RESUMO
Lowe syndrome (LS) is an oculocerebrorenal syndrome of Lowe (OCRL1) genetic disorder resulting in a defect of the OCRL protein, a phosphatidylinositol-4,5-bisphosphate 5-phosphatase containing various domains including a Rho GTPase-activating protein (RhoGAP) homology domain catalytically inactive. We previously reported surgery-associated bleeding in patients with LS, suggestive of platelet dysfunction, accompanied with a mild thrombocytopenia in several patients. To decipher the role of OCRL in platelet functions and in megakaryocyte (MK) maturation, we conducted a case-control study on 15 patients with LS (NCT01314560). While all had a drastically reduced expression of OCRL, this deficiency did not affect platelet aggregability, but resulted in delayed thrombus formation on collagen under flow conditions, defective platelet spreading on fibrinogen and impaired clot retraction. We evidenced alterations of the myosin light chain phosphorylation (P-MLC), with defective Rac1 activity and, inversely, elevated active RhoA. Altered cytoskeleton dynamics was also observed in cultured patient MKs showing deficient proplatelet extension with increased P-MLC that was confirmed using control MKs transfected with OCRL-specific small interfering(si)RNA (siOCRL). Patients with LS also had an increased proportion of circulating barbell-shaped proplatelets. Our present study establishes that a deficiency of the OCRL protein results in a defective actomyosin cytoskeleton reorganisation in both MKs and platelets, altering both thrombopoiesis and some platelet responses to activation necessary to ensure haemostasis.
Assuntos
Plaquetas/citologia , Megacariócitos/citologia , Síndrome Oculocerebrorrenal/genética , Monoéster Fosfórico Hidrolases/fisiologia , Trombopoese/fisiologia , Actomiosina/análise , Adolescente , Adulto , Anemia/etiologia , Coagulação Sanguínea , Plaquetas/ultraestrutura , Estudos de Casos e Controles , Forma Celular , Criança , Colágeno , Citoesqueleto/ultraestrutura , Feminino , Inativação Gênica , Humanos , Masculino , Megacariócitos/ultraestrutura , Pessoa de Meia-Idade , Mutação , Cadeias Leves de Miosina/metabolismo , Síndrome Oculocerebrorrenal/sangue , Síndrome Oculocerebrorrenal/patologia , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Fosforilação , Domínios Proteicos , Processamento de Proteína Pós-Traducional , RNA Interferente Pequeno/genética , Transdução de Sinais , Trombocitopenia/etiologia , Adulto JovemRESUMO
BACKGROUND AND OBJECTIVES: Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). MATERIALS AND METHODS: The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. RESULTS: Proliferation of stem cells isolated out of UCB was significantly higher (P < 0·0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44 ± 9% versus 76 ± 11%, respectively (P < 0·0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7·4 platelet-like particles per input cell from PB compared to 4·2 from UCB (P = 0·02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. CONCLUSION: This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.
Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Células-Tronco Hematopoéticas/citologia , Dispositivos Lab-On-A-Chip , Citoesqueleto de Actina/metabolismo , Antígenos CD34/metabolismo , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Citometria de Fluxo , Humanos , Integrina beta3/metabolismo , Megacariócitos/citologia , Microscopia , Fenótipo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , RefrigeraçãoRESUMO
Platelets originate from megakaryocytes (MKs) by cytoplasmic elongation into proplatelets. Direct platelet release is not seen in bone marrow hematopoietic islands. It was suggested that proplatelet fragmentation into platelets can occur intravascularly, yet evidence of its dependence on hydrodynamic forces is missing. Therefore, we investigated whether platelet production from MKs could be up-regulated by circulatory forces. Human mature MKs were perfused at a high shear rate on von Willebrand factor. Cells were observed in real time by videomicroscopy, and by confocal and electron microscopy after fixation. Dramatic cellular modifications followed exposure to high shear rates: 30% to 45% adherent MKs were converted into proplatelets and released platelets within 20 minutes, contrary to static conditions that required several hours, often without platelet release. Tubulin was present in elongated proplatelets and platelets, thus ruling out membrane tethers. By using inhibitors, we demonstrated the fundamental roles of microtubule assembly and MK receptor GPIb. Secretory granules were present along the proplatelet shafts and in shed platelets, as shown by P-selectin labeling. Platelets generated in vitro were functional since they responded to thrombin by P-selectin expression and cytoskeletal reorganization. In conclusion, MK exposure to high shear rates promotes platelet production via GPIb, depending on microtubule assembly and elongation.
Assuntos
Plaquetas/citologia , Plaquetas/metabolismo , Megacariócitos/citologia , Megacariócitos/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Citometria de Fluxo/métodos , Humanos , Microscopia Confocal/métodos , Microscopia Eletrônica/métodos , Microscopia de Vídeo/métodos , Modelos Biológicos , Perfusão , Estresse Mecânico , Trombopoese/imunologia , Tubulina (Proteína)/metabolismo , Fator de von Willebrand/metabolismoRESUMO
Blood platelets are essential for controlling hemostasis. They are released by megakaryocytes (MKs) located in the bone marrow, upon extension of cytoplasmic protrusions into the lumen of bone marrow sinusoids. Their number increases in postpulmonary capillaries, suggesting a role for oxygen gradient in thrombopoiesis (ie, platelet biogenesis). In this study, we show that initiation of thrombopoiesis from human mature MKs was enhanced under hyperoxia or during pro-oxidant treatments, whereas antioxidants dampened it. Quenching mitochondrial reactive oxygen species (mtROS) with MitoTEMPO decreased thrombopoiesis, whereas genetically enhancing mtROS by deacetylation-null sirtuin-3 expression increased it. Blocking cytosolic ROS production by NOX inhibitors had no impact. Classification according to the cell roundness index delineated 3 stages of thrombopoiesis in mature MKs. Early-stage round MKs exhibited the highest index, which correlated with low mtROS levels, a mitochondrial tubular network, and the mitochondrial recruitment of the fission activator Drp1. Intermediate MKs at the onset of thrombopoiesis showed high mtROS levels and small, well-delineated mitochondria. Terminal MKs showed the lowest roundness index and long proplatelet extensions. Inhibiting Drp1-dependent mitochondrial fission of mature MKs by Mdivi-1 favored a tubular mitochondrial network and lowered both mtROS levels and intermediate MKs proportion, whereas enhancing Drp1 activity genetically had opposite effects. Reciprocally, quenching mtROS limited mitochondrial fission in round MKs. These data demonstrate a functional coupling between ROS and mitochondrial fission in MKs, which is crucial for the onset of thrombopoiesis. They provide new molecular cues that control initiation of platelet biogenesis and may help elucidate some unexplained thrombocytopenia.
Assuntos
Megacariócitos , Trombopoese , Plaquetas , Humanos , Dinâmica Mitocondrial , Espécies Reativas de OxigênioRESUMO
OBJECTIVES: The goal of this study was to identify differences in shear-induced platelet aggregation (SIPA) between patients who did or did not experience subacute stent thrombosis (SAT). BACKGROUND: Despite dual antiplatelet therapy, SAT after coronary stenting occurs in approximately 1% of patients. There is no accepted platelet function test to identify patients at risk. METHODS: We analyzed platelet aggregation in 10 patients who had experienced SAT (cases), 22 stented patients without SAT (controls), and 17 healthy volunteers (normals). All patients except normals were treated with both aspirin and clopidogrel. RESULTS: Shear-induced platelet aggregation was higher in cases than in controls at both shear rates of 200 s(-1) (40.9 +/- 12.2% vs. 18.2 +/- 18%, p = 0.013) and 4,000 s(-1) (57.4 +/- 16.4% vs. 23.4 +/- 21.2%, p = 0.009). Moreover, SIPA in cases was significantly higher than in normals both at 200 s(-1) (p = 0.013) and 4,000(-1) (p = 0.009). CONCLUSIONS: Shear-induced platelet aggregation is increased in patients experiencing SAT compared with controls receiving dual antiplatelet therapy and to normals receiving no antiplatelet therapy, which suggests increased intrinsic patient-related platelet reactivity in patients with SAT. The predictive value of SIPA for SAT requires prospective investigation.
