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Patients with colorectal cancer (CRC) are at increased risk of venous thrombosis, but the precise mechanisms of thrombogenesis in CRC remain largely unknown. We aimed to identify the novel role of neutrophil extracellular traps (NETs) in the induction of procoagulant activity (PCA) in CRC, and to evaluate its interactions with platelets and endothelial cells (ECs). In this study, we first showed that the levels of NETs in the peripheral blood of CRC patients were increased in parallel with cancer progression and reached significance in stage II patients compared to healthy subjects. In addition, neutrophils from CRC patients were more prone to produce NETs, resulting in shortened coagulation time, significantly increased thrombin-antithrombin (TAT) complexes and fibrin fibrils compared to healthy controls. Furthermore, platelets from CRC patients stimulated healthy neutrophils to extrude NETs, which could be inhibited by the depletion of HMGB1. Conversely, NETs from CRC patients could also induce the exposure of PS on platelets, leading to markedly enhanced PCA. Importantly, ECs were also converted to a procoagulant phenotype when exposed to NETs from CRC patients. The PCA of NETs-activated platelets or ECs could be inhibited either by the cleavage of NETs with DNase1 or the blockage of histone with activated protein C (APC). Our results reveal the complex interactions between neutrophils, platelets and ECs and their potential role in the hypercoagulable state in CRC. We propose that NETs may provide new therapeutic targets to combat the thrombotic consequences of CRC.
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Plaquetas/metabolismo , Neoplasias Colorrectales/complicaciones , Trampas Extracelulares/metabolismo , Activación Plaquetaria , Trombosis/etiología , Anciano , Coagulación Sanguínea , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/metabolismo , Células Endoteliales/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trombosis/sangre , Trombosis/metabolismoRESUMEN
Relatively little information is known about the definitive role of phosphatidylserine (PS) in the hypercoagulability of heart failure (HF). Our objectives were to assess the levels of PS exposure on microparticles (MPs) and blood cells (BCs) in each group of HF patients and to evaluate their procoagulant activity (PCA). HF patients in each NYHA functional class II-IV (II n = 30, III n = 30, IV n = 30) and healthy controls (n = 25) were enrolled in the present study. PS exposure on MPs, BCs was analyzed with flow cytometry. MPs were classified based on their cellular origin: platelets (CD41a+), neutrophils (CD66b+), endothelial cells (CD31+CD41a-), erythrocytes (CD235a+), monocytes (CD14+), T lymphocytes (CD3+), and B lymphocytes (CD19+). PCA was evaluated by clotting time, extrinsic/intrinsic FXa and prothrombinase production assays, as well as fibrin formation assays. Inhibition assays of PCA of PS+ BCs and MPs were performed by lactadherin. There was no significant difference in MP cellular origin between healthy and HF subjects. However, the total number of PS+ MPs was significantly increased in HF patients compared with healthy controls. In addition, circulating PS+ BCs cooperated with PS+ MPs to markedly shorten coagulation time and dramatically increase FXa/thrombin generation and fibrin formation in each HF group. Moreover, blockade of exposed PS on BCs and MPs with lactadherin inhibited PCA by approximately 80%. Our results lead us to believe that exposing PS on the injured BCs and MPs played a pivotal role in the hypercoagulability state in HF patients.
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Micropartículas Derivadas de Células/fisiología , Células Endoteliales/fisiología , Insuficiencia Cardíaca/sangre , Fosfatidilserinas/metabolismo , Trombofilia/etiología , Adulto , Anciano , Células Sanguíneas/patología , Coagulación Sanguínea , Pruebas de Coagulación Sanguínea , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana EdadRESUMEN
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
RESUMEN
Multiple myeloma (MM) is characterized by an increased incidence of thromboembolic events, particularly when treated with immunomodulatory drugs (IMiDs) in combination with dexamethasone. The optimal prophylactic strategy to prevent the hypercoagulable state of patients with MM is still debated. The aim of the current study was to investigate the definitive role of phosphatidylserine (PS) in supporting procoagulant activity (PCA) in patients with MM. Patients with MM (n=20) and healthy subjects (n=15) were recruited for the present study. PS analyses were performed by flow cytometry and confocal microscopy. The PCA was evaluated by clotting time, purified coagulation complex assays and fibrin production assays. The percentage of PS+ blood cells was significantly higher in patients with MM than in healthy subjects. Additionally, the patient serum induced more PS exposure on endothelial cells (ECs) in vitro than serum from healthy subjects. Isolated blood cells from patients with MM and ECs cultured with patient serum in vitro demonstrated significantly shortened coagulation time, greatly intrinsic/extrinsic factor Xa generation and increased thrombin formation. In addition, the levels of PS+ erythrocytes, platelets, leukocytes, and ECs incubated with IMiDs and dexamethasone were higher than with IMiDs alone. The findings support the hypothesis that increased PS exposure on blood cells and ECs participates in the hypercoagulable state in patients with MM. Thus, blocking PS may be a novel therapeutic target for the prevention of thrombosis in these patients.
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Factor Xa/metabolismo , Mieloma Múltiple/complicaciones , Fosfatidilserinas/sangre , Tromboembolia Venosa/sangre , Adulto , Anciano , Coagulación Sanguínea , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Masculino , Persona de Mediana Edad , Mieloma Múltiple/sangre , Mieloma Múltiple/metabolismo , Células Tumorales Cultivadas , Tromboembolia Venosa/metabolismoRESUMEN
Background: Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with diabetic kidney disease (DKD). This study was designed to evaluate whether exposed PS on microparticles (MPs) and MP-origin cells were involved in the hypercoagulability in DKD patients. Methods: DKD patients (n = 90) were divided into three groups based on urinary albumin excretion rate, defined as normoalbuminuria (No-A) (<30 mg/24 h), microalbuminuria (Mi-A) (30-299 mg/24 h) or macroalbuminuria (Ma-A) (>300 mg/24 h), and compared with healthy controls (n = 30). Lactadherin was used to quantify PS exposure on MPs and their original cells. Healthy blood cells (BCs) and human umbilical vein endothelial cells (HUVECs) were treated with 25, 5 or 2.5 mmol/L glucose as well as 3-12 mg/dL uric acid and cells were evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure and fibrin strands were observed using confocal microscopy. Results: Using flow cytometry, we found that PS+ MPs (derived from platelets, erythrocytes, HUVECs, neutrophils, monocytes and lymphocytes) and BCs were significantly higher in patients than in controls. Furthermore, the number of PS+ MPs and BCs in patients with Ma-A was significantly higher than in patients with No-A. Similarly, we observed markedly elevated PS exposure on HUVECs cultured with serum from patients with Ma-A versus serum from patients with Mi-A or normoalbuminuria. In addition, circulating PS+ MPs cooperated with PS+ cells, contributing to markedly shortened coagulation time and dramatically increased FXa/thrombin generation and fibrin formation in each DKD group. Confocal microscopy images demonstrated colocalization of fibrin with PS on HUVECs. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by â¼80%. In vitro, BCs and endothelial cells exposed more PS in hypoglycemia or hyperglycemia. Interestingly, reconstitution experiments showed that hypoglycemia-treated cells could be further activated or injured when recovery is obtained reaching hyperglycemia. Moreover, uric acid induced PS exposure on cells (excluding platelets) at concentrations >6 mg/dL. Linear regression analysis showed that levels of PS+ BCs and microparticles were positively correlated with uric acid and proteinuria, but negatively correlated with glomerular filtration rate. Conclusions: Our results suggest that PS+ MPs and MP-origin cells play procoagulant roles in patients with DKD. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.
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Coagulación Sanguínea/efectos de los fármacos , Micropartículas Derivadas de Células/metabolismo , Nefropatías Diabéticas/patología , Fosfatidilserinas/farmacología , Trombofilia/patología , Trombosis/epidemiología , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Trombofilia/tratamiento farmacológico , Trombofilia/metabolismoRESUMEN
BACKGROUND: The definitive role of phosphatidylserine (PS) in the prothrombotic state of non-valvular atrial fibrillation (NVAF) remains unclear. Our objectives were to study the PS exposure on blood cells and microparticles (MPs) in NVAF, and evaluate their procoagulant activity (PCA). METHODS: NVAF patients without (nâ¯=â¯60) and with left atrial thrombi (nâ¯=â¯18) and controls (nâ¯=â¯36) were included in our study. Exposed PS was analyzed with flow cytometry and confocal microscopy. PCA was evaluated using clotting time, factor Xa (FXa), thrombin and fibrin formation. RESULTS: PS+ blood cells and MPs were significantly higher in NVAF patients without and with left atrial thrombi (both Pâ¯<â¯0.01) than in controls. Patients with left atrial thrombi showed increased PS+ platelets, neutrophils, erythrocytes and MPs compared with patients without thrombi (all Pâ¯<â¯0.05). Moreover, in patients with left atrial thrombi, MPs primarily originated from platelets (56.1%) followed by leukocytes (21.9%, including MPs from neutrophils, monocytes and lymphocytes), erythrocytes (12.2%) and endothelial cells (8.9%). Additionally, PS+ blood cells and MPs contributed to markedly shortened coagulation time and dramatically increased FXa/thrombin/fibrin (all Pâ¯<â¯0.001) generation in both NVAF groups. Furthermore, blockade of exposed PS on blood cells and MPs with lactadherin inhibited PCA by approximately 80%. Lastly, we found that the amount of PS+ platelets and MPs was positively correlated with thrombus diameter (all pâ¯<â¯0.005). CONCLUSIONS: Our results suggest that exposed PS on blood cells and MPs play a procoagulant role in NVAF patients. Blockade of PS prior to thrombus formation might be a novel therapeutic approach in these patients.
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Fibrilación Atrial/sangre , Fibrilación Atrial/diagnóstico por imagen , Células Sanguíneas/metabolismo , Coagulación Sanguínea/fisiología , Micropartículas Derivadas de Células/metabolismo , Fosfatidilserinas/metabolismo , Anciano , Fibrilación Atrial/fisiopatología , Células Sanguíneas/química , Pruebas de Coagulación Sanguínea/métodos , Micropartículas Derivadas de Células/química , Ecocardiografía/métodos , Electrocardiografía/métodos , Femenino , Humanos , Masculino , Microscopía Confocal/métodos , Persona de Mediana Edad , Fosfatidilserinas/análisisRESUMEN
The mechanisms of thrombogenicity in essential thrombocythemia (ET) are complex and not well defined. Our objective was to explore whether phosphatidylserine (PS) exposure on blood cells and endothelial cells (ECs) can account for the increased thrombosis and distinct thrombotic risks among mutational subtypes in ET. Using flow cytometry and confocal microscopy, we found that the levels of PS-exposing erythrocytes, platelets, leukocytes, and serum-cultured ECs were significantly higher in each ET group [JAK2, CALR, and triple-negative (TN) (all P < 0.001)] than those in controls. Among ET patients, those with JAK2 mutations showed higher levels of PS-positive erythrocytes, platelets, neutrophils, and serum-cultured ECs than TN patients or those with CALR mutations, which show similar levels. Coagulation function assays showed that higher levels of PS-positive blood cells and serum-cultured ECs led to markedly shortened coagulation time and dramatically increased levels of FXa, thrombin, and fibrin production. This procoagulant activity could be largely blocked by addition of lactadherin (approx. 70% inhibition). Confocal microscopy showed that the FVa/FXa complex and fibrin fibrils colocalized with PS on ET serum-cultured ECs. Additionally, we found a relationship between D-dimer, prothrombin fragment F1 + 2, and PS exposure. Our study reveals a previously unrecognized link between hypercoagulability and exposed PS on cells, which might also be associated with distinct thrombotic risks among mutational subtypes in ET. Thus, blocking PS-binding sites may represent a new therapeutic target for preventing thrombosis in ET.
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Plaquetas/patología , Endotelio Vascular/patología , Eritrocitos/patología , Leucocitos/patología , Fosfatidilserinas/metabolismo , Trombocitemia Esencial/fisiopatología , Trombosis/etiología , Adulto , Anciano , Sustitución de Aminoácidos , Plaquetas/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Células Cultivadas , China/epidemiología , Endotelio Vascular/metabolismo , Eritrocitos/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana/citología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Janus Quinasa 2/genética , Janus Quinasa 2/metabolismo , Leucocitos/metabolismo , Masculino , Persona de Mediana Edad , Mutación , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismo , Riesgo , Propiedades de Superficie , Trombocitemia Esencial/genética , Trombocitemia Esencial/metabolismo , Trombocitemia Esencial/patología , Trombosis/epidemiologíaRESUMEN
Despite the high efficacy and safety of arsenic trioxide (ATO) in treating acute promyelocytic leukemia (APL) and eradicating APL leukemia-initiating cells (LICs), the mechanism underlying its selective cytotoxicity remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, through autophagy. However, the role of ETosis in ATO-induced APL LIC eradication remains unclear. For this study, we evaluated the effects of ATO on ETosis and the contributions of drug-induced ETosis to APL LIC eradication. In NB4 cells, ATO primarily increased ETosis at moderate concentrations (0.5-0.75 µM) and stimulated apoptosis at higher doses (1.0-2.0 µM). Furthermore, ATO induced ETosis through mammalian target of rapamycin (mTOR)-dependent autophagy, which was partially regulated by reactive oxygen species. Additionally, rapamycin-enhanced ATO-induced ETosis in NB4 cells and APL cells from newly diagnosed and relapsed patients. In contrast, rapamycin had no effect on apoptosis in these cells. We also noted that PML/RARA oncoprotein was effectively cleared with this combination. Intriguingly, activation of autophagy with rapamycin-enhanced APL LIC eradication clearance by ATO in vitro and in a xenograft APL model, while inhibition of autophagy spared clonogenic cells. Our current results show that ATO exerts antileukemic effects at least partially through ETosis and targets LICs primarily through ETosis. Addition of drugs that target the ETotic pathway could be a promising therapeutic strategy to further eradicate LICs and reduce relapse.
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Trióxido de Arsénico/farmacología , Autofagia/efectos de los fármacos , Leucemia Promielocítica Aguda/patología , Serina-Treonina Quinasas TOR/metabolismo , Adolescente , Adulto , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Masculino , Ratones SCID , Persona de Mediana Edad , Recurrencia Local de Neoplasia/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Sirolimus/farmacología , Carga Tumoral , Adulto JovenRESUMEN
The present study aimed to evaluate the procoagulant effects of phosphatidylserine (PS) exposure on blood cells and microparticles (MPs), and examine its role in predicting early recurrence atrial fibrillation (ERAF) in patients with atrial fibrillation (AF) treated with pulmonary vein isolation (PVI). Blood samples were obtained from 40 healthy controls and 56 patients with AF at baseline (prior to PVI), and 0, 1 h, 1 day, 3 days and 7 days following PVI. The exposure of PS (PS+) to blood cells (platelets, erythrocytes and leukocytes) and MPs was detected using flow cytometry. The procoagulant activity was evaluated by coagulation time, and the formation of factor Xa (FXa) and thrombin. In addition, independent factors associated with PS+ blood cells and MPs, and significant predictors of ERAF following PVI were investigated by statistical analyses. The numbers of PS+ blood cells and MPs were significantly increased by PVI (P<0.01). A significant decrease in coagulation time, and increases in FXa and thrombin were exhibited in the PS+ blood cells and MPs from patients with AF treated with PVI, whereas these alterations were inhibited by either lactadherin or antitissue factor (P<0.01). The maximum power of the PVI was significantly associated with plateletderived MPs, and highsensitivity Creactive protein (hsCRP) was closely associated with leukocytederived MPs and endothelialderived MPs (EMPs) (P<0.01). In addition, hsCRP and EMPs >355/µl were identified as independent predictors of ERAF (P<0.05). The increased numbers of PS+ platelets, erythrocytes, leukocytes and MPs contributed to the procoagulant state of AF, and hsCRP and EMPs were able to predict ERAF following PVI.
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Fibrilación Atrial/sangre , Fibrilación Atrial/metabolismo , Células Sanguíneas/metabolismo , Coagulación Sanguínea , Micropartículas Derivadas de Células/metabolismo , Fosfatidilserinas/metabolismo , Venas Pulmonares/metabolismo , Plaquetas/metabolismo , Eritrocitos/metabolismo , Factor X/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , RecurrenciaRESUMEN
The procoagulant status of patients with non-small cell lung cancer (NSCLC) after chemotherapy is poorly characterized and the role of platelets in hypercoagulative state of NSCLC is unknown. The aim of this study was to evaluate the procoagulant activity (PCA) of platelets in NSCLC before and after chemotherapy. The subjects were 52 patients newly diagnosed with NSCLC. The patients had decreased clotting time compared with healthy subjects, and the thrombin-antithrombin complex increased 2.5-fold after chemotherapy. Platelets in the patients after chemotherapy had enhanced phosphatidylserine (PS) exposure, and shortened coagulation time as well as increased thrombin and fibrin formation of platelets compared with those before chemotherapy. Platelet-derived microparticles increased 2-fold at day 1 and peaked at day 2 post-chemotherapy. Treatment of cisplatin in vitro also resulted in upregulated intrinsic FXa and thrombin formation on platelets with a dose-dependent manner. Platelets treated with aspirin significantly decreased PCA. However, lactadherin blocked PS and inhibited the PCA approximately by 70%. Seven days after chemotherapy, PCA of platelets restored to the baseline as that before chemotherapy, indicating that within a week of chemotherapy patient platelets are highly procoagulant and effective intervention should be taken in case of thrombosis. Our results suggested that platelets after chemotherapy had elevated PCA and may contribute to the hypercoagulative state of NSCLC. Prophylactic anti-coagulant combined with anti-platelet therapy may play an inhibitory role in thrombotic complications in NSCLC.
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Coagulación Sanguínea/efectos de los fármacos , Plaquetas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Trombosis/prevención & control , Anciano , Antígenos de Superficie/metabolismo , Aspirina/farmacología , Aspirina/uso terapéutico , Tiempo de Sangría , Plaquetas/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/sangre , Micropartículas Derivadas de Células/efectos de los fármacos , Femenino , Humanos , Neoplasias Pulmonares/sangre , Masculino , Persona de Mediana Edad , Proteínas de la Leche/metabolismo , Fosfatidilserinas/metabolismo , Inhibidores de Agregación Plaquetaria/farmacología , Trombina/metabolismo , Tromboplastina/metabolismo , Trombosis/sangre , Regulación hacia ArribaRESUMEN
The mechanisms that eliminate activated platelets in inflammation-induced disseminated intravascular coagulation (DIC) in micro-capillary circulation are poorly understood. This study explored an alternate pathway for platelet disposal mediated by endothelial cells (ECs) through phosphatidylserine (PS) and examined the effect of platelet clearance on procoagulant activity (PCA) in sepsis. Platelets in septic patients demonstrated increased levels of surface activation markers and apoptotic vesicle formation, and also formed aggregates with leukocytes. Activated platelets adhered were and ultimately digested by ECs in vivo and in vitro. Blocking PS on platelets or αvß3 integrin on ECs attenuated platelet clearance resulting in increased platelet count in a mouse model of sepsis. Furthermore, platelet removal by ECs resulted in a corresponding decrease in platelet-leukocyte complex formation and markedly reduced generation of factor Xa and thrombin on platelets. Pretreatment with lactadherin significantly increased phagocytosis of platelets by approximately 2-fold, diminished PCA by 70%, prolonged coagulation time, and attenuated fibrin formation by 50%. Our results suggest that PS-mediated clearance of activated platelets by the endothelium results in an anti-inflammatory, anticoagulant, and antithrombotic effect that contribute to maintaining platelet homeostasis during acute inflammation. These results suggest a new therapeutic target for impeding the development of DIC.
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Plaquetas/citología , Células Endoteliales/citología , Fosfatidilserinas/farmacología , Activación Plaquetaria/efectos de los fármacos , Sepsis/metabolismo , Adulto , Coagulación Sanguínea , Plaquetas/efectos de los fármacos , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Femenino , Células Endoteliales de la Vena Umbilical Humana , Humanos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Leucocitos/metabolismo , Masculino , Persona de Mediana EdadRESUMEN
Despite routine treatment of unselected acute promyelocytic leukemia (APL) with all-trans-retinoic acid (ATRA), early death because of hemorrhage remains unacceptably common, and the mechanism underlying this complication remains elusive. We have recently demonstrated that APL cells undergo a novel cell death program, termed ETosis, which involves release of extracellular chromatin. However, the role of promyelocytic extracellular chromatin in APL-associated coagulation remains unclear. Our objectives were to identify the novel role of ATRA-promoted extracellular chromatin in inducing a hypercoagulable and hyperfibrinolytic state in APL and to evaluate its interaction with fibrin and endothelial cells (ECs). Results from a series of coagulation assays have shown that promyelocytic extracellular chromatin increases thrombin and plasmin generation, causes a shortening of plasma clotting time of APL cells, and increases fibrin formation. DNase I but not anti-tissue factor antibody could inhibit these effects. Immunofluorescence staining showed that promyelocytic extracellular chromatin and phosphatidylserine on APL cells provide platforms for fibrin deposition and render clots more resistant to fibrinolysis. Additionally, coincubation assays revealed that promyelocytic extracellular chromatin is cytotoxic to ECs, converting them to a procoagulant phenotype. This cytotoxity was blocked by DNase I by 20% or activated protein C by 31%. Our current results thus delineate the pathogenic role of promyelocytic extracellular chromatin in APL coagulopathy. Furthermore, the remaining coagulation disturbance in high-risk APL patients after ATRA administration may be treatable by intrinsic pathway inhibition via accelerating extracellular chromatin degradation.
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Coagulación Sanguínea , Cromatina/patología , Cromatina/fisiología , Fibrinólisis , Leucemia Promielocítica Aguda/complicaciones , Células Cultivadas , Cromatina/ultraestructura , Células Endoteliales , Fibrina/metabolismo , Células Precursoras de Granulocitos/patología , Humanos , Leucemia Promielocítica Aguda/sangre , Tretinoina/farmacología , Células Tumorales CultivadasRESUMEN
The role of phosphatidylserine (PS)-mediated procoagulant activity (PCA) in stroke remains unclear. To ascertain this role, early dynamic evolution of PS exposure on blood cells and released microparticles (MPs) and the corresponding PCA were evaluated in patients with acute ischemic stroke (AIS). Flow cytometry analyses revealed that initial levels of PS exposure on erythrocyte, platelet, and leukocyte were 2.40-, 1.36-, and 1.38-fold higher, respectively, in AIS than the risk factor-matched (RF) controls. Concomitantly, total PS+ MPs were increased in AIS (1949 ± 483/µl) compared with the RF group (1674 ± 387/µl; P = 0.019) and healthy controls (1052 ± 179/µl; P < 0.001). Specifically, PS+ erythrocytes gradually increased within 1 week. PS+ platelets and MPs peaked at 24 h and declined at 7 days, while PS+ leukocytes were markedly elevated at 24 h. Further, PS exposure on blood cells and MPs in stroke resulted in shortened clotting time with an accompanying increase in FXa and thrombin formation significantly. Treatment with lactadherin, a PS antagonist, delayed the coagulation time by approximately 20 % and blocked the generation of FXa and thrombin by about 50 %. Furthermore, initial counts of PS+ platelets and platelet MPs significantly correlated with stroke severity. Thrombin generation promoted by platelets and MPs at 12 h was significantly higher in patients with cardioembolism than in patients without. The thrombophilic susceptibility of AIS patients can be partly ascribed to PS exposure on blood cells and the release of MPs. Our studies identify PS exposure as a potentially novel therapeutic target in the treatment of AIS.
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Factores de Coagulación Sanguínea/metabolismo , Coagulación Sanguínea , Isquemia Encefálica/sangre , Fosfatidilserinas/sangre , Accidente Cerebrovascular/sangre , Anciano , Pruebas de Coagulación Sanguínea , Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Eritrocitos/metabolismo , Femenino , Citometría de Flujo , Humanos , Masculino , Persona de Mediana Edad , Trombina/metabolismoRESUMEN
OBJECTIVE Phosphatidylserine (PS) is a major component of the inner leaflet of membrane bilayers. During cell activation or apoptosis, PS is externalized to the outer membrane, providing an important physiological signal necessary for the release of the microparticles (MPs) that are generated through the budding of cellular membranes. MPs express PS and membrane antigens that reflect their cellular origin. PS exposure on the cell surface and the release of MPs provide binding sites for factor Xa and prothrombinase complexes that promote thrombin formation. Relatively little is known about the role of PS exposure on blood cells and MPs in patients with internal carotid artery (ICA) stenosis who have undergone carotid artery stenting (CAS). The authors aimed to investigate the extent of PS exposure on blood cells and MPs and to define its role in procoagulant activity (PCA) in the 7 days following CAS. METHODS The study included patients with ICA stenosis who had undergone CAS (n = 70), matched patients who had undergone catheter angiography only (n = 30), and healthy controls (n = 30). Blood samples were collected from all patients just before the procedure after an overnight fast and at 2, 6, 24, 48, and 72 hours and 7 days after the CAS procedure. Blood was collected from healthy controls after an overnight fast. Phosphatidylserine-positive (PS+) MPs and blood cells were analyzed by flow cytometry, while PCA was assessed with clotting time analysis, purified coagulation complex assays, and fibrin formation assays. RESULTS The authors found that levels of PS+ blood cells and PS+ blood cell-derived MPs (platelets and platelet-derived MPs [PMPs], neutrophils and neutrophil-derived MPs [NMPs], monocytes and monocyte-derived MPs [MMPs], erythrocytes and erythrocyte-derived MPs [RMPs], and endothelial cells and endothelial cell-derived MPs [EMPs]) were increased in the 7 days following the CAS procedure. Specifically, elevation of PS exposure on platelets/PMPs, neutrophils/NMPs, and monocytes/MMPs was detected within 2 hours of CAS, whereas PS exposure was delayed on erythrocytes/RMPs and EMPs, with an increase detected 24 hours after CAS. In addition, PS+ platelets/PMPs peaked at 2 hours, while PS+ neutrophils/NMPs, monocytes/MMPs, and erythrocytes/RMPs peaked at 48 hours. After their peak, all persisted at levels above baseline for 7 days post-CAS. Moreover, the level of PS+ blood cells/MPs was correlated with shortened coagulation time and significantly increased intrinsic and extrinsic Xase, thrombin generation, and fibrin formation. Pretreatment of blood cells with lactadherin at their peak time point after CAS blocked PS, resulting in prolonged coagulation times, decreased procoagulant enzyme activation, and fibrin production. CONCLUSIONS The results of this study suggest that increased exposure of PS on blood cells and MPs may contribute to enhanced PCA in patients with ICA stenosis who have undergone CAS, explaining the risk of perioperative thromboembolic complications in these patients. PS on blood cells and MPs may serve as an important biomarker for predicting, and as a pivotal target for monitoring and treating, acute postoperative complications after CAS. â CLASSIFICATION OF EVIDENCE Type of question: association; study design: prospective cohort trial; evidence: Class I.
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Células Sanguíneas/metabolismo , Coagulación Sanguínea/fisiología , Estenosis Carotídea/cirugía , Micropartículas Derivadas de Células/metabolismo , Fosfatidilserinas/metabolismo , Stents , Anciano , Estenosis Carotídea/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Procedimientos Quirúrgicos VascularesRESUMEN
BACKGROUND: Relatively little is known about the role of phosphatidylserine (PS) in procoagulant activity (PCA) in patients with non-ST-elevated myocardial infarction (NSTEMI) after stent implantation. This study was designed to evaluate whether exposed PS on microparticles (MPs) and blood cells were involved in the hypercoagulable state in NSTEMI patients with stent implantation. METHODS: NSTEMI patients (n=90) and healthy controls (n=20) were included in our study. PS exposure on MPs and blood cells was analyzed with flow cytometer and confocal microscope. PCA was evaluated by clotting time, purified coagulation complex assays and fibrin production assays. RESULTS: Baseline levels of MPs and PS+ blood cells were significantly higher (P<0.001) in the patients than in controls. After stent implantation, a remarkable increase was observed in both MPs and PS+ blood cells. Specifically, PS+ MPs, PS+ platelets and erythrocytes peaked at 18h following stent implantation, while PS+ leukocytes peaked on day 2. In addition, circulating MPs (mostly derived from platelets, leukocytes, erythrocytes and endothelial cells) cooperating with PS+ blood cells, contributed to markedly shortened coagulation time and markedly increased FXa/thrombin/fibrin (all P<0.01) generation in patient group. Moreover, blockade of exposed PS on MPs and cells with lactadherin inhibited PCA by approximately 70%. CONCLUSIONS: Our results suggest that PS+ MPs and blood cells play a procoagulant role in NSTEMI patients following stent implantation. Blockade of PS could become a novel therapeutic modality for the prevention of thrombosis in these patients.
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Células Sanguíneas , Micropartículas Derivadas de Células/metabolismo , Infarto del Miocardio sin Elevación del ST , Intervención Coronaria Percutánea , Fosfatidilserinas/análisis , Trombofilia , Anciano , Aspirina/uso terapéutico , Células Sanguíneas/metabolismo , Células Sanguíneas/patología , Coagulación Sanguínea/fisiología , Clopidogrel , Femenino , Humanos , Masculino , Persona de Mediana Edad , Infarto del Miocardio sin Elevación del ST/sangre , Infarto del Miocardio sin Elevación del ST/complicaciones , Infarto del Miocardio sin Elevación del ST/tratamiento farmacológico , Infarto del Miocardio sin Elevación del ST/cirugía , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/instrumentación , Intervención Coronaria Percutánea/métodos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Estadística como Asunto , Stents , Trