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1.
Arterioscler Thromb Vasc Biol ; 41(3): 1092-1104, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33472402

RESUMO

OBJECTIVE: GPVI (glycoprotein VI) is a key molecular player in collagen-induced platelet signaling and aggregation. Recent evidence indicates that it also plays important role in platelet aggregation and thrombus growth through interaction with fibrin(ogen). However, there are discrepancies in the literature regarding whether the monomeric or dimeric form of GPVI binds to fibrinogen at high affinity. The mechanisms of interaction are also not clear, including which region of fibrinogen is responsible for GPVI binding. We aimed to gain further understanding of the mechanisms of interaction at molecular level and to identify the regions on fibrinogen important for GPVI binding. Approach and Results: Using multiple surface- and solution-based protein-protein interaction methods, we observe that dimeric GPVI binds to fibrinogen with much higher affinity and has a slower dissociation rate constant than the monomer due to avidity effects. Moreover, our data show that the highest affinity interaction of GPVI is with the αC-region of fibrinogen. We further show that GPVI interacts with immobilized fibrinogen and fibrin variants at a similar level, including a nonpolymerizing fibrin variant, suggesting that GPVI binding is independent of fibrin polymerization. CONCLUSIONS: Based on the above findings, we conclude that the higher affinity of dimeric GPVI over the monomer for fibrinogen interaction is achieved by avidity. The αC-region of fibrinogen appears essential for GPVI binding. We propose that fibrin polymerization into fibers during coagulation will cluster GPVI through its αC-region, leading to downstream signaling, further activation of platelets, and potentially stimulating clot growth. Graphic Abstract: A graphic abstract is available for this article.


Assuntos
Fibrinogênio/metabolismo , Fragmentos de Peptídeos/sangue , Glicoproteínas da Membrana de Plaquetas/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Produtos de Degradação da Fibrina e do Fibrinogênio/química , Produtos de Degradação da Fibrina e do Fibrinogênio/metabolismo , Fibrinogênio/química , Humanos , Técnicas In Vitro , Camundongos , Microscopia de Força Atômica , Fragmentos de Peptídeos/química , Peptídeos/química , Peptídeos/metabolismo , Agregação Plaquetária/fisiologia , Glicoproteínas da Membrana de Plaquetas/química , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Transdução de Sinais , Ressonância de Plasmônio de Superfície
2.
Angiology ; 72(1): 16-23, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32705876

RESUMO

Structural aspects of red blood cells have been associated with cardiovascular disease. No data linking mean corpuscular volume (MCV) to clinical outcomes and on-treatment platelet reactivity in patients with peripheral artery disease (PAD) are available. We investigated a composite of atherothrombotic events and target vessel restenosis or reocclusion following infrainguinal stenting for stable PAD. Residual platelet reactivity was measured by light transmission aggregometry (LTA) and the VerifyNow assays. We included 104 patients receiving dual antiplatelet therapy (DAPT) with aspirin and clopidogrel. In receiver-operating characteristic analysis, MCV effectively discriminated between patients with and without adverse outcomes and identified a MCV ≤90.8 fL as optimal cutoff. Adverse outcomes occurred significantly more often in patients with low MCV (log-rank P = .002). In univariable Cox regression analysis, low MCV was associated with an increased risk of future adverse outcomes (hazard ratio [HR]: 2.662 [95%CI: 1.304-5.434]; P = .007) and remained significantly associated after adjustment (HR: 2.591 [95%CI: 1.242-5.403]; P = .011). Mean corpuscular volume was inversely correlated with arachidonic acid (AA)- and adenosine diphosphate (ADP)-inducible platelet reactivity by LTA and with the VerifyNow aspirin assay. Low MCV is associated with adverse outcomes over 2 years following infrainguinal stenting. Mean corpuscular volume correlates inversely with AA- and ADP-inducible platelet reactivity during DAPT.


