse HPS models that lack adaptor protein (AP)-3 or biogenesis of lysosome-related organelles complex (BLOC)-3 or BLOC-1. We show that α granule secretion elicited by low agonist doses is impaired in all 3 HPS models. High agonist doses or supplemental adenosine 5'-diphosphate (ADP) restored normal α granule secretion, suggesting that the impairment is secondary to absent dense granule content release. Intravital microscopy following laser-induced vascular injury showed that defective hemostatic thrombus formation in HPS mice largely reflected reduced total platelet accumulation and affirmed a reduced area of α granule secretion. Agonist-induced lysosome secretion ex vivo was also impaired in all 3 HPS models but was incompletely rescued by high agonist doses or excess ADP. Our results imply that (1) AP-3, BLOC-1, and BLOC-3 facilitate protein sorting to lysosomes to support ultimate secretion; (2) impaired secretion of α granules in HPS, and to some degree of lysosomes, is secondary to impaired dense granule secretion; and (3) diminished α granule and lysosome secretion might contribute to pathology in HPS.


Assuntos
Plaquetas/fisiologia , Síndrome de Hermanski-Pudlak/sangue , Complexo 3 de Proteínas Adaptadoras/deficiência , Complexo 3 de Proteínas Adaptadoras/genética , Complexo 3 de Proteínas Adaptadoras/fisiologia , Difosfato de Adenosina/farmacologia , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Degranulação Celular/fisiologia , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina , Síndrome de Hermanski-Pudlak/etiologia , Síndrome de Hermanski-Pudlak/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/deficiência , Lectinas/genética , Lectinas/fisiologia , Lisossomos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Selectina-P/sangue , Proteínas SNARE/sangue , Vesículas Secretórias/fisiologia , Trombina/farmacologia , Trombose/sangue , Trombose/etiologia , Proteínas de Transporte Vesicular/deficiência , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/fisiologia
11.
Blood ; 124(11): 1808-15, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24951424

RESUMO

Hemostatic thrombi develop a characteristic architecture in which a core of highly activated platelets is covered by a shell of less-activated platelets. Here we have used a systems biology approach to examine the interrelationship of this architecture with transport rates and agonist distribution in the gaps between platelets. Studies were performed in mice using probes for platelet accumulation, packing density, and activation plus recently developed transport and thrombin activity probes. The results show that intrathrombus transport within the core is much slower than within the shell. The region of slowest transport coincides with the region of greatest packing density and thrombin activity, and appears prior to full platelet activation. Deleting the contact-dependent signaling molecule, Sema4D, delays platelet activation, but not the emergence of the low transport region. Collectively, these results suggest a timeline in which initial platelet accumulation and the narrowing gaps between platelets create a region of reduced transport that facilitates local thrombin accumulation and greater platelet activation, whereas faster transport rates within the shell help to limit thrombin accumulation and growth of the core. Thus, from a systems perspective, platelet accumulation produces an altered microenvironment that shapes thrombus architecture, which in turn affects agonist distribution and subsequent thrombus growth.


Assuntos
Coagulação Sanguínea , Modelos Cardiovasculares , Ativação Plaquetária , Trombina/metabolismo , Animais , Humanos , Camundongos , Transporte Proteico
12.
Blood ; 124(11): 1816-23, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24951425

RESUMO

Hemostatic thrombi formed after a penetrating injury have a heterogeneous architecture in which a core of highly activated, densely packed platelets is covered by a shell of less-activated, loosely packed platelets. In the first manuscript in this series, we show that regional differences in intrathrombus protein transport rates emerge early in the hemostatic response and are preserved as the thrombus develops. Here, we use a theoretical approach to investigate this process and its impact on agonist distribution. The results suggest that hindered diffusion, rather than convection, is the dominant mechanism responsible for molecular movement within the thrombus. The analysis also suggests that the thrombus core, as compared with the shell, provides an environment for retaining soluble agonists such as thrombin, affecting the extent of platelet activation by establishing agonist-specific concentration gradients radiating from the site of injury. This analysis accounts for the observed weaker activation and relative instability of platelets in the shell and predicts that a failure to form a tightly packed thrombus core will limit thrombin accumulation, a prediction tested by analysis of data from mice with a defect in clot retraction.


Assuntos
Coagulação Sanguínea , Simulação por Computador , Modelos Cardiovasculares , Ativação Plaquetária , Trombina/metabolismo , Animais , Humanos , Camundongos , Transporte Proteico
13.
Blood ; 124(11): 1824-31, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-24951426

RESUMO

Hemostatic thrombi formed after a penetrating injury have a distinctive structure in which a core of highly activated, closely packed platelets is covered by a shell of less-activated, loosely packed platelets. We have shown that differences in intrathrombus molecular transport emerge in parallel with regional differences in platelet packing density and predicted that these differences affect thrombus growth and stability. Here we test that prediction in a mouse vascular injury model. The studies use a novel method for measuring thrombus contraction in vivo and a previously characterized mouse line with a defect in integrin αIIbß3 outside-in signaling that affects clot retraction ex vivo. The results show that the mutant mice have a defect in thrombus consolidation following vascular injury, resulting in an increase in intrathrombus transport rates and, as predicted by computational modeling, a decrease in thrombin activity and platelet activation in the thrombus core. Collectively, these data (1) demonstrate that in addition to the activation state of individual platelets, the physical properties of the accumulated mass of adherent platelets is critical in determining intrathrombus agonist distribution and platelet activation and (2) define a novel role for integrin signaling in the regulation of intrathrombus transport rates and localization of thrombin activity.


Assuntos
Coagulação Sanguínea , Modelos Cardiovasculares , Ativação Plaquetária , Trombina/metabolismo , Animais , Humanos , Camundongos , Transporte Proteico
15.
Arterioscler Thromb Vasc Biol ; 35(3): 645-54, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25614284

RESUMO

OBJECTIVE: Biological and physical factors interact to modulate blood response in a wounded vessel, resulting in a hemostatic clot or an occlusive thrombus. Flow and pressure differential (ΔP) across the wound from the lumen to the extravascular compartment may impact hemostasis and the observed core/shell architecture. We examined physical and biological factors responsible for regulating thrombin-mediated clot growth. APPROACH AND RESULTS: Using factor XIIa-inhibited human whole blood perfused in a microfluidic device over collagen/tissue factor at controlled wall shear rate and ΔP, we found thrombin to be highly localized in the P-selectin(+) core of hemostatic clots. Increasing ΔP from 9 to 29 mm Hg (wall shear rate=400 s(-1)) reduced P-selectin(+) core size and total clot size because of enhanced extravasation of thrombin. Blockade of fibrin polymerization with 5 mmol/L Gly-Pro-Arg-Pro dysregulated hemostasis by enhancing both P-selectin(+) core size and clot size at 400 s(-1) (20 mm Hg). For whole-blood flow (no Gly-Pro-Arg-Pro), the thickness of the P-selectin-negative shell was reduced under arterial conditions (2000 s(-1), 20 mm Hg). Consistent with the antithrombin-1 activity of fibrin implicated with Gly-Pro-Arg-Pro, anti-γ'-fibrinogen antibody enhanced core-localized thrombin, core size, and overall clot size, especially at venous (100 s(-1)) but not arterial wall shear rates (2000 s(-1)). Pathological shear (15 000 s(-1)) and Gly-Pro-Arg-Pro synergized to exacerbate clot growth. CONCLUSIONS: Hemostatic clotting was dependent on core-localized thrombin that (1) triggered platelet P-selectin display and (2) was highly regulated by fibrin and the transclot ΔP. Also, γ'-fibrinogen had a role in venous but not arterial conditions.


Assuntos
Colágeno Tipo I/sangue , Fibrina/metabolismo , Fibrinogênios Anormais/metabolismo , Hemostasia , Trombina/metabolismo , Tromboplastina/metabolismo , Trombose/sangue , Lesões do Sistema Vascular/sangue , Animais , Artérias/metabolismo , Artérias/patologia , Artérias/fisiopatologia , Velocidade do Fluxo Sanguíneo , Modelos Animais de Doenças , Humanos , Dispositivos Lab-On-A-Chip , Masculino , Mecanotransdução Celular , Camundongos , Selectina-P/sangue , Polimerização , Pressão , Fluxo Sanguíneo Regional , Estresse Mecânico , Trombose/patologia , Trombose/fisiopatologia , Fatores de Tempo , Lesões do Sistema Vascular/patologia , Lesões do Sistema Vascular/fisiopatologia , Veias/metabolismo , Veias/patologia , Veias/fisiopatologia
16.
Blood ; 121(10): 1875-85, 2013 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-23303817

