RESUMO
ABSTRACT: Glycoprotein Ibα (GPIbα) is expressed on the surface of platelets and megakaryocytes (MKs) and anchored to the membrane skeleton by filamin A (flnA). Although GPIb and flnA have fundamental roles in platelet biogenesis, the nature of this interaction in megakaryocyte biology remains ill-defined. We generated a mouse model expressing either human wild-type (WT) GPIbα (hGPIbαWT) or a flnA-binding mutant (hGPIbαFW) and lacking endogenous mouse GPIbα. Mice expressing the mutant GPIbα transgene exhibited macrothrombocytopenia with preserved GPIb surface expression. Platelet clearance was normal and differentiation of MKs to proplatelets was unimpaired in hGPIbαFW mice. The most striking abnormalities in hGPIbαFW MKs were the defective formation of the demarcation membrane system (DMS) and the redistribution of flnA from the cytoplasm to the peripheral margin of MKs. These abnormalities led to disorganized internal MK membranes and the generation of enlarged megakaryocyte membrane buds. The defective flnA-GPIbα interaction also resulted in misdirected release of buds away from the vasculature into bone marrow interstitium. Restoring the linkage between flnA and GPIbα corrected the flnA redistribution within MKs and DMS ultrastructural defects as well as restored normal bud size and release into sinusoids. These studies define a new mechanism of macrothrombocytopenia resulting from dysregulated MK budding. The link between flnA and GPIbα is not essential for the MK budding process, however, it plays a major role in regulating the structure of the DMS, bud morphogenesis, and the localized release of buds into the circulation.
Assuntos
Megacariócitos , Complexo Glicoproteico GPIb-IX de Plaquetas , Trombocitopenia , Animais , Humanos , Camundongos , Plaquetas/metabolismo , Citoplasma/metabolismo , Filaminas/genética , Filaminas/metabolismo , Megacariócitos/metabolismo , Morfogênese , Complexo Glicoproteico GPIb-IX de Plaquetas/genética , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Trombocitopenia/genética , Trombocitopenia/metabolismoRESUMO
Microvascular thrombosis and inflammation (thromboinflammation) are major causes of morbidity and mortality in critically ill patients with limited therapeutic options. Platelets are central to thromboinflammation, and microvascular platelet thrombi are highly effective at recruiting and activating leukocytes at sites of endothelial injury. Whilst parallel-plate flow chambers, microslides and straight microchannel assays have been widely used to recapitulate leukocyte adhesive behavior on 2-dimensional (2D) surfaces, none of these methods achieve high fidelity 3-dimensional (3D) geometries emulating microvascular platelet thrombi. As a result, the role of hydrodynamic factors in regulating leukocyte interactions with platelet thrombi remains ill-defined. Here, we report a microfluidic post model that allows visualization and analysis of neutrophil-platelet interactions in a 3D flow field. We have utilized the unique mechanosensitive features of platelets to enable selective micropatterning of the 3D posts with human or mouse platelets. By modulating the activation status of platelets, our method enables precise control of platelet surface reactivity and neutrophil recruitment. In addition, our microfluidic post assay accurately recapitulated the rolling versus stationary adhesion behavior of single neutrophils and demonstrated the efficacy of the P-selectin and Mac-1 blocking antibodies to reduce neutrophil recruitment and stationary adhesion, respectively. Moreover, the geometry of posts had a major influence on the efficiency of neutrophil recruitment and adhesion stability. This new post method highlights the importance of platelet 3D geometries in facilitating efficient, localized neutrophil recruitment. These findings have potentially important implications for the potent proinflammatory function of microvascular platelet thrombi.
Assuntos
Plaquetas , Trombose , Animais , Adesão Celular , Humanos , Inflamação , Leucócitos , Camundongos , Microfluídica , NeutrófilosRESUMO
BACKGROUND: Continuous agitation during storage slows down the platelet storage lesions. However, in special circumstances, manual-mixing can be alternatively used to store products for short time periods without compromising platelet quality. Based on this finding, and given the role of shear stress in modulating receptor expression, we were interested in comparing the levels of platelet adhesion receptor, GPVI and platelet adhesion capacity under each storage condition. METHODS: Platelet concentrates (PCs) were divided into three groups: continuously-agitated PCs (CAG-PCs) with or without PP2 (Src kinase inhibitor) and manually-mixed PCs (MM-PCs). Platelet count/MPV, swirling, GPVI and P-selectin expression, GPVI shedding, platelet adhesion/spreading to collagen were examined during 5 days of storage. RESULTS: While MM- and CAG-PCs showed similar levels of P-selectin expression, GPVI expression was significantly elevated in MM-PCs with lower GPVI shedding/expression ratios, enhanced platelet adhesion/spreading and swirling in manually-mixed PCs. Of note, CAG-PCs treated with PP2 also demonstrated lower P-selectin expression and GPVI shedding, higher GPVI expression and attenuated swirling and spreading capability. CONCLUSION: Given the comparable platelet activation state in MM and CAG-PCs as indicated by P-selectin expression, enhanced platelet adhesion/spreading in MM-PCs, along with relatively higher GPVI expression here, supports previous studies demonstrating a role for biomechanical forces in modulating GPVI-dependent function. Thus, lower GPVI expression in CAG-PCs may be due to shear forces induced by agitation, which keeps this receptor down-regulated while also attenuating platelet adhesion/spreading capacities during storage. Low platelet function in PP2-CAG-PCs also highlights the importance of Src-kinases threshold activity in maintaining platelets quality.
RESUMO
Thrombosis with associated inflammation (thromboinflammation) occurs commonly in a broad range of human disorders. It is well recognized clinically in the context of superficial thrombophlebitis (thrombosis and inflammation of superficial veins); however, it is more dangerous when it develops in the microvasculature of injured tissues and organs. Microvascular thrombosis with associated inflammation is well recognized in the context of sepsis and ischemia-reperfusion injury; however, it also occurs in organ transplant rejection, major trauma, severe burns, the antiphospholipid syndrome, preeclampsia, sickle cell disease, and biomaterial-induced thromboinflammation. Central to thromboinflammation is the loss of the normal antithrombotic and anti-inflammatory functions of endothelial cells, leading to dysregulation of coagulation, complement, platelet activation, and leukocyte recruitment in the microvasculature. α-Thrombin plays a critical role in coordinating thrombotic and inflammatory responses and has long been considered an attractive therapeutic target to reduce thromboinflammatory complications. This review focuses on the role of basic aspects of coagulation and α-thrombin in promoting thromboinflammatory responses and discusses insights gained from clinical trials on the effects of various inhibitors of coagulation on thromboinflammatory disorders. Studies in sepsis patients have been particularly informative because, despite using anticoagulant approaches with different pharmacological profiles, which act at distinct points in the coagulation cascade, bleeding complications continue to undermine clinical benefit. Future advances may require the development of therapeutics with primary anti-inflammatory and cytoprotective properties, which have less impact on hemostasis. This may be possible with the growing recognition that components of blood coagulation and platelets have prothrombotic and proinflammatory functions independent of their hemostatic effects.
Assuntos
Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Inflamação/prevenção & controle , Trombose/prevenção & controle , Humanos , Inflamação/complicações , Inflamação/imunologia , Trombose/complicações , Trombose/imunologiaRESUMO
The circulating life span of blood platelets is regulated by the prosurvival protein BCL-XL It restrains the activity of BAK and BAX, the essential prodeath mediators of intrinsic apoptosis. Disabling the platelet intrinsic apoptotic pathway in mice by deleting BAK and BAX results in a doubling of platelet life span and concomitant thrombocytosis. Apoptotic platelets expose phosphatidylserine (PS) via a mechanism that is distinct from that driven by classical agonists. Whether there is any role for apoptotic PS in platelet function in vivo, however, is unclear. Apoptosis has also been associated with the platelet storage lesion (PSL), the constellation of biochemical deteriorations that occur during blood bank storage. In this study, we investigated the role of BAK/BAX-mediated apoptosis in hemostasis and thrombosis and in the development of the PSL. We show that although intrinsic apoptosis is rapidly induced during storage at 37°C, it is not detected when platelets are kept at the standard storage temperature of 22°C. Remarkably, loss of BAK and BAX did not prevent the development of the PSL at either temperature. BAK/BAX-deficient mice exhibited increased bleeding times and unstable thrombus formation. This phenotype was not caused by impaired PS exposure, but was associated with a defect in granule release from aged platelets. Strikingly, rejuvenation of BAK/BAX-deficient platelets in vivo completely rescued the observed hemostatic defects. Thus, apoptotic culling of old platelets from the bloodstream is essential to maintain a functional, hemostatically reactive platelet population. Inhibiting intrinsic apoptosis in blood banked platelets is unlikely to yield significant benefit.