Assuntos
Arteriopatias Oclusivas/terapia , Índices de Eritrócitos/fisiologia , Doença Arterial Periférica/fisiopatologia , Agregação Plaquetária/fisiologia , Stents , Idoso , Arteriopatias Oclusivas/diagnóstico por imagem , Arteriopatias Oclusivas/fisiopatologia , Estudos de Coortes , Terapia Antiplaquetária Dupla , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/terapia , Recidiva , Ultrassonografia Doppler de Pulso
3.
Int J Mol Sci ; 21(24)2020 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-33352749

RESUMO

The expansion of nanotechnology for drug delivery applications has raised questions regarding the safety of nanoparticles (NPs) due to their potential for interacting at molecular and cellular levels. Although polymeric NPs for drug delivery are formulated using FDA-approved polymers such as lactide- and glycolide-based polymers, their interactions with blood constituents, remain to be identified. The aim of this study was to determine the impact of size-selected Poly-lactide-co-glycolide-polyethylene glycol (PLGA-PEG) NPs on platelet activity. The NPs of 113, 321, and 585 nm sizes, were formulated and their effects at concentrations of 0-2.2 mg/mL on the activation and aggregation of platelet-rich plasma (PRP) were investigated. The results showed that NPs of 113 nm did not affect adenosine diphosphate (ADP)-induced platelet aggregation at any NP concentration studied. The NPs of 321 and 585 nm, at concentrations ≥0.25 mg/mL, reduced ADP-activated platelet aggregation. The platelet activation profile remained unchanged in the presence of investigated NPs. Confocal microscopy revealed that NPs were attached to or internalised by platelets in both resting and activated states, with no influence on platelet reactivity. The results indicate minimal risks of interference with platelet function for PLGA-PEG NPs and that these NPs can be explored as nanocarriers for targeted drug delivery to platelets.


Assuntos
Plaquetas/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Nanopartículas/administração & dosagem , Agregação Plaquetária/efeitos dos fármacos , Plasma Rico em Plaquetas/efeitos dos fármacos , Polietilenoglicóis/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Plaquetas/fisiologia , Portadores de Fármacos/química , Humanos , Nanopartículas/química , Agregação Plaquetária/fisiologia , Plasma Rico em Plaquetas/fisiologia
4.
PLoS One ; 15(12): e0244792, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382840

RESUMO

Blood coagulation is central to myocardial ischemia and reperfusion (IR) injury. Studies on the light elicited circadian rhythm protein Period 2 (PER2) using whole body Per2-/- mice found deficient platelet function and reduced clotting which would be expected to protect from myocardial IR-injury. In contrast, intense light induction of PER2 protected from myocardial IR-injury while Per2 deficiency was detrimental. Based on these conflicting data, we sought to evaluate the role of platelet specific PER2 in coagulation and myocardial ischemia and reperfusion injury. We demonstrated that platelets from mice with tissue-specific deletion of Per2 in the megakaryocyte lineage (Per2loxP/loxP-PF4-CRE) significantly clot faster than platelets from control mice. We further found increases in infarct sizes or plasma troponin levels in Per2loxP/loxP-PF4-CRE mice when compared to controls. As intense light increases PER2 protein in human tissues, we also performed translational studies and tested the effects of intense light therapy on coagulation in healthy human subjects. Our human studies revealed that intense light therapy repressed procoagulant pathways in human plasma samples and significantly reduced the clot rate. Based on these results we conclude that intense light elicited PER2 has an inhibitory function on platelet aggregation in mice. Further, we suggest intense light as a novel therapy to prevent or treat clotting in a clinical setting.


Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/metabolismo , Isquemia Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas Circadianas Period/metabolismo , Fototerapia , Animais , Humanos , Luz , Masculino , Camundongos , Isquemia Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/sangue , Proteínas Circadianas Period/genética , Agregação Plaquetária/fisiologia , Proteômica
5.
PLoS One ; 15(7): e0235392, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726315

RESUMO

Platelets upregulate the generation of thrombin and reinforce the fibrin clot which increases the incidence risk of venous thromboembolism (VTE). However, the role of platelets in the pathogenesis of venous cardiovascular diseases remains hard to quantify. An experimentally validated model of thrombin generation dynamics is formulated. The model predicts that a high platelet count increases the peak value of generated thrombin as well as the endogenous thrombin potential (ETP) as reported in experimental data. To investigate the effects of platelets density, shear rate, and wound size on the initiation of blood coagulation, we calibrate a previously developed model of venous thrombus formation and implement it in 3D using a novel cell-centered finite-volume solver. We conduct numerical simulations to reproduce in vitro experiments of blood coagulation in microfluidic capillaries. Then, we derive a reduced one-equation model of thrombin distribution from the previous model under simplifying hypotheses and we use it to determine the conditions of clotting initiation on the platelet count, the shear rate, and the plasma composition. The initiation of clotting also exhibits a threshold response to the size of the wounded region in good agreement with the reported experimental findings.