RESUMO

Achieving hemostasis following vascular injury requires the rapid accumulation of platelets and fibrin. Here we used a combination of confocal intravital imaging, genetically engineered mice, and antiplatelet agents to determine how variations in the extent of platelet activation following vascular injury arise from the integration of different elements of the platelet-signaling network. Two forms of penetrating injury were used to evoke the hemostatic response. Both produced a hierarchically organized structure in which a core of fully activated platelets was overlaid with an unstable shell of less-activated platelets. This structure emerged as hemostasis was achieved and persisted for at least 60 minutes following injury, its organization at least partly reflecting agonist concentration gradients. Thrombin activity and fibrin formation were found primarily in the innermost core. As proposed previously, greater packing density in the core facilitated contact-dependent signaling and limited entry of plasma-borne molecules visualized with fluorophores coupled to dextran and albumin. Blocking contact-dependent signaling or inhibiting thrombin reduced the size of the core, while the shell was heavily influenced by adenosine 5'-diphosphate and regulators of Gi2-mediated signaling. Thus, the hemostatic response is shown to produce a hierarchical structure arising, in part, from distinct elements of the platelet-signaling network.


Assuntos
Plaquetas/fisiologia , Hemostasia/fisiologia , Músculo Esquelético/metabolismo , Transdução de Sinais , Trombina/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Animais , Antígenos CD/fisiologia , Plaquetas/ultraestrutura , Fibrina/metabolismo , Subunidade alfa Gi2 de Proteína de Ligação ao GTP/metabolismo , Hemostasia/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/lesões , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12/química , Receptores Purinérgicos P2Y12/metabolismo , Semaforinas/fisiologia , Trombina/antagonistas & inibidores
17.
Blood ; 121(20): 4221-30, 2013 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-23564909

RESUMO

Semaphorin 4D (Sema4D) is a transmembrane protein that supports contact-dependent amplification of platelet activation by collagen before being gradually cleaved by the metalloprotease ADAM17, as we have previously shown. Cleavage releases a soluble 120-kDa exodomain fragment for which receptors exist on platelets and endothelial cells. Here we have examined the mechanism that regulates Sema4D exodomain cleavage. The results show that the membrane-proximal cytoplasmic domain of Sema4D contains a binding site for calmodulin within the polybasic region Arg762-Lys779. Coprecipitation studies show that Sema4D and calmodulin are associated in resting platelets, forming a complex that dissociates upon platelet activation by the agonists that trigger Sema4D cleavage. Inhibiting calmodulin with W7 or introducing a membrane-permeable peptide corresponding to the calmodulin-binding site is sufficient to trigger the dissociation of Sema4D from calmodulin and initiate cleavage. Conversely, deletion of the calmodulin-binding site causes constitutive shedding of Sema4D. These results show that (1) Sema4D is a calmodulin-binding protein with a site of interaction in its membrane-proximal cytoplasmic domain, (2) platelet agonists cause dissociation of the calmodulin-Sema4D complex, and (3) dissociation of the complex is sufficient to trigger ADAM17-dependent cleavage of Sema4D, releasing a bioactive fragment.


Assuntos
Antígenos CD/química , Antígenos CD/metabolismo , Plaquetas/metabolismo , Calmodulina/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Semaforinas/química , Semaforinas/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Células CHO , Calmodulina/antagonistas & inibidores , Cricetinae , Cricetulus , Inibidores Enzimáticos/farmacologia , Humanos , Dados de Sequência Molecular , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/genética , Ligação Proteica/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Semaforinas/genética , Sulfonamidas/farmacologia
18.
Curr Opin Hematol ; 21(5): 410-7, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25023471

RESUMO

PURPOSE OF REVIEW: Several decades of work by many investigators have elucidated the major signaling pathways responsible for platelet activation. Still to be fully understood is how these pathways are integrated into a single network and how changing conditions within a growing thrombus affect that network. In this review we will consider some of the recent studies that address these issues and describe a model that provides insights into platelet activation as it occurs in vivo. RECENT FINDINGS: Genetic and pharmacologic studies performed in vivo have demonstrated that platelet activation during hemostasis and thrombosis is heterogeneous. Those studies indicate that distinct platelet activation pathways are not merely redundant, but are coordinated in time and space to achieve an optimal response. This coordination is achieved at least in part by the evolving distribution of platelet agonists and changes in solute transport within a hemostatic plug. SUMMARY: Studies examining the coordination of platelet signaling in time and space continue to increase our understanding of hemostasis and thrombosis. In addition to helping to decipher platelet biology, the results have implications for the understanding of new and existing antiplatelet agents and their potential risks.