Assuntos
Doença das Coronárias/terapia , Agregação Plaquetária/fisiologia , Stents/efeitos adversos , Trombose/fisiopatologia , Ticlopidina/análogos & derivados , Idoso , Angioplastia Coronária com Balão , Aspirina/farmacologia , Aspirina/uso terapêutico , Estudos de Casos e Controles , Clopidogrel , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ativação Plaquetária/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/uso terapêutico , Estudos Prospectivos , Trombose/prevenção & controle , Ticlopidina/farmacologia , Ticlopidina/uso terapêuticoRESUMO
The vascular endothelium is a thromboresistant surface, allowing the free flowing of blood cell elements. Platelets are predominantly involved in the rapid response to a vascular lesion, exposing the underlying thrombogenic subendothelium and leading to initiation of thrombus formation. Thrombus growth requires on the one hand, the recruitment of circulating platelets to the luminal side of the thrombus and on the other hand, the assembly of the proteins of the blood coagulation cascade on the platelet catalytic surface leading to thrombin formation. High shear forces are necessary for the dual role of von Willebrand factor (VWF) in the initiation of platelet thrombus formation and in its growth and stabilization. In hemodynamic conditions, platelet adhesion depends on the interaction between VWF and the platelet receptor glycoprotein Ib (GPIb). This interaction is the only one able to resist to the high shear rates that prevail in arterioles, the microcirculation or stenosed arteries. Thereafter, the interaction between VWF and the alphaIIbbeta3 integrin allows the definitive arrest of platelets and induces thrombus formation. Thus, high shear forces by themselves are able to induce platelet activation/aggregation, without added exogenous agonist. VWF is synthesised by endothelial cells as a series of multimers of different sizes. The multimers with the highest molecular weight, the so-called ultra-large multimers, are strongly thrombogenic by their increased ability to bind platelet GPIb and to induce the formation of circulating aggregates. These ultra-large multimers are normally cleaved by the ADAMTS13 metalloprotease into smaller multimers which are also less thrombogenic. The in vivo proteolysis of VWF by ADAMTS13 depends on the high shear rates, which increase the opening of multimers anchored to the endothelial cell layer and the exposure of the cleavage site of VWF by ADAMTS13. An ADAMTS13 deficiency thus likely would result in the accumulation of ultra-large multimers on the endothelial surface, which retains platelets on the activated endothelium and results in micro-thrombi formation, as seen in thrombotic thrombocytopenic purpura. Platelet-VWF interactions are also involved in inflammation. Activation of endothelial cells induces the release of VWF from the Weibel-Palade bodies as well as the surface expression of VWF and P-selectin. These molecules allow leukocyte and platelet rolling on endothelial cells, and expression of E-selectin, VCAM-1 and other adhesion molecules. Recently, it has been shown that activated platelets allow transient activation of intact, non stimulated endothelial cells, thus increasing the inflammation process. VWF and platelet P-selectin have been shown to be essential to this process. Thus, platelet--vessel wall interactions are involved in thrombosis and inflammation essentially via VWF.
Assuntos
Plaquetas/fisiologia , Vasos Sanguíneos/fisiologia , Endotélio Vascular/fisiologia , Animais , HumanosRESUMO
We present a new, rapid method for producing blood platelets in vitro from cultured megakaryocytes based on a microfluidic device. This device consists in a wide array of VWF-coated micropillars. Such pillars act as anchors on megakaryocytes, allowing them to remain trapped in the device and subjected to hydrodynamic shear. The combined effect of anchoring and shear induces the elongation of megakaryocytes and finally their rupture into platelets and proplatelets. This process was observed with megakaryocytes from different origins and found to be robust. This original bioreactor design allows to process megakaryocytes at high throughput (millions per hour). Since platelets are produced in such a large amount, their extensive biological characterisation is possible and shows that platelets produced in this bioreactor are functional.
Assuntos
Plaquetas/citologia , Sangue Fetal/citologia , Dispositivos Lab-On-A-Chip , Megacariócitos/citologia , Modelos Biológicos , Antígenos CD/fisiologia , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Biomimética , Reatores Biológicos , Plaquetas/fisiologia , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Sangue Fetal/fisiologia , Expressão Gênica , Humanos , Megacariócitos/fisiologia , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Contagem de Plaquetas , Reologia , Estresse MecânicoRESUMO
von Willebrand disease (VWD)-type 2B is characterized by gain-of-function mutations in the von Willebrand factor (VWF) A1-domain, leading to increased affinity for its platelet-receptor, glycoprotein Ibα. We engineered the first knock-in (KI) murine model for VWD-type 2B by introducing the p.V1316M mutation in murine VWF. Homozygous KI-mice replicated human VWD-type 2B with macrothrombocytopenia (platelet counts reduced by 55%, platelet volume increased by 44%), circulating platelet-aggregates and a severe bleeding tendency. Also, vessel occlusion was deficient in the FeCl3-induced thrombosis model. Platelet aggregation induced by thrombin or collagen was defective for KI-mice at all doses. KI-mice manifested a loss of high molecular weight multimers and increased multimer degradation. In a model of VWF-string formation, the number of platelets/string and string-lifetime were surprisingly enhanced in KI-mice, suggesting that proteolysis of VWF/p.V1316M is differentially regulated in the circulation versus the endothelial surface. Furthermore, we observed increased leukocyte recruitment during an inflammatory response induced by the reverse passive Arthus reaction. This points to an active role of VWF/p.V1316M in the exfiltration of leukocytes under inflammatory conditions. In conclusion, our genetically-engineered VWD-type 2B mice represent an original model to study the consequences of spontaneous VWF-platelet interactions and the physiopathology of this human disease.