Assuntos
Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Modelos Teóricos , Contagem de Plaquetas/métodos , Testes de Coagulação Sanguínea , Plaquetas/metabolismo , Fibrina/metabolismo , Humanos , Agregação Plaquetária/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Resistência ao Cisalhamento/fisiologia , Trombina/metabolismo , Tromboplastina/metabolismo , Trombose/metabolismo , Trombose/fisiopatologia , Veias/metabolismo , Veias/fisiologia
6.
Nat Commun ; 11(1): 3479, 2020 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-32661250

RESUMO

Genetic factors contribute to the risk of thrombotic diseases. Recent genome wide association studies have identified genetic loci including SLC44A2 which may regulate thrombosis. Here we show that Slc44a2 controls platelet activation and thrombosis by regulating mitochondrial energetics. We find that Slc44a2 null mice (Slc44a2(KO)) have increased bleeding times and delayed thrombosis compared to wild-type (Slc44a2(WT)) controls. Platelets from Slc44a2(KO) mice have impaired activation in response to thrombin. We discover that Slc44a2 mediates choline transport into mitochondria, where choline metabolism leads to an increase in mitochondrial oxygen consumption and ATP production. Platelets lacking Slc44a2 contain less ATP at rest, release less ATP when activated, and have an activation defect that can be rescued by exogenous ADP. Taken together, our data suggest that mitochondria require choline for maximum function, demonstrate the importance of mitochondrial metabolism to platelet activation, and reveal a mechanism by which Slc44a2 influences thrombosis.


Assuntos
Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Ativação Plaquetária/fisiologia , Trombose/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Estudo de Associação Genômica Ampla , Masculino , Espectrometria de Massas , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Ativação Plaquetária/genética , Agregação Plaquetária/genética , Agregação Plaquetária/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Trombose/genética
7.
Crit Care ; 24(1): 360, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32552865

RESUMO

Thrombotic complications and coagulopathy frequently occur in COVID-19. However, the characteristics of COVID-19-associated coagulopathy (CAC) are distinct from those seen with bacterial sepsis-induced coagulopathy (SIC) and disseminated intravascular coagulation (DIC), with CAC usually showing increased D-dimer and fibrinogen levels but initially minimal abnormalities in prothrombin time and platelet count. Venous thromboembolism and arterial thrombosis are more frequent in CAC compared to SIC/DIC. Clinical and laboratory features of CAC overlap somewhat with a hemophagocytic syndrome, antiphospholipid syndrome, and thrombotic microangiopathy. We summarize the key characteristics of representative coagulopathies, discussing similarities and differences so as to define the unique character of CAC.


Assuntos
Betacoronavirus , Transtornos da Coagulação Sanguínea/diagnóstico , Transtornos da Coagulação Sanguínea/epidemiologia , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/epidemiologia , Pneumonia Viral/diagnóstico , Pneumonia Viral/epidemiologia , Transtornos da Coagulação Sanguínea/sangue , Testes de Coagulação Sanguínea/métodos , Infecções por Coronavirus/sangue , Humanos , Mediadores da Inflamação/sangue , Pandemias , Agregação Plaquetária/fisiologia , Pneumonia Viral/sangue
8.
Clin Hemorheol Microcirc ; 75(4): 467-474, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32390610

RESUMO

BACKGROUND: Platelet activation is associated with abdominal obesity and exercise training is an important modulator of body weight. OBJECTIVE: We investigated the effects of two high intensity interval exercise (HIIE) protocols of different intensity and duration on platelet indices and platelet aggregation in overweight men. METHODS: Ten overweight men performed 6 intervals of 30s exercise at 110% of peak power output (PPO) interspersed by 3 : 30 min active recovery (1/7 protocol) at 40% of PPO and 6 intervals of 2 min exercise at 85% of PPO interspersed by 2 min active recovery (1/1 protocol) at 30% of PPO in two separate sessions. Platelet indices and platelet aggregation were measured before and immediately after both HIIEs. RESULTS: Platelet indices increased significantly following HIIE (P < 0.05), though, significant differences between the two protocols were only detected for platelet count, which was markedly increased following 1/1 protocol. Platelet aggregation increased significantly (P < 0.05) in response to the two HIIE protocols, with no significant difference being observed between the two protocols (P > 0.05). CONCLUSIONS: It is concluded that HIIE leads to transient increases in markers of thrombus formation and that work to rest ratio is an important factor when investigating the changes in thrombocytosis following HIIE.


Assuntos
Difosfato de Adenosina/metabolismo , Exercício Físico/fisiologia , Treinamento Intervalado de Alta Intensidade/métodos , Sobrepeso/sangue , Agregação Plaquetária/fisiologia , Contagem de Plaquetas/métodos , Adulto , Humanos , Masculino , Adulto Jovem
9.
PLoS Comput Biol ; 16(4): e1007709, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32343724

RESUMO

Mechanical interactions between flowing and coagulated blood (thrombus) are crucial in dictating the deformation and remodeling of a thrombus after its formation in hemostasis. We propose a fully-Eulerian, three-dimensional, phase-field model of thrombus that is calibrated with existing in vitro experimental data. This phase-field model considers spatial variations in permeability and material properties within a single unified mathematical framework derived from an energy perspective, thereby allowing us to study effects of thrombus microstructure and properties on its deformation and possible release of emboli under different hemodynamic conditions. Moreover, we combine this proposed thrombus model with a particle-based model which simulates the initiation of the thrombus. The volume fraction of a thrombus obtained from the particle simulation is mapped to an input variable in the proposed phase-field thrombus model. The present work is thus the first computational study to integrate the initiation of a thrombus through platelet aggregation with its subsequent viscoelastic responses to various shear flows. This framework can be informed by clinical data and potentially be used to predict the risk of diverse thromboembolic events under physiological and pathological conditions.


Assuntos
Vasos Sanguíneos/fisiologia , Trombose/fisiopatologia , Biofísica/métodos , Coagulação Sanguínea/fisiologia , Plaquetas/fisiologia , Simulação por Computador , Humanos , Modelos Biológicos , Adesividade Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Trombose/sangue
10.
Transfusion ; 60(5): 1050-1059, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32187695

RESUMO

BACKGROUND: Our previous study showed that ultraviolet C (UVC) from xenon (Xe) flash without any photoreactive compounds inactivated bacteria in platelet concentrates (PCs) with less damage to platelets (PLTs) as compared with Xe flash containing ultraviolet A, ultraviolet B, and visible light. Here, we report a UVC irradiation system for PCs under flow conditions consisting of a flow path-irradiation sheet, a peristaltic pump, and a collection bag. STUDY DESIGN AND METHODS: Platelet concentrates containing Ringer's solution (R-PCs) inoculated with bacteria were injected into a flow path sheet using a peristaltic pump, being irradiated with UVC from Xe flash. The quality of the irradiated PCs containing platelet additive solution (PAS-PCs) was assessed based on PC variables, PLT surface markers, and aggregation ability. RESULTS: Streptococcus dysgalactiae (12 tests) and Escherichia coli (11) were all negative on bacterial culture, while Staphylococcus aureus (12) and Klebsiella pneumoniae (14) grew in one and two R-PCs, respectively. Bacillus cereus spores were inactivated in 7 of 12 R-PCs. PC variables became significantly different between irradiated and nonirradiated PAS-PCs. P-selectin, first procaspase-activating compound (PAC-1) binding, and phosphatidylserine increased by irradiation. Aggregability stimulated by adenosine diphosphate, collagen, or thromboxane A2 increased in the irradiated PAS-PCs, while that by thrombin became smaller compared with nonirradiated controls. CONCLUSION: This newly developed system inactivated bacteria including spores in R-PCs. PAS-PCs irradiated by this system retained acceptable in vitro quality and aggregability. Usage of a peristaltic pump instead of agitator during irradiation may enable this system to be directly combined with an apheresis blood cell separator.


Assuntos
Plaquetas/citologia , Preservação de Sangue , Desinfecção/instrumentação , Viabilidade Microbiana , Raios Ultravioleta , Xenônio/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/fisiologia , Bacillus cereus/efeitos da radiação , Bactérias/efeitos dos fármacos , Bactérias/efeitos da radiação , Remoção de Componentes Sanguíneos , Plaquetas/efeitos dos fármacos , Plaquetas/efeitos da radiação , Preservação de Sangue/instrumentação , Preservação de Sangue/métodos , Segurança do Sangue/instrumentação , Segurança do Sangue/métodos , Desinfecção/métodos , Contaminação de Medicamentos/prevenção & controle , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Escherichia coli/efeitos da radiação , Humanos , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/fisiologia , Klebsiella pneumoniae/efeitos da radiação , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Viabilidade Microbiana/efeitos da radiação , Soluções para Preservação de Órgãos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Agregação Plaquetária/efeitos da radiação , Controle de Qualidade , Solução de Ringer/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia , Staphylococcus aureus/efeitos da radiação , Streptococcus/efeitos dos fármacos , Streptococcus/fisiologia , Streptococcus/efeitos da radiação
11.
Anesth Analg ; 131(2): 622-630, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32102014

RESUMO

BACKGROUND: Heparin-induced thrombocytopenia (HIT) can put cardiac surgery patients at a high risk of lethal complications. If anti-PF4/heparin antibodies (anti-PF4/Hep Abs) are present, 2 strategies exist to prevent intraoperative aggregation during bypass surgery: first, using an alternative anticoagulant, and second, using heparin combined with an antiaggregant. The new P2Y12 inhibitor, cangrelor, could be an attractive candidate for the latter strategy; several authors have reported its successful use. The present in vitro study evaluated cangrelor's ability to inhibit heparin-induced platelet aggregation in the presence of anti-PF4/Hep Abs. METHODS: Platelet-poor plasma (PPP) from 30 patients with functional anti-PF4/Hep Abs was mixed with platelet-rich plasma (PRP) from 5 healthy donors.Light transmission aggregometry was used to measure platelet aggregation after adding 0.5 IU·mL of heparin (HIT) to the plasma, and this was compared with samples spiked with normal saline (control) and samples spiked with cangrelor 500 ng·mL and heparin 0.5 IU·mL (treatment). Friedman test with post hoc Dunn-Bonferroni test was used for between-group comparisons. RESULTS: Heparin 0.5 IU·mL triggered aggregation in 22 of 44 PPP-PRP mixtures, with a median aggregation of 86% (interquartile range [IQR], 69-91). The median aggregation of these 22 positive samples' respective control tests was 22% (IQR, 16-30) (P < .001). Median aggregation in the cangrelor-treated samples was 29% (IQR, 19-54) and significantly lower than the HIT samples (P < .001). Cangrelor inhibited heparin-induced aggregation by a median of 91% (IQR, 52-100). Cangrelor only reduced heparin-induced aggregation by >95% in 10 of the 22 positive samples (45%). Cangrelor inhibited heparin-induced aggregation by <50% in 5 of the 22 positive samples (22%) and by <10% in 3 samples (14%). CONCLUSIONS: This in vitro study found that cangrelor was an unreliable inhibitor of heparin-induced aggregation in the presence of anti-PF4/Hep Abs. We conclude that cangrelor should not be used as a standard antiaggregant for cardiac patients affected by HIT during surgery. Unless cangrelor's efficacy in a particular patient has been confirmed in a presurgery aggregation test, other strategies should be chosen.


Assuntos
Monofosfato de Adenosina/análogos & derivados , Anticoagulantes/efeitos adversos , Procedimentos Cirúrgicos Cardíacos/métodos , Heparina/efeitos adversos , Agregação Plaquetária/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Purinérgicos P2Y12 , Monofosfato de Adenosina/sangue , Monofosfato de Adenosina/farmacologia , Anticoagulantes/sangue , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Heparina/sangue , Humanos , Agregação Plaquetária/fisiologia , Antagonistas do Receptor Purinérgico P2Y/sangue , Receptores Purinérgicos P2Y12/sangue
13.
Biochem Pharmacol ; 174: 113827, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31987853

RESUMO

Adenosine analogues have high affinity and selectivity for adenosine receptors (AR), and exhibit anti-platelet activity. Plasma proteins play an important role in the regulation of platelet function and may influence the action of anti-platelet compounds. Little is known about the interactions of AR agonists with plasma proteins. This study investigates the interplay between AR agonists and plasma proteins and the consequences of those interactions. Surface plasmon resonance was employed together with molecular docking study to determine the binding kinetics of four selected ARagonists (PSB0777, Cl-Ado, MRE0094, UK432097) to several carrier proteins and to clarify the nature of these interactions. The influence of a whole plasma and of some plasma components on the effectiveness of ARagonists in the inhibition of platelet function was assessed by flow cytometry (platelet activation) and ELISA (platelet adhesion). Plasma proteins remarkably diminished the effectiveness of ARagonists in inhibiting platelet activation and adhesion in vitro. ARagonists were found to strongly bind to human serum albumin (HSA) and the protein components of lipoproteins - apolipoproteins; HSA was essential for the binding of water-soluble PSB0777, whereas apolipoproteins were needed for interactions with poorly-water soluble compounds such as UK432097 and MRE0094. In addition, HSA was shown to significantly reduce the effectiveness of PSB0777 in inhibiting ADP-induced platelet activation. In conclusion, HSA and lipoproteins are important carriers for ARagonists, which can affect pharmacodynamics of ARagonists used as platelet inhibitors.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Adenosina/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação de Plaquetas/farmacologia , Adenosina/análogos & derivados , Adenosina/química , Agonistas do Receptor A2 de Adenosina/química , Adulto , Feminino , Furanos/química , Furanos/farmacologia , Humanos , Masculino , Simulação de Acoplamento Molecular/métodos , Ativação Plaquetária/fisiologia , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Receptores Purinérgicos P1/metabolismo , Adulto Jovem
14.
AJNR Am J Neuroradiol ; 41(1): 140-146, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31896567

RESUMO

BACKGROUND AND PURPOSE: Vascular devices generating high shear stress can cause type 2A acquired von Willebrand disease, which is characterized by low von Willebrand factor activity accompanied by hemorrhagic complications. The braided mesh structure of flow-diverting stents with a relatively small strut size can create abnormally high shear stress while arterial blood flows through the stent struts into the aneurysm, and flow-diverting stent may be associated with reduced von Willebrand factor activity. MATERIALS AND METHODS: Aneurysmal morphologic parameters and patient data were examined retrospectively among patients who had an unruptured intracranial aneurysm treated with a flow-diverting stent. The RISTOtest (test for whole blood ristocetin-induced platelet aggregation) for von Willebrand factor activity, as well as tests for aspirin and clopidogrel/prasugrel effectiveness, were performed immediately before the endovascular procedure and 24 hours later by multiple electrode aggregometry. RESULTS: A total of 39 patients with 56 aneurysms were recruited, and statistical analyses were performed in 32 patents with 49 aneurysms. Compared with the baseline values, von Willebrand factor activity was reduced in 16 patients but increased in 23 patients. Aneurysmal variables (eg, neck area, volume, volume-to-neck area ratio, size ratio, and morphologic index) clearly distinguished patients with reduced von Willebrand factor activity from those with nonreduced von Willebrand factor activity. The receiver operating characteristic curve showed that the morphologic index and volume had the highest discriminative power, with an area under the curve of 0.99. CONCLUSIONS: In high-volume/large-neck aneurysms, flow-diverting stent implantation can cause reduced von Willebrand factor activity, which may be linked causally to acquired von Willebrand disease.


Assuntos
Procedimentos Endovasculares/instrumentação , Aneurisma Intracraniano/terapia , Stents/efeitos adversos , Doença de von Willebrand Tipo 2/etiologia , Fator de von Willebrand/metabolismo , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Agregação Plaquetária/fisiologia , Estudos Retrospectivos , Adulto Jovem
15.
Int J Artif Organs ; 43(3): 208-214, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31674867

RESUMO

Assessing the platelets' functional status during surgery on cardiopulmonary bypass is challenging. This study used multiple electrode impedance aggregometry (Multiplate®) to create a timeline of platelet aggregation changes as induced by cardiopulmonary bypass in antiplatelet-naive patients undergoing elective surgery for mitral valve regurgitation. We performed six consecutive measurements (T1: pre-operatively, T2: after heparinization, T3: 3 min after establishment of cardiopulmonary bypass, T4: immediately after administration of cardioplegia, T5: 5 min after administration of cardioplegia, and T6: 45 min after administration of cardioplegia). Platelet aggregation was determined after stimulation with 3.2-µg/mL collagen, 6.4-µM adenosine diphosphate, and 32-µM thrombin receptor activating peptide. Five patients were included (age: 64 ± 10 years, one female). We observed a decrease in hematocrit levels by -17.1% ± 3.7% (T1 vs T6) with a drop after establishment of cardiopulmonary bypass (T2 vs T3) and slightly decreasing platelet counts by -6.2% ± 7.7% (T1 vs T6). Immediately after establishment of cardiopulmonary bypass (T2 vs T3), we observed reduced platelet aggregation responses for stimulation with adenosine diphosphate (-19.7% ± 12.8%) and thrombin receptor activating peptide (-19.3% ± 6.3%). Interestingly, we found augmented platelet aggregation for all stimuli 45 min after administration of cardioplegia (T5 vs T6) with the strongest increase for collagen (+83.4% ± 42.8%; adenosine diphosphate: +39.0% ± 37.2%; thrombin receptor activating peptide: +34.5% ± 18.5%). Thus, after an initial drop due to hemodilution upon establishment of cardiopulmonary bypass, platelet reactivity increased over time which was not outweighed by decreasing platelet counts due to mechanical platelet destruction and absorption. These findings have implications for rational transfusion, peri-operative antiplatelet therapy, and for the management of patients on other extracorporeal support, such as extracorporeal life support or extracorporeal membrane oxygenation.


Assuntos
Ponte Cardiopulmonar , Cuidados Intraoperatórios , Insuficiência da Valva Mitral/sangue , Agregação Plaquetária , Testes de Função Plaquetária , Idoso , Procedimentos Cirúrgicos Cardíacos/instrumentação , Procedimentos Cirúrgicos Cardíacos/métodos , Ponte Cardiopulmonar/efeitos adversos , Ponte Cardiopulmonar/instrumentação , Ponte Cardiopulmonar/métodos , Circulação Extracorpórea , Feminino , Humanos , Cuidados Intraoperatórios/instrumentação , Cuidados Intraoperatórios/métodos , Cinética , Masculino , Teste de Materiais , Pessoa de Meia-Idade , Insuficiência da Valva Mitral/cirurgia , Projetos Piloto , Agregação Plaquetária/efeitos dos fármacos , Agregação Plaquetária/fisiologia , Testes de Função Plaquetária/instrumentação , Testes de Função Plaquetária/métodos
16.
Acta Diabetol ; 57(4): 389-399, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31679079

RESUMO

AIMS: Platelets are pivotal in arterial thrombosis, and platelet hyperresponsiveness may contribute to the increased incidence of cardiovascular events in diabetes mellitus. Consequently, we hypothesized that increased in vitro platelet aggregation responses exist in men with diabetes mellitus. METHODS: The Danish Cardiovascular Screening Trial (DANCAVAS) is a community-based cardiovascular screening trial including men aged 65-74 years. Platelet aggregation was tested using 96-well light transmission aggregometry with thrombin receptor-activating peptide (TRAP), adenosine diphosphate, collagen type 1, arachidonic acid and protease-activated receptor-4 in three concentrations. Further, cardiovascular risk factors and coronary artery calcification (CAC), estimated by CT scans and ankle-brachial index, were obtained. RESULTS: Included were 720 men aged 65-74 years, 110 with diabetes mellitus. Overall, there was no difference in platelet aggregation among men with versus without diabetes mellitus when adjusting for or excluding platelet inhibitor treatment and men with established cardiovascular disease (CVD). This was true for all agonists, e.g., 10 µM TRAP-induced platelet aggregation of median 69% (IQR 53-75) versus 70% (IQR 60-76) in men with versus without diabetes mellitus. Platelet aggregation did not correlate with HbA1c or CAC. Men with diabetes mellitus displayed higher CAC, median 257 Agatston units (IQR 74-1141) versus median 111 Agatston units (IQR 6-420) in the remaining individuals, p < 0.0001. CONCLUSIONS: Among outpatients with diabetes mellitus, but no CVD and no platelet inhibitor treatment, neither are platelets hyperresponsive in diabetes mellitus, nor is platelet aggregation associated with glycemic status or with the degree of coronary atherosclerosis. TRIAL REGISTRATION: ISRCTN12157806.


Assuntos
Doenças Cardiovasculares/diagnóstico , Diabetes Mellitus Tipo 2/sangue , Agregação Plaquetária/fisiologia , Idoso , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/complicações , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/diagnóstico , Estudos Transversais , Dinamarca/epidemiologia , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Incidência , Masculino , Programas de Rastreamento/métodos , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios X
17.
Cell Mol Life Sci ; 77(5): 885-901, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31278420

RESUMO

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.


Assuntos
Células Endoteliais/metabolismo , Endotélio Vascular/crescimento & desenvolvimento , Neovascularização Fisiológica/fisiologia , Receptores Purinérgicos P2Y2/metabolismo , Angiopoietina-2/biossíntese , Antígenos CD34/biossíntese , Células Cultivadas , Humanos , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Fosforilação/fisiologia , Agregação Plaquetária/fisiologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptores CXCR4/biossíntese , Receptores Purinérgicos P2Y2/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/biossíntese
18.
Int J Mol Sci ; 20(23)2019 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-31783490

RESUMO

Dysregulation of platelet function can contribute to the disease progression in sepsis. The proteasome represents a critical and vital element of cellular protein metabolism in platelets and its proteolytic activity has been associated with platelet function. However, the role of the platelet proteasome as well as its response to infection under conditions of sepsis have not been studied so far. We measured platelet proteasome activity by fluorescent substrates, degradation of poly-ubiquitinated proteins and cleavage of the proteasome substrate Talin-1 in the presence of living E. coli strains and in platelets isolated from sepsis patients. Upregulation of the proteasome activator PA28 (PSME1) was assessed by quantitative real-time PCR in platelets from sepsis patients. We show that co-incubation of platelets with living E. coli (UTI89) results in increased degradation of poly-ubiquitinated proteins and cleavage of Talin-1 by the proteasome. Proteasome activity and cleavage of Talin-1 was significantly increased in α-hemolysin (HlyA)-positive E. coli strains. Supporting these findings, proteasome activity was also increased in platelets of patients with sepsis. Finally, the proteasome activator PA28 (PSME1) was upregulated in this group of patients. In this study we demonstrate for the first time that the proteasome in platelets is activated in the septic milieu.


Assuntos
Plaquetas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Sepse/metabolismo , Sepse/microbiologia , Regulação para Cima/fisiologia , Escherichia coli/patogenicidade , Proteínas Hemolisinas/metabolismo , Humanos , Proteínas Musculares/metabolismo , Ativação Plaquetária/fisiologia , Agregação Plaquetária/fisiologia , Talina/metabolismo , Ativação Transcricional/fisiologia
19.
Sci Rep ; 9(1): 15932, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31685838

RESUMO

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIbα-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies.


Assuntos
Plaquetas/fisiologia , Neutrófilos/fisiologia , Agregação Plaquetária/fisiologia , Trombose/patologia , Animais , Plaquetas/metabolismo , Antígenos CD40/deficiência , Antígenos CD40/genética , Ligante de CD40/deficiência , Ligante de CD40/genética , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microfluídica/instrumentação , Microfluídica/métodos , Microscopia de Fluorescência , Microvasos/efeitos dos fármacos , Microvasos/patologia , Neutrófilos/imunologia , Adesividade Plaquetária/efeitos dos fármacos , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Reologia , Trombose/metabolismo , Fator de von Willebrand/metabolismo
20.
Clinics (Sao Paulo) ; 74: e1222, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31576918

RESUMO

OBJECTIVES: Ischemic stroke (IS) or transient ischemic attack (TIA) history is present in 4-17% of patients with coronary artery disease (CAD). This subgroup of patients is at high risk for both ischemic and bleeding events. The aim of this study was to determine the role of platelet aggregability, coagulation and endogenous fibrinolysis in patients with CAD and previous IS or TIA. METHODS: A prospective case-control study that included 140 stable CAD patients divided into two groups: the CASE group (those with a previous IS/TIA, n=70) and the CONTROL group (those without a previous IS/TIA, n=70). Platelet aggregability (VerifyNow Aspirin® and VerifyNow P2Y12®), coagulation (fibrinogen and thromboelastography by Reorox®) and endogenous fibrinolysis (D dimer and plasminogen activator inhibitor-1) were evaluated. RESULTS: Patients in the CASE group presented significantly higher systolic blood pressure levels (135.84±16.09 vs 123.68±16.11, p<0.01), significantly more previous CABG (25.71% vs 10%, p=0.015) and significantly higher calcium channel blocker usage (42.86% vs 24.29%, p=0.02) than those in the control group. In the adjusted models, low triglyceride values, low hemoglobin values and higher systolic blood pressure were significantly associated with previous IS/TIA (CASE group). Most importantly, platelet aggregability, coagulation and fibrinolysis tests were not independently associated with previous cerebrovascular ischemic events (CASE group). CONCLUSION: Platelet aggregability, coagulation and endogenous fibrinolysis showed similar results among CAD patients with and without previous IS/TIA. Therefore, it remains necessary to identify other targets to explain the higher bleeding risk presented by these patients.


Assuntos
Coagulação Sanguínea/fisiologia , Doença da Artéria Coronariana/sangue , Fibrinólise/fisiologia , Ataque Isquêmico Transitório/sangue , Agregação Plaquetária/fisiologia , Acidente Vascular Cerebral/sangue , Idoso , Testes de Coagulação Sanguínea , Estudos de Casos e Controles , Doença da Artéria Coronariana/fisiopatologia , Feminino , Humanos , Ataque Isquêmico Transitório/fisiopatologia , Masculino , Pessoa de Meia-Idade , Testes de Função Plaquetária , Estudos Prospectivos , Acidente Vascular Cerebral/fisiopatologia
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