Assuntos
Plaquetas/citologia , Lesões do Sistema Vascular/patologia , Animais , Plaquetas/metabolismo , Forma Celular , Humanos , Ativação Plaquetária , Transdução de Sinais , Lesões do Sistema Vascular/metabolismo
19.
Blood ; 119(14): 3352-60, 2012 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-22271446

RESUMO

Mounting evidence suggests that agonist-initiated signaling in platelets is closely regulated to avoid excessive responses to injury. A variety of physiologic agonists induce a cascade of signaling events termed as inside-out signaling that culminate in exposure of high-affinity binding sites on integrin α(IIb)ß(3). Once platelet activation has occurred, integrin α(IIb)ß(3) stabilizes thrombus formation by providing agonist-independent "outside-in" signals mediated in part by contractile signaling. Junctional adhesion molecule A (JAM-A), a member of the cortical thymocyte marker of the Xenopus (CTX) family, was initially identified as a receptor for a platelet stimulatory mAb. Here we show that JAM-A in resting platelets functions as an endogenous inhibitor of platelet function. Genetic ablation of Jam-A in mice enhances thrombotic function of platelets in vivo. The absence of Jam-A results in increase in platelet aggregation ex vivo. This gain of function is not because of enhanced inside-out signaling because granular secretion, Thromboxane A2 (TxA2) generation, as well as fibrinogen receptor activation, are normal in the absence of Jam-A. Interestingly, integrin outside-in signaling such as platelet spreading and clot retraction is augmented in Jam-A-deficient platelets. We conclude that JAM-A normally limits platelet accumulation by inhibiting integrin outside-in signaling thus preventing premature platelet activation.


Assuntos
Plaquetas/metabolismo , Moléculas de Adesão Celular/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Receptores de Superfície Celular/metabolismo , Trombose/etiologia , Animais , Tempo de Sangramento , Moléculas de Adesão Celular/genética , Retração do Coágulo/genética , Técnicas de Inativação de Genes , Estudos de Associação Genética , Humanos , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Adesividade Plaquetária/genética , Embolia Pulmonar/genética , Embolia Pulmonar/mortalidade , Embolia Pulmonar/patologia , Receptores de Superfície Celular/genética , Transdução de Sinais , Trombose/genética , Trombose/prevenção & controle
20.
Blood ; 119(8): 1935-45, 2012 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-22210881

RESUMO

Platelets are essential for normal hemostasis, but close regulation is required to avoid the destructive effects of either inappropriate platelet activation or excessive responses to injury. Here, we describe a novel complex comprising the scaffold protein, spinophilin (SPL), and the tyrosine phosphatase, SHP-1, and show that it can modulate platelet activation by sequestering RGS10 and RGS18, 2 members of the regulator of G protein signaling family. We also show that SPL/RGS/SHP1 complexes are present in resting platelets where constitutive phosphorylation of SPL(Y398) creates an atypical binding site for SHP-1. Activation of the SHP-1 occurs on agonist-induced phosphorylation of SHP-1(Y536), triggering dephosphorylation and decay of the SPL/RGS/SHP1 complex. Preventing SHP-1 activation blocks decay of the complex and produces a gain of function. Conversely, deleting spinophilin in mice inhibits platelet activation. It also attenuates the rise in platelet cAMP normally caused by endothelial prostacyclin (PGI(2)). Thus, we propose that the role of the SPL/RGS/SHP1 complex in platelets is time and context dependent. Before injury, the complex helps maintain the quiescence of circulating platelets by maximizing the impact of PGI(2). After injury, the complex gradually releases RGS proteins, limiting platelet activation and providing a mechanism for temporal coordination of pro thrombotic and antithrombotic inputs.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ativação Plaquetária , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas RGS/metabolismo , Animais , Sítios de Ligação/genética , Plaquetas/metabolismo , Western Blotting , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Modelos Biológicos , Mutação , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas RGS/genética , Transdução de Sinais , Transfecção , Tirosina/genética , Tirosina/metabolismo
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