Assuntos
Apoptose , Plaquetas/metabolismo , Suscetibilidade a Doenças , Animais , Apoptose/genética , Biomarcadores , Tempo de Sangramento , Contagem de Células Sanguíneas , Coagulação Sanguínea , Caspases/metabolismo , Sobrevivência Celular/genética , Feminino , Genótipo , Masculino , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Transdução de Sinais , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo , Proteína bcl-X/genética , Proteína bcl-X/metabolismoRESUMO
Clot retraction refers to the process whereby activated platelets transduce contractile forces onto the fibrin network of a thrombus, which over time increases clot density and decreases clot size. This process is considered important for promoting clot stability and maintaining blood vessel patency. Insights into the mechanisms regulating clot retraction at sites of vascular injury have been hampered by a paucity of in vivo experimental models. By pairing localized vascular injury with thrombin microinjection in the mesenteric circulation of mice, we have demonstrated that the fibrin network of thrombi progressively compacts over a 2-hour period. This was a genuine retraction process, as treating thrombi with blebbistatin to inhibit myosin IIa-mediated platelet contractility prevented shrinkage of the fibrin network. Real-time confocal analysis of fibrinolysis after recombinant tissue-type plasminogen activator (tPA) administration revealed that incomplete proteolysis of fibrin polymers markedly facilitated clot retraction. Similarly, inhibiting endogenous fibrinolysis with tranexamic acid reduced retraction of fibrin polymers in vivo. In vitro clot retraction experiments indicated that subthreshold doses of tPA facilitated clot retraction through a plasmin-dependent mechanism. These effects correlated with changes in the elastic modulus of fibrin clots. These findings define the endogenous fibrinolytic system as an important regulator of clot retraction, and show that promoting clot retraction is a novel and complementary means by which fibrinolytic enzymes can reduce thrombus size.
Assuntos
Retração do Coágulo , Fibrinólise , Actomiosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Fibrina/metabolismo , Fibrinólise/efeitos dos fármacos , Humanos , Masculino , Camundongos , Miosina não Muscular Tipo IIA/metabolismo , Trombose/diagnóstico por imagem , Trombose/metabolismo , Trombose/patologia , Ativador de Plasminogênio Tecidual/metabolismo , Ativador de Plasminogênio Tecidual/farmacologia , Ácido Tranexâmico/farmacologiaRESUMO
The Dok proteins are a family of adaptor molecules that have a well defined role in regulating cellular migration, immune responses, and tumor progression. Previous studies have demonstrated that Doks-1 to 3 are expressed in platelets and that Dok-2 is tyrosine-phosphorylated downstream of integrin αIIbß3, raising the possibility that it participates in integrin αIIbß3 outside-in signaling. We demonstrate that Dok-2 in platelets is primarily phosphorylated by Lyn kinase. Moreover, deficiency of Dok-2 leads to dysregulated integrin αIIbß3-dependent cytosolic calcium flux and phosphatidylinositol(3,4)P2 accumulation. Although agonist-induced integrin αIIbß3 affinity regulation was unaltered in Dok-2(-/-) platelets, Dok-2 deficiency was associated with a shear-dependent increase in integrin αIIbß3 adhesive function, resulting in enhanced platelet-fibrinogen and platelet-platelet adhesive interactions under flow. This increase in adhesion was restricted to discoid platelets and involved the shear-dependent regulation of membrane tethers. Dok-2 deficiency was associated with an increased rate of platelet aggregate formation on thrombogenic surfaces, leading to accelerated thrombus growth in vivo. Overall, this study defines an important role for Dok-2 in regulating biomechanical adhesive function of discoid platelets. Moreover, they define a previously unrecognized prothrombotic mechanism that is not detected by conventional platelet function assays.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Resistência ao Cisalhamento , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fibrinogênio/farmacologia , Hemorreologia/efeitos dos fármacos , Humanos , Proteínas Imobilizadas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoproteínas/deficiência , Adesividade Plaquetária/efeitos dos fármacos , Resistência ao Cisalhamento/efeitos dos fármacos , Trombose/metabolismo , Trombose/patologia , Trombose/fisiopatologia , Fatores de TempoRESUMO
