RESUMO
BACKGROUND: Thrombin (via PAR [protease-activated receptor]-1 and PAR-4) and ADP (via P2Y12 receptors) are potent endogenous platelet activators implicated in the development of cardiovascular disease. We aimed to assess whether platelet pathways alter with aging. METHODS: We characterized platelet activity in community-dwelling volunteers (n=174) in the following age groups: (1) 20 to 30 (young); (2) 40 to 55 (middle-aged); (3) ≥70 years (elderly). Platelet activity was assessed by aggregometry; flow cytometry (surface markers [P-selectin: alpha granule release, CD63: dense granule release, PAC-1: measure of conformationally active GPIIb/IIIa at the fibrinogen binding site]) measured under basal conditions and after agonist stimulation [ADP, thrombin, PAR-1 agonist or PAR-4 agonist]); receptor cleavage and quantification; fluorometry; calcium flux; ELISA. RESULTS: The elderly had higher basal platelet activation than the young, evidenced by increased expression of P-selectin, CD63, and PAC-1, which correlated with increasing inflammation (IL [interleukin]-1ß/IL-6). The elderly demonstrated higher P2Y12 receptor density, with greater ADP-induced platelet aggregation (P<0.05). However, elderly subjects were resistant to thrombin, achieving less activation in response to thrombin (higher EC50) and to selective stimulation of both PAR-1 and PAR-4, with higher basal PAR-1/PAR-4 cleavage and less inducible PAR-1/PAR-4 cleavage (all P<0.05). Thrombin resistance was attributable to a combination of reduced thrombin orienting receptor GPIbα (glycoprotein Ibα), reduced secondary ADP contribution to thrombin-mediated activation, and blunted calcium flux. D-Dimer, a marker of in situ thrombin generation, correlated with platelet activation in the circulation, ex vivo thrombin resistance, and circulating inflammatory mediators (TNF [tumor necrosis factor]-α/IL-6). CONCLUSIONS: Aging is associated with a distinctive platelet phenotype of increased basal activation, ADP hyperreactivity, and thrombin resistance. In situ thrombin generation associated with systemic inflammation may be novel target to prevent cardiovascular disease in the elderly.
Assuntos
Doenças Cardiovasculares , Receptor PAR-1 , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Idoso , Plaquetas/metabolismo , Cálcio/metabolismo , Doenças Cardiovasculares/metabolismo , Humanos , Inflamação/metabolismo , Interleucina-6/metabolismo , Selectina-P/metabolismo , Fenótipo , Ativação Plaquetária , Agregação Plaquetária , Receptor PAR-1/metabolismo , Receptores de Trombina/metabolismo , Trombina/metabolismoRESUMO
OBJECTIVE: Platelets are critical in mediating both rapid responses to injury and the development and progression of coronary disease. Several studies have shown that, after prolonged exposure to agonists, they produce and release inflammatory mediators including interleukin-1ß (IL-1ß), via the classical pathway (NLRP3 inflammasome and caspase-1 cleavage to release active IL-1ß) as described for leukocytes. This study aimed to determine whether there is rapid release of IL-1ß in response to soluble platelet agonists and whether such rapid release is NLRP3- and caspase-1-dependent. METHODS AND RESULTS: Using flow cytometry to detect platelet activation (and release of α and dense granule contents) and the combination of Western blotting, enzyme-linked-immunosorbent assay, and immunogold labeling transmission electron and immunofluorescence microscopy, we identified that resting human platelets contain mature IL-1ß. Platelets release IL-1ß within minutes in response to adenosine diphosphate (ADP), collagen, and thrombin receptor agonists, but not in response to conventional NLRP3 inflammasome agonists-lipopolysaccharide and adenosine triphosphate. The rapid release of IL-1ß in response to ADP and thrombin receptor agonists was independent of caspases (including caspase-1) and NLRP3. Immature and mature IL-1ß were identified as low-abundance proteins on transmission electron microscopy of human platelets, and were localized to the platelet cytosol, open canalicular system, and the periphery of α granules. CONCLUSION: Unlike monocytes and neutrophils, human platelets are capable of rapid agonist- and time-dependent release of IL-1ß by a mechanism which is independent of caspase-1 and NLRP3.
Assuntos
Inflamassomos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Difosfato de Adenosina , Plaquetas/metabolismo , Caspase 1/metabolismo , Caspases , Humanos , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptores de TrombinaRESUMO
The high occurrence of cancer-associated thrombosis is associated with elevated thrombin generation. Tumour cells increase the potential for thrombin generation both directly, through the expression and release of procoagulant factors, and indirectly, through signals that activate other cell types (including platelets, leukocytes and erythrocytes). Furthermore, cancer treatments can worsen these effects. Coagulation factors, including tissue factor, and inhibitors of coagulation are altered and extracellular vesicles (EVs), which can promote and support thrombin generation, are released by tumour and other cells. Some phosphatidylserine-expressing platelet subsets and platelet-derived EVs provide the surface required for the assembly of coagulation factors essential for thrombin generation in vivo. This review will explore the causes of increased thrombin production in cancer, and the availability and utility of tests and biomarkers. Increased thrombin production not only increases blood coagulation, but also promotes tumour growth and metastasis and as a consequence, thrombin and its contributors present opportunities for treatment of cancer-associated thrombosis and cancer itself.
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Platelets play an important role in diseases such as cardiovascular disease and cancer, especially through their release of extracellular vesicles (EVs) and role in thrombosis. The effects of the anti-inflammatory drug colchicine on platelets are not well understood. We investigated the effect of colchicine on the release of pro-coagulant EVs from platelets under low-level activation. Citrated platelet-rich plasma (PRP) from healthy donors was incubated with 2 mM colchicine or vehicle at 37°C for 30 minutes with gentle rotation. The incubation conditions caused mild platelet activation (expression of CD62P and increased surface lactadherin binding) and release of EVs expressing phosphatidylserine (PS, measured by binding of lactadherin), CD61 and CD62P, both of which were attenuated by colchicine. The incubation conditions shortened the delay to fibrin generation and this correlated with elevated levels of PS+/CD61+ EVs. Removal of EVs from plasma abrogated clot formation in the overall haemostatic potential (OHP) assay. Colchicine decreased levels of both PS+/CD61+ and CD62P+ EVs and abrogated the shortened delay to fibrin generation achieved by platelet activation. Similar results were observed after incubation of PRP with 200 µM vinblastine, suggesting a microtubular effect. An alternative method of platelet activation using platelet agonists 20 µM ADP or 10 µM epinephrine also increased CD62P+ EV levels, and this too was attenuated by prior incubation with colchicine. Our novel findings demonstrate procoagulant effects of low-level platelet activation and EV formation which are inhibited by colchicine.
Assuntos
Coagulação Sanguínea/efeitos dos fármacos , Coagulantes/farmacologia , Colchicina/farmacologia , Ativação Plaquetária/efeitos dos fármacos , Antígenos de Superfície/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Epinefrina/administração & dosagem , Hemostasia/efeitos dos fármacos , Humanos , Microtúbulos/metabolismo , Proteínas do Leite/metabolismo , Selectina-P/metabolismo , Fosfatidilserinas/metabolismo , Agregação Plaquetária , Plasma Rico em Plaquetas/metabolismo , Moduladores de Tubulina/farmacologia , Vimblastina/administração & dosagemRESUMO
OBJECTIVES: The balance between hyper- and hypocoagulable states is critical after coronary artery surgery both with (coronary artery bypass grafting [CABG]) and without (off-pump coronary artery bypass [OPCAB]) cardiopulmonary bypass to prevent thrombotic or bleeding complications. We aimed to quantify novel parameters of coagulation, fibrinolysis, and overall hemostasis ≤6 months after CABG and OPCAB and to determine the influences on these parameters. METHODS: A total of 63 patients (30 CABG, 33 OPCAB) had blood collected before and at various points ≤6 months after surgery. Fibrin and fibrinolysis time curves were generated by measuring the absorption of 405 nm each minute for 100 minutes after the addition of tissue factor and tissue plasminogen activator to cell-free plasma. The parameters were compared with those from a group of healthy controls. RESULTS: The patients' preoperative prothrombotic assay parameters were compared with those from healthy controls. Both CABG and OPCAB patients were hypercoagulable until at least day 10 after surgery, with elevation of fibrin generation (CABG, peak day 3, +28.9%; OPCAB, peak day 1, +16.3% vs preoperative baseline) and impairment of fibrinolysis capacity (CABG, day 1, -58.4%; OPCAB, day 1, -22.6%). Surgical revascularization resulted in resolution of preoperative hypercoagulability by 6 months postoperatively. Patients with preoperative myocardial infarction (MI) had prolonged hypercoagulability after surgery that was most exaggerated after CABG (overall hemostatic potential day 5, no MI, +64.1% vs with MI, +128.9% compared with baseline; P = .013). CONCLUSIONS: Patients will be vulnerable to thrombotic events for ≤6 weeks after coronary surgery yet will have resolution of hypercoagulability by 6 months. Preoperative factors, such as MI, could require individualized management of thrombosis prophylaxis in the postoperative period.