Assuntos
Doença de von Willebrand Tipo 2/sangue , Doença de von Willebrand Tipo 2/patologia , Substituição de Aminoácidos , Animais , Modelos Animais de Doenças , Feminino , Engenharia Genética , Hemostasia/genética , Humanos , Inflamação/genética , Inflamação/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/sangue , Proteínas Mutantes/química , Proteínas Mutantes/genética , Adesividade Plaquetária , Contagem de Plaquetas , Doença de von Willebrand Tipo 2/genética , Fator de von Willebrand/química , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
von Willebrand disease type 2B (VWD-type 2B) is characterized by gain-of-function mutations of von Willebrand factor (vWF) that enhance its binding to platelet glycoprotein Ibα and alter the protein's multimeric structure. Patients with VWD-type 2B display variable extents of bleeding associated with macrothrombocytopenia and sometimes with thrombopathy. Here, we addressed the molecular mechanism underlying the severe macrothrombocytopenia both in a knockin murine model for VWD-type 2B by introducing the p.V1316M mutation in the murine Vwf gene and in a patient bearing this mutation. We provide evidence of a profound defect in megakaryocyte (MK) function since: (a) the extent of proplatelet formation was drastically decreased in 2B MKs, with thick proplatelet extensions and large swellings; and (b) 2B MKs presented actin disorganization that was controlled by upregulation of the RhoA/LIM kinase (LIMK)/cofilin pathway. In vitro and in vivo inhibition of the LIMK/cofilin signaling pathway rescued actin turnover and restored normal proplatelet formation, platelet count, and platelet size. These data indicate, to our knowledge for the first time, that the severe macrothrombocytopenia in VWD-type 2B p.V1316M is due to an MK dysfunction that originates from a constitutive activation of the RhoA/LIMK/cofilin pathway and actin disorganization. This suggests a potentially new function of vWF during platelet formation that involves regulation of actin dynamics.
Assuntos
Fatores de Despolimerização de Actina/genética , Quinases Lim/genética , Trombocitopenia/fisiopatologia , Doença de von Willebrand Tipo 2/fisiopatologia , Fator de von Willebrand/genética , Animais , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Mutação , Transdução de Sinais , Proteínas rho de Ligação ao GTP , Proteína rhoA de Ligação ao GTP , Doença de von Willebrand Tipo 2/enzimologiaRESUMO
Hematopoietic stem cells (HSC) differentiate into megakaryocytes (MK), whose function is to release platelets. Attempts to improve in vitro platelet production have been hampered by the low amplification of MK. Providing HSC with an optimal three-dimensional (3D) architecture may favor MK differentiation by mimicking some crucial functions of the bone marrow structure. To this aim, porous hydrogel scaffolds were used to study MK differentiation from HSC as well as platelet production. Flow cytometry, qPCR and perfusion studies showed that 3D was suitable for longer kinetics of CD34+ cell proliferation and for delayed megakaryocytic differentiation far beyond the limited shelf-life observed in liquid culture but also increased production of functional platelets. We provide evidence that these 3D effects were related to 1) persistence of MK progenitors and precursors and 2) prolongation of expression of EKLF and c-myb transcription factors involved in early MK differentiation. In addition, presence of abundant mature MK with increased ploidy and impressive cytoskeleton elongations was in line with expression of NF-E2 transcription factor involved in late MK differentiation. Platelets produced in flow conditions were functional as shown by integrin αIIbß3 activation following addition of exogenous agonists. This study demonstrates that spatial organization and biological cues synergize to improve MK differentiation and platelet production. Thus, 3D environment constitutes a powerful tool for unraveling the physiological mechanisms of megakaryopoiesis and thrombopoiesis in the bone marrow environment, potentially leading to an improved amplification of MK and platelet production.
Assuntos
Plaquetas/metabolismo , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Megacariócitos/metabolismo , Trombopoese , Alicerces Teciduais/química , Antígenos CD34/metabolismo , Plaquetas/citologia , Células Cultivadas , Feminino , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Humanos , Hidrogéis/química , Integrina alfa2/biossíntese , Integrina beta3/biossíntese , Fatores de Transcrição Kruppel-Like/biossíntese , Masculino , Megacariócitos/citologia , Subunidade p45 do Fator de Transcrição NF-E2/biossíntese , Proteínas Proto-Oncogênicas c-myb/biossínteseRESUMO
Agonist-stimulated platelet activation triggers conformational changes of integrin αIIbß3, allowing fibrinogen binding and platelet aggregation. We have previously shown that an octapeptide, p1YMESRADR8, corresponding to amino acids 313-320 of the ß-ribbon extending from the ß-propeller domain of αIIb, acts as a potent inhibitor of platelet aggregation. Here we have performed in silico modelling analysis of the interaction of this peptide with αIIbß3 in its bent and closed (not swing-out) conformation and show that the peptide is able to act as a substitute for the ß-ribbon by forming a clasp restraining the ß3 hybrid and ßI domains in a closed conformation. The involvement of species-specific residues of the ß3 hybrid domain (E356 and K384) and the ß1 domain (E297) as well as an intrapeptide bond (pE315-pR317) were confirmed as important for this interaction by mutagenesis studies of αIIbß3 expressed in CHO cells and native or substituted peptide inhibitory studies on platelet functions. Furthermore, NMR data corroborate the above results. Our findings provide insight into the important functional role of the αIIb ß-ribbon in preventing integrin αIIbß3 head piece opening, and highlight a potential new therapeutic approach to prevent integrin ligand binding.
Assuntos
Integrina alfa2/metabolismo , Agregação Plaquetária/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Fibrinogênio/metabolismo , Humanos , Ativação Plaquetária , Ligação ProteicaRESUMO
Von Willebrand factor (vWF) cleaving metalloprotease activity represents an important factor in understanding the pathophysiology of thrombotic microangiopathies (TMA). Thrombotic events, leading to dramatic complications, occur preferentially in the microvasculature. Our aim was to determine the ability of TMA plasma with a complete deficiency of protease activity to promote shear-induced platelet aggregation (SIPA) at high shear rates using a coaxial cylinder shearing device. We have demonstrated the ability of four out of seven TMA plasma to aggregate normal washed platelets at 4,000 s(-1). In these four TMA plasma, significant SIPA was obtained, whereas control plasma did not induce more SIPA than buffer. The % of unusually large (UL) multimers in TMA plasma varied from 1 to 18%. Surprisingly, the extent of SIPA in TMA plasma appeared independent of the percentage of UL multimers. Interestingly, anti-GPIb antibody 6D1 could block completely this pathological aggregation, opening new therapeutic possibilities. In contrast, none of eight other TMA patients' plasma with a normal protease activity, exhibited any increase of SIPA compared with control plasma. These results strongly suggest that the deficiency of vWF-cleaving protease appears to be necessary, but not sufficient for elevated SIPA. In contrast, the % of UL is not associated with increased SIPA.
Assuntos
Síndrome Hemolítico-Urêmica/enzimologia , Metaloendopeptidases/deficiência , Agregação Plaquetária , Púrpura Trombocitopênica Trombótica/enzimologia , Proteínas ADAM , Proteína ADAMTS13 , Adolescente , Adulto , Idoso , Feminino , Síndrome Hemolítico-Urêmica/sangue , Hemorreologia , Humanos , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Trombótica/sangue , Estresse MecânicoRESUMO
It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s(-1)) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf (-/-) mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf (-/-) and Vwf (+/+) blood, exposed to VWF at 1800 s(-1) in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf(-/-) mice than from Vwf(+/+) mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf(-/-) mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s(-1) on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.
Assuntos
Plaquetas/fisiologia , Trombopoese , Fator de von Willebrand/metabolismo , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Plaquetas/patologia , Citoesqueleto/metabolismo , Humanos , Megacariócitos/citologia , Megacariócitos/metabolismo , Camundongos , Microtúbulos/metabolismo , Perfusão , Adesividade Plaquetária , Estresse Mecânico , Trombina/metabolismo , Trombocitopenia/sangue , Trombocitopenia/induzido quimicamente , Doença de von Willebrand Tipo 3/sangue , Doença de von Willebrand Tipo 3/metabolismoRESUMO
Neutrophil beta1 integrin expression and contribution to cell adhesion were revisited in this study. alpha9beta1 and alpha5beta1 appeared here as the main beta1 integrins expressed on the membrane of resting platelet-depleted neutrophils-alpha6beta1 representing <15% and alpha2beta1 undetectable. Neutrophil activation slightly enhanced alpha5 expression, did not change alpha6, but resulted in a two- to threefold increase of alpha9beta1, which then became the major beta1 integrin of the neutrophil membrane. alpha9beta1 was the only beta1 integrin to be up-regulated after transendothelial migration across TNF-alpha-activated HUVECs. As alpha9beta1 binds VCAM-1, we analyzed its participation to neutrophil adhesion to TNF-alpha-activated endothelial cells. Blocking anti-alpha9 mAb had little effect on neutrophil static adhesion, contrasting with the strong inhibition by anti-beta2 mAb. Under flow conditions, the anti-alpha9 mAb had no effect by itself on neutrophil adhesion to activated HUVECs but enhanced the blocking effect of anti-beta2 antibodies significantly and further enhanced the velocity of beta2-blocked rolling neutrophils. In conclusion, we describe here for the first time a nearly exclusive up-regulation of alpha9beta1 expression among all beta1 integrins during neutrophil activation and transendothelial migration and a possibly important synergy between alpha9beta1 and beta2 integrins in stabilizing neutrophil adhesion to endothelium under flow conditions.
Assuntos
Antígenos CD18/metabolismo , Adesão Celular , Endotélio Vascular/citologia , Integrinas/metabolismo , Ativação de Neutrófilo , Quimiotaxia de Leucócito , Endotélio Vascular/metabolismo , Humanos , Integrinas/biossíntese , Neutrófilos , Perfusão , Regulação para Cima/genéticaRESUMO
The highly negatively charged membrane sialoglycoprotein leukosialin, CD43, is shed during neutrophil activation. This is generally thought to enhance cell adhesion. We here describe two novel consequences of this shedding, during neutrophil activation by phorbol esters or by chemoattractants after TNF-alpha priming. CD43 proteolysis was investigated by Western blotting, using a polyclonal antibody to CD43 intracellular domain. Our data emphasize the importance of a juxtamembranous cleavage of about 50% of membrane CD43 molecules by cathepsin G. Indeed, it is inhibited by alpha1-antichymotrypsin and cathepsin G inhibitor I and is reproduced by exogenous purified cathepsin G. The resulting membrane-anchored C-terminal fragment, CD43-CTF, becomes susceptible to presenilin/gamma-secretase, which releases CD43 intracytoplasmic domain: preincubation with three different gamma-secretase inhibitors, before PMN treatment by agonists or by purified cathepsin G, results in the accumulation of CD43-CTF. Because CD43 binds E-selectin, we also investigated the effect of the soluble extracellular domain CD43s, released by cathepsin G juxtamembranous cleavage, on neutrophil adhesion to endothelial cells. A recombinant CD43s-Fc fusion protein inhibited neutrophil E selectindependent adhesion to endothelial cells under flow conditions, while it had no effect on neutrophil static adhesion. We thus propose that, in addition to its potential pro-adhesive role, CD43 proteolysis results in: (i) the release, by cathepsin G, of CD43 extracellular domain, able to inhibit the adhesion of flowing neutrophils on endothelial cells and thus to participate to the natural control of inflammation; (ii) the release and/or the clearance, by presenilin/gamma-secretase, of CD43 intracellular domain, thereby regulating CD43-mediated signaling.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Catepsinas/metabolismo , Leucossialina/metabolismo , Neutrófilos/metabolismo , Presenilinas/metabolismo , Serina Endopeptidases/metabolismo , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Catepsina G , Catepsinas/antagonistas & inibidores , Catepsinas/genética , Adesão Celular/fisiologia , Selectina E/fisiologia , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/metabolismo , Leucossialina/genética , Ativação de Neutrófilo/fisiologia , Presenilinas/genética , Inibidores de Proteases/farmacologia , Estrutura Terciária de Proteína/fisiologia , Serina Endopeptidases/genética , Transdução de Sinais/fisiologiaRESUMO
OBJECTIVE: The inability to regulate the inflammatory response initiated upon infection leads to severe sepsis, characterized by widespread microvascular injury and thrombosis, organ ischemia, and dysfunction. A disintegrin-like and metalloprotease with thrombospondin type 1 repeats (ADAMTS)-13 regulates primary hemostasis by proteolyzing von Willebrand factor (VWF). Decreased ADAMTS-13 has been reported in disseminated intravascular coagulation due to severe sepsis. The present study investigates whether the sepsis-related dysregulation of endothelial activation leads to specific changes of ADAMTS-13. DESIGN: Case-control study. SETTING: Adult intensive care unit in a university hospital. PATIENTS/SUBJECTS: Three groups were studied: a case group of 30 patients with severe sepsis, a control group of 29 patients with comparable organ failure unrelated to sepsis, and 30 age- and gender-matched healthy subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Significantly lower ADAMTS-13 activity was observed in patients with severe sepsis (43.2%; interquartile range, 32.7, 67.0) than in patients with organ failure unrelated to sepsis (67.8%; 57.4, 87.9; p < .05) and healthy subjects (105.6%; 87.2, 125.6; p < .001). Accordingly, ADAMTS-13 antigen was more decreased in patients with severe sepsis than in patients with organ failure unrelated to sepsis and healthy subjects. VWF antigen was higher in patients with severe sepsis than in patients with organ failure unrelated to sepsis and healthy subjects. We found strong negative correlations in severe sepsis but not in organ failure unrelated to sepsis, between ADAMTS-13 activity and 1) VWF antigen; 2) thrombomodulin; 3) interleukin-6; 4) Acute Physiology and Chronic Health Evaluation II score; 5) shock; 6) acute renal injury. Moreover, patients above the median of ADAMTS-13 activity presented a higher survival compared with those below the median in the patients with severe sepsis but not in the patients with organ failure unrelated to sepsis. In contrast, there was no significant association between VWF antigen and survival in either the severe sepsis group or the group with organ failure unrelated to sepsis. CONCLUSIONS: We observed low ADAMTS-13 activity and antigen in severe sepsis and in other conditions associated with organ dysfunction. ADAMTS-13 levels were significantly associated with differences in morbidity, mortality, and variables of inflammation and endothelial dysregulation only in severe sepsis patients. This suggests that ADAMTS-13 deficiency may have a pathophysiological relevance specific to severe sepsis.
Assuntos
Proteínas ADAM/sangue , Insuficiência de Múltiplos Órgãos/etiologia , Sepse/sangue , Sepse/complicações , Proteína ADAMTS13 , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Insuficiência de Múltiplos Órgãos/mortalidade , Prognóstico , Sepse/mortalidade , Índice de Gravidade de DoençaRESUMO
Shear stress encountered in stenosed human arteries is able to induce a certain range of platelet activation. In order to determine the extent of platelet shape change induced by high shear rate conditions, we used electron microscopy (EM) and immuno-EM to study platelet ultrastructure from blood flowing in vivo through stenosed arteries. Then it was compared with platelets from healthy controls exposed in vitro to a shear rate of 4000 s(-1). Six patients with stenosed arteries (iliac, femoral and renal) were investigated at the time of transcutaneous angiography. Blood was harvested from the same catheter in the stenosed artery and in the abdominal aortic artery (control sample), each patient being its own control. The percentage of platelets with shape changes (loss of discoid form, pseudopod emission, organelle centralisation) significantly increased in samples from stenosed arteries. Shape change was concomitant with the membrane glycoprotein IIb-IIIa distribution at the pseudopod extremities. These activated platelets had not completed secretion and were maintained in a reversible activation state. Similar results were obtained on platelets from healthy donors submitted in vitro to a high shear rate. In conclusion, this study shows that the high shear rate encountered in human stenosed arteries is able to induce shape change and reversible activation of platelets in vivo.
Assuntos
Artérias/patologia , Plaquetas/patologia , Constrição Patológica/sangue , Ativação Plaquetária , Glicoproteínas da Membrana de Plaquetas/metabolismo , Idoso , Plaquetas/ultraestrutura , Estudos de Casos e Controles , Forma Celular , Feminino , Humanos , Masculino , Microscopia Eletrônica , Transporte Proteico , Estresse MecânicoRESUMO
We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin alphaIIbbeta3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT-treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non-treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce alphaIIbbeta3-dependent cell adhesion to immobilized VWF, while Fg-dependent cell adhesion occurred independently of the activation state of alphaIIbbeta3. By comparing the effects of two potent platelet alphaIIbbeta3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated alphaIIbbeta3. In particular, AP2 prevented VWF-dependent but not Fg-dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated alphaIIbbeta3-dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT-activated alphaIIbbeta3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.
Assuntos
Fibrinogênio/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fator de von Willebrand/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Adesão Celular/efeitos dos fármacos , Agregação Celular/efeitos dos fármacos , Cricetinae , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Epitopos/análise , Humanos , Agregação Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Dithiothreitol (DTT) is known to induce an active conformation of alphaIIbbeta3 integrin and to promote the aggregation of Chinese hamster ovary (CHO)-alphaIIbbeta3 cells in the presence of soluble fibrinogen (Fg). The aim of this study was to compare adhesion and spreading with Fg or von Willebrand factor (VWF) of CHO-alphaIIbbeta3 cells in the presence or absence of DTT. Our results indicate that DTT treatment was required to induce cell spreading on VWF. In contrast, CHO-alphaIIbbeta3 cell spreading on Fg was already optimal in the absence of DTT. We used a small perfusion chamber coupled to videomicroscopy to demonstrate that CHO-alphaIIbbeta3 cells that were adherent and spread on VWF required DTT activation to resist to detachment under increasing shear rates (50-1600/s). In contrast, untreated or DTT-treated cells spread on Fg were able to resist to extremely high flow rates. These data provide novel evidence that activated alphaIIbbeta3 is absolutely required for spread cells to resist detachment and strengthens the importance of the alphaIIbbeta3 activation step for adhesion and spreading to VWF.
Assuntos
Complexo Glicoproteico GPIIb-IIIa de Plaquetas/fisiologia , Fator de von Willebrand/metabolismo , Animais , Células CHO/metabolismo , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Separação Celular/métodos , Cricetinae , Cultura em Câmaras de Difusão , Ditiotreitol/farmacologia , Relação Dose-Resposta a Droga , Fibrinogênio/metabolismo , Citometria de Fluxo , Integrina alfaVbeta3/metabolismo , Integrina alfaVbeta3/fisiologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , ReologiaRESUMO
Shear-induced platelet aggregation (SIPA) involves the sequential interaction of von Willebrand factor (VWF) with both glycoprotein Ib (GPIb) and alphaIIbbeta3 receptors. Type 2B recombinant VWF (2B-rVWF), characterized by an increased affinity for GPIb, induces strong SIPA at a high shear rate (4000 s-1). Despite the increased affinity of 2B-rVWF for GPIb, patients with type 2B von Willebrand disease have a paradoxical bleeding disorder, which is not well understood. The purpose of this study was to determine if SIPA induced by 2B-rVWF was associated with alphaIIbbeta3-dependent platelet activation. To this end, we have addressed the influence of 2B-rVWF (Val553Met substitution) on SIPA-dependent variations of tyrosine protein phosphorylation (P-Tyr) and the effect of alphaIIbbeta3 blockers. At a high shear rate, 2B-rVWF induced a strong SIPA, as shown by a 92.7% +/- 0.4% disappearance of single platelets (DSP) after 4.5 minutes. In these conditions, increased P-Tyr of proteins migrating at positions 64 kd, 72 kd, and 125 kd were observed. The band at 125 kd was identified as pp125FAK using anti-phospho-FAK antibody. This effect, which required a high level of SIPA (> 70% DSP), was observed at 4000 s-1 but not at 200 s-1. Monoclonal antibodies (MoAbs) 6D1 (anti-GPIb) and 328 (anti-VWF A1 domain), completely abolished SIPA and p125FAK phosphorylation mediated by 2B-rVWF. In contrast, neither RGDS peptide nor MoAb 7E3, both known to block alphaIIbbeta3 engagement, had any effect on SIPA and pp125FAK. The size of aggregates formed at a high shear rate in the presence of 2B-rVWF was decreased by genistein, demonstrating the biologic relevance of pp125FAK. These findings provide a unique mechanism whereby the enhanced interaction of 2B-rVWF with GPIb, without engagement of alphaIIbbeta3, is sufficient to induce SIPA but does not lead to stable thrombus formation.