Thrombosis promotes leukocyte infiltration into inflamed tissues, leading to organ injury in a broad range of diseases; however, the mechanisms by which thrombi guide leukocytes to sites of vascular injury remain ill-defined. Using mouse models of endothelial injury (traumatic or ischemia reperfusion), we demonstrate a distinct process of leukocyte recruitment, termed "directed intravascular migration," specifically mediated by platelet thrombi. Single adherent platelets and platelet aggregates stimulated leukocyte shape change at sites of endothelial injury; however, only thrombi were capable of inducing directed intravascular leukocyte migration. Leukocyte recruitment and migration induced by platelet thrombi occurred most prominently in veins but could also occur in arteries following ischemia-reperfusion injury. In vitro studies demonstrated a major role for platelet-derived NAP-2 (CXCL-7) and its CXCR1/2 receptor in regulating leukocyte polarization and motility. In vivo studies demonstrated the presence of an NAP-2 chemotactic gradient within the thrombus body. Pharmacologic blockade of CXCR1/2 as well as genetic deletion of NAP-2 markedly reduced leukocyte shape change and intrathrombus migration. These studies define a distinct process of leukocyte migration that is initiated by homotypic adhesive interactions between platelets, leading to the development of an NAP-2 chemotactic gradient within the thrombus body that guides leukocytes to sites of vascular injury.
Assuntos
Plaquetas/citologia , Quimiocinas CXC/metabolismo , Leucócitos/citologia , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/metabolismo , Trombose/imunologia , Animais , Plaquetas/imunologia , Plaquetas/metabolismo , Adesão Celular/imunologia , Movimento Celular/imunologia , Polaridade Celular/imunologia , Proteínas de Fluorescência Verde/genética , Leucócitos/imunologia , Artérias Mesentéricas/imunologia , Artérias Mesentéricas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ferimentos Penetrantes Produzidos por Agulha/imunologia , Ferimentos Penetrantes Produzidos por Agulha/patologia , Neutrófilos/citologia , Neutrófilos/imunologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologiaRESUMO
Apoptotic caspases, including caspase-9, are thought to facilitate platelet shedding by megakaryocytes. They are known to be activated during platelet apoptosis, and have also been implicated in platelet hemostatic responses. However, the precise requirement for, and the regulation of, apoptotic caspases have never been defined in either megakaryocytes or platelets. To establish the role of caspases in platelet production and function, we generated mice lacking caspase-9 in their hematopoietic system. We demonstrate that both megakaryocytes and platelets possess a functional apoptotic caspase cascade downstream of Bcl-2 family-mediated mitochondrial damage. Caspase-9 is the initiator caspase, and its loss blocks effector caspase activation. Surprisingly, steady-state thrombopoiesis is unperturbed in the absence of caspase-9, indicating that the apoptotic caspase cascade is not required for platelet production. In platelets, loss of caspase-9 confers resistance to the BH3 mimetic ABT-737, blocking phosphatidylserine (PS) exposure and delaying ABT-737-induced thrombocytopenia in vivo. Despite this, steady-state platelet lifespan is normal. Casp9(-/-) platelets are fully capable of physiologic hemostatic responses and functional regulation of adhesive integrins in response to agonist. These studies demonstrate that the apoptotic caspase cascade is required for the efficient death of megakaryocytes and platelets, but is dispensable for their generation and function.
Assuntos
Apoptose/fisiologia , Plaquetas/citologia , Caspase 9/fisiologia , Megacariócitos/citologia , Trombopoese/fisiologia , Animais , Compostos de Bifenilo/farmacologia , Compostos de Bifenilo/toxicidade , Plaquetas/enzimologia , Caspase 9/deficiência , Caspase 9/genética , Linhagem da Célula , Hemostasia/efeitos dos fármacos , Hemostasia/fisiologia , Hirudinas/farmacologia , Fígado/embriologia , Transplante de Fígado , Megacariócitos/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrofenóis/farmacologia , Nitrofenóis/toxicidade , Piperazinas/farmacologia , Piperazinas/toxicidade , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Quimera por Radiação , Sulfonamidas/farmacologia , Sulfonamidas/toxicidade , Trombocitopenia/induzido quimicamente , Proteína X Associada a bcl-2/deficiênciaRESUMO
A large variety of dietary phytochemicals has been shown to improve thrombosis and stroke outcomes in preclinical studies. Many of these compounds feature electrophilic functionalities that potentially undergo covalent addition to the sulfhydryl side chain of cysteine residues within proteins. However, the impact of such covalent modifications on the platelet activity and function remains unclear. This study explores the irreversible engagement of 23 electrophilic phytochemicals with platelets, unveiling the unique antiplatelet selectivity of sulforaphane (SFN). SFN impairs platelet responses to adenosine diphosphate (ADP) and a thromboxane A2 receptor agonist while not affecting thrombin and collagen-related peptide activation. It also substantially reduces platelet thrombus formation under arterial flow conditions. Using an alkyne-integrated probe, protein disulfide isomerase A6 (PDIA6) was identified as a rapid kinetic responder to SFN. Mechanistic profiling studies revealed SFN's nuanced modulation of PDIA6 activity and substrate specificity. In an electrolytic injury model of thrombosis, SFN enhanced the thrombolytic activity of recombinant tissue plasminogen activator (rtPA) without increasing blood loss. Our results serve as a catalyst for further investigations into the preventive and therapeutic mechanisms of dietary antiplatelets, aiming to enhance the clot-busting power of rtPA, currently the only approved therapeutic for stroke recanalization that has significant limitations.
RESUMO
BH3 mimetics are a new class of proapo-ptotic anticancer agents that have shown considerable promise in preclinical animal models and early-stage human trials. These agents act by inhibiting the pro-survival function of one or more Bcl-2-related proteins. Agents that inhibit Bcl-x(L) induce rapid platelet death that leads to thrombocytopenia; however, their impact on the function of residual circulating platelets remains unclear. In this study, we demonstrate that the BH3 mimetics, ABT-737 or ABT-263, induce a time- and dose-dependent decrease in platelet adhesive function that correlates with ectodomain shedding of the major platelet adhesion receptors, glycoprotein Ibα and glycoprotein VI, and functional down-regulation of integrin α(IIb)ß(3). Analysis of platelets from mice treated with higher doses of BH3 mimetics revealed the presence of a subpopulation of circulating platelets undergoing cell death that have impaired activation responses to soluble agonists. Functional analysis of platelets by intravital microscopy revealed a time-dependent defect in platelet aggregation at sites of vascular injury that correlated with an increase in tail bleeding time. Overall, these studies demonstrate that Bcl-x(L)-inhibitory BH3 mimetics not only induce thrombocytopenia but also a transient thrombocytopathy that can undermine the hemostatic function of platelets.
Assuntos
Plaquetas/fisiologia , Hemostasia/fisiologia , Trombocitopenia/fisiopatologia , Proteína bcl-X/metabolismo , Compostos de Anilina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo/farmacologia , Plaquetas/metabolismo , Plaquetas/ultraestrutura , Western Blotting , Colágeno/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Hemostasia/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Varredura , Nitrofenóis/farmacologia , Fosfatidilserinas/metabolismo , Piperazinas/farmacologia , Adesividade Plaquetária/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Contagem de Plaquetas , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas , Glicoproteínas da Membrana de Plaquetas/metabolismo , Sulfonamidas/farmacologia , Trombocitopenia/induzido quimicamente , Fatores de Tempo , Proteína bcl-X/antagonistas & inibidoresAssuntos
Cáusticos/toxicidade , Cloretos/toxicidade , Modelos Animais de Doenças , Compostos Férricos/toxicidade , Oxidantes/toxicidade , Trombose/induzido quimicamente , Animais , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Agregação Eritrocítica/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Camundongos , Oxidantes/farmacologia , Ativação Plaquetária , Trombose/prevenção & controleRESUMO
Apoptosis and necrosis represent distinct cell death processes that regulate mammalian development, physiology and disease. Apoptosis characteristically leads to the silent destruction and removal of cells in the absence of an inflammatory response. In contrast, necrotic cell death can induce physiologic inflammatory responses linked to tissue defense and repair. Although anucleate, platelets undergo programmed cell death, with apoptosis playing an important role in clearing effete platelets from the circulation. While it has long been recognized that procoagulant platelets exhibit characteristic features of dying cells, recent studies have demonstrated that platelet procoagulant function can occur independent of apoptosis. A growing body of evidence suggest that the biochemical, morphologic and functional changes underlying agonist-induced platelet procoagulant function are broadly consistent with cell necrosis, raising the possibility that distinct death pathways regulate platelet function and survival. In this article, we will discuss the mechanisms underlying apoptotic and necrotic cell death pathways and examine the evidence linking these pathways to the platelet procoagulant response. We will also discuss the potential contribution of these pathways to the platelet storage lesion and propose a simplified nomenclature to describe procoagulant platelets.
Assuntos
Plaquetas/patologia , Necrose/sangue , Trombofilia/sangue , Apoptose , Coagulação Sanguínea , Humanos , Inflamação/sangue , Deficiência do Pool Plaquetário/etiologiaRESUMO
Platelet activation at sites of vascular injury is essential for the arrest of bleeding; however, excessive platelet accumulation at regions of atherosclerotic plaque rupture can result in the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and ischemic stroke. Rheological disturbances (high shear stress) have an important role in promoting arterial thrombosis by enhancing the adhesive and signaling function of platelet integrin alpha(IIb)beta(3) (GPIIb-IIIa). In this study we have defined a key role for the Type Ia phosphoinositide 3-kinase (PI3K) p110beta isoform in regulating the formation and stability of integrin alpha(IIb)beta(3) adhesion bonds, necessary for shear activation of platelets. Isoform-selective PI3K p110beta inhibitors have been developed which prevent formation of stable integrin alpha(IIb)beta(3) adhesion contacts, leading to defective platelet thrombus formation. In vivo, these inhibitors eliminate occlusive thrombus formation but do not prolong bleeding time. These studies define PI3K p110beta as an important new target for antithrombotic therapy.
Assuntos
Artérias/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Adesividade Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Trombose/metabolismo , Animais , Tempo de Sangramento , Plaquetas/metabolismo , Citometria de Fluxo , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Inibidores de Fosfoinositídeo-3 Quinase , Reologia , Serotonina/metabolismo , Trombose/patologia , Proteínas rap de Ligação ao GTP/metabolismoRESUMO
Recanalization with restored cerebral perfusion is the primary goal of thrombolytic therapy in acute ischemic stroke. The identification of adjunctive therapies that can be safely used to enhance thrombolysis in stroke remains an elusive goal. We report here the development of a mouse in situ carotid artery thrombolysis (iCAT) stroke model involving graded cerebral ischemia to induce unihemispheric infarction after thrombotic occlusion of the common carotid artery (CCA). Electrolytic-induced thrombotic occlusion of the left CCA enabled real-time assessment of recanalization and rethrombosis events after thrombolysis with recombinant tissue-type plasminogen activator (rtPA). Concurrent transient stenosis of the right CCA induced unihemispheric hypoperfusion and infarction in the left middle cerebral artery territory. Real-time assessment of thrombolysis revealed recanalization rates <30% in rtPA-treated animals with high rates of rethrombosis. Addition of the direct thrombin inhibitor argatroban increased recanalization rates to 50% and reduced rethrombosis. Paradoxically, this was associated with increased cerebral ischemia and stroke-related mortality (25%-42%). Serial analysis of carotid and cerebral blood flow showed that coadministration of argatroban with rtPA resulted in a marked increase in carotid artery embolization, leading to distal obstruction of the middle cerebral artery. Real-time imaging of carotid thrombi revealed that adjunctive anticoagulation destabilized platelet-rich thrombi at the vessel wall, leading to dislodgement of large platelet emboli. These studies confirm the benefits of anticoagulants in enhancing thrombolysis and large artery recanalization; however, at high levels of anticoagulation (â¼3-fold prolongation of activated partial thromboplastin time), this effect is offset by increased incidence of carotid artery embolization and distal middle cerebral artery occlusion. The iCAT stroke model should provide important new insight into the effects of adjunctive antithrombotic agents on real-time thrombus dynamics during thrombolysis and their correlation with stroke outcomes.
Assuntos
Isquemia Encefálica , AVC Isquêmico , Acidente Vascular Cerebral , Tromboembolia , Animais , Anticoagulantes/uso terapêutico , Antitrombinas/uso terapêutico , Arginina/análogos & derivados , Isquemia Encefálica/complicações , Isquemia Encefálica/tratamento farmacológico , Artéria Carótida Primitiva , Fibrinolíticos/farmacologia , Fibrinolíticos/uso terapêutico , Infarto da Artéria Cerebral Média/complicações , Infarto da Artéria Cerebral Média/tratamento farmacológico , Camundongos , Ácidos Pipecólicos , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/etiologia , Sulfonamidas , Terapia Trombolítica/efeitos adversos , Terapia Trombolítica/métodos , Ativador de Plasminogênio Tecidual/farmacologia , Ativador de Plasminogênio Tecidual/uso terapêutico , Resultado do TratamentoRESUMO
Phosphoinositide (PI) 3-kinase (PI3K) signaling processes play an important role in regulating the adhesive function of integrin alpha(IIb)beta(3), necessary for platelet spreading and sustained platelet aggregation. PI3K inhibitors are effective at reducing platelet aggregation and thrombus formation in vivo and as a consequence are currently being evaluated as novel antithrombotic agents. PI3K regulation of integrin alpha(IIb)beta(3) activation (affinity modulation) primarily occurs downstream of G(i)-coupled and tyrosine kinase-linked receptors linked to the activation of Rap1b, AKT, and phospholipase C. In the present study, we demonstrate an important role for PI3Ks in regulating the avidity (strength of adhesion) of high affinity integrin alpha(IIb)beta(3) bonds, necessary for the cellular transmission of contractile forces. Using knock-out mouse models and isoform-selective PI3K inhibitors, we demonstrate that the Type Ia p110 beta isoform plays a major role in regulating thrombin-stimulated fibrin clot retraction in vitro. Reduced clot retraction induced by PI3K inhibitors was not associated with defects in integrin alpha(IIb)beta(3) activation, actin polymerization, or actomyosin contractility but was associated with a defect in integrin alpha(IIb)beta(3) association with the contractile cytoskeleton. Analysis of integrin alpha(IIb)beta(3) adhesion contacts using total internal reflection fluorescence microscopy revealed an important role for PI3Ks in regulating the stability of high affinity integrin alpha(IIb)beta(3) bonds. These studies demonstrate an important role for PI3K p110 beta in regulating the avidity of high affinity integrin alpha(IIb)beta(3) receptors, necessary for the cellular transmission of contractile forces. These findings may provide new insight into the potential antithrombotic properties of PI3K p110 beta inhibitors.
Assuntos
Plaquetas/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Plaquetas/efeitos dos fármacos , Classe I de Fosfatidilinositol 3-Quinases , Retração do Coágulo/efeitos dos fármacos , Retração do Coágulo/fisiologia , Citoesqueleto/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinases/genética , Estresse Mecânico , Trombina/farmacologia , Tromboxano A2/metabolismoRESUMO
Procoagulant platelets exhibit hallmark features of apoptotic cells, including membrane blebbing, microvesiculation, and phosphatidylserine (PS) exposure. Although platelets possess many well-known apoptotic regulators, their role in regulating the procoagulant function of platelets is unclear. To clarify this, we investigated the consequence of removing the essential mediators of apoptosis, Bak and Bax, or directly inducing apoptosis with the BH3 mimetic compound ABT-737. Treatment of platelets with ABT-737 triggered PS exposure and a marked increase in thrombin generation in vitro. This increase in procoagulant function was Bak/Bax- and caspase-dependent, but it was unaffected by inhibitors of platelet activation or by chelating extracellular calcium. In contrast, agonist-induced platelet procoagulant function was unchanged in Bak(-/-)Bax(-/-) or caspase inhibitor-treated platelets, but it was completely eliminated by extracellular calcium chelators or inhibitors of platelet activation. These studies show the existence of 2 distinct pathways regulating the procoagulant function of platelets.
Assuntos
Coagulação Sanguínea , Plaquetas/fisiologia , Fosfatidilserinas/metabolismo , Plaquetas/citologia , Cálcio , Caspases , Células Cultivadas , Quelantes/farmacologia , Humanos , Inibidores da Agregação Plaquetária/farmacologia , Trombina/biossíntese , Proteína Killer-Antagonista Homóloga a bcl-2 , Proteína X Associada a bcl-2RESUMO
Hemopoietic cells express relatively high levels of the type I phosphoinositide (PI) 3-kinase isoforms, with p110δ and γ exhibiting specialized signaling functions in neutrophils, monocytes, mast cells, and lymphocytes. In platelets, p110ß appears to be the dominant PI 3-kinase isoform regulating platelet activation, irrespective of the nature of the primary platelet activating stimulus. Based on findings with isoform-selective p110ß pharmacological inhibitors and more recently with p110ß-deficient platelets, p110ß appears to primarily signal downstream of G(i)- and tyrosine kinase-coupled receptors. Functionally, inhibition of p110ß kinase function leads to a marked defect in integrin α(IIb)ß3 adhesion and reduced platelet thrombus formation in vivo. This defect in platelet adhesive function is not associated with increased bleeding, suggesting that therapeutic targeting of p110ß may represent a safe approach to reduce thrombotic complications in patients with cardiovascular disease.
Assuntos
Plaquetas/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Animais , Fibrinolíticos/farmacologia , Humanos , Inibidores de Fosfoinositídeo-3 Quinase , Ativação Plaquetária , Adesividade Plaquetária , Transdução de SinaisRESUMO
PI3Ks (phosphoinositide 3-kinases) play a critical role in platelet functional responses. PI3Ks are activated upon P2Y12 receptor stimulation and generate pro-aggregatory signals. P2Y12 receptor has been shown to play a key role in the platelet aggregation and thromboxane A2 generation caused by co-stimulation with Gq or Gz, or super-stimulation of Gi pathways. In the present study, we evaluated the role of specific PI3K isoforms alpha, beta, gamma and delta in platelet aggregation, thromboxane A2 generation and ERK (extracellular-signal-regulated kinase) activation. Our results show that loss of the PI3K signal impaired the ability of ADP to induce platelet aggregation, ERK phosphorylation and thromboxane A2 generation. We also show that Gq plus Gi- or Gi plus Gz-mediated platelet aggregation, ERK phosphorylation and thromboxane A2 generation in human platelets was inhibited by TGX-221, a PI3Kbeta-selective inhibitor, but not by PIK75 (a PI3Kalpha inhibitor), AS252424 (a PI3Kgamma inhibitor) or IC87114 (a PI3Kdelta inhibitor). TGX-221 also showed a similar inhibitory effect on the Gi plus Gz-mediated platelet responses in platelets from P2Y1-/- mice. Finally, 2MeSADP (2-methyl-thio-ADP)-induced Akt phosphorylation was significantly inhibited in the presence of TGX-221, suggesting a critical role for PI3Kbeta in Gi-mediated signalling. Taken together, our results demonstrate that PI3Kbeta plays an important role in ADP-induced platelet aggregation. Moreover, PI3Kbeta mediates ADP-induced thromboxane A2 generation by regulating ERK phosphorylation.