Assuntos
Coagulação Sanguínea , Ponte de Artéria Coronária/efeitos adversos , Fibrinólise , Infarto do Miocárdio/cirurgia , Trombofilia/etiologia , Biomarcadores/sangue , Testes de Coagulação Sanguínea , Estudos de Casos e Controles , Ponte de Artéria Coronária sem Circulação Extracorpórea/efeitos adversos , Fibrina/metabolismo , Humanos , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico , Hemorragia Pós-Operatória/sangue , Hemorragia Pós-Operatória/etiologia , Fatores de Risco , Trombofilia/sangue , Trombose/sangue , Trombose/etiologia , Fatores de Tempo , Resultado do TratamentoRESUMO
Elastin is predominantly comprised of crosslinked tropoelastin. For many years elastin was considered to serve a solely structural role but is now being increasingly identified as causal in cell signaling, development and repair. We introduced tropoelastin into an in vitro model in which airway smooth muscle cells (ASMCs) were stimulated with transforming growth factor (TGF)-ß1 to examine the modulatory effect of this modular elastin sequence on release of angiogenic factors and matrix metalloproteinases (MMPs). Human ASMCs were presented to surfaces coated with tropoelastin or collagen and controls, then stimulated with TGF-ß1. Transcript levels of vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) were quantified 4 and 24 h after TGF-ß1 stimulation. Protein VEGF release from cells and CTGF sequestered at cell surfaces were measured by ELISA at 24 and 48 h. TGF-ß1 increased VEGF mRNA 2.4 fold at 4 h and 5 fold at 24 h, accompanied by elevated cognate protein release 3 fold at 24 h and 2.5 fold at 48 h. TGF-ß1 stimulation increased CTGF mRNA 6.9 fold at 4 h and 11.8 fold at 24 h, accompanied by increased sequestering of its protein counterpart 1.2 fold at 24 h and 1.4 fold at 48 h. Pre-incubation of cells with tropoelastin did not modulate VEGF or CTGF mRNA expression, but combined with TGF-ß1 stimulation it led to enhanced VEGF release 5.1-fold at 24h and 4.4-fold at 48 h. Pre-incubation with tropoelastin decreased CTGF sequestering 0.6-fold at 24 and 48 h, and increased MMP-2 production. Collagen pre-incubation under the same conditions displayed no effect on TGF-ß1 stimulation apart from a slightly decreased (0.9 fold) sequestered CTGF at 48 h. As CTGF is known to anchor VEGF to the matrix and inhibit its angiogenic activity, a process which can be reversed by digestion with MMP-2, these findings reveal that elastin sequences can disrupt the balance of angiogenic factors, with implications for aberrant angiogenesis. The results suggest a model of molecular crosstalk and support an active role for elastin in vascular remodeling.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Regulação da Expressão Gênica/fisiologia , Miócitos de Músculo Liso/fisiologia , Fator de Crescimento Transformador beta1/metabolismo , Tropoelastina/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Colágeno/metabolismo , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Miócitos de Músculo Liso/metabolismo , Sistema Respiratório/citologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Persistent alteration to host polymorphonuclear cell (PMN) physiology has been demonstrated after cardiac surgery performed with cardiopulmonary bypass (CPB). However, to date, PMN physiology and function beyond the first 24 h have not been investigated after cardiac surgery performed without CPB (off-pump coronary artery bypass grafting [OPCAB]). Blood samples of 15 patients were collected preoperatively and on days 1, 3, and 5 after OPCAB. Expression of CD11b, CD18, CBRM1/5, and CD62L were assessed by flow cytometry under resting conditions and after stimulation with formyl methionyl-leucyl-phenylalanine (fMLF), and respiratory burst activity was also measured. Under resting conditions, PMN CD11b, CBRM1/5, and CD62L expressions were minimally altered by surgery. Compared with the response of preoperative PMNs, PMNs assayed on days 3 and 5 after OPCAB demonstrated a significantly blunted increase in the expression of CD11b and CBRM1/5 after fMLF, significantly diminished shedding of CD62L in response to platelet-activating factor and fMLF, and diminished superoxide production after stimulation on day 3. The alteration of PMN function after OPCAB implies that cardiac surgical trauma without CPB directly modulates host PMN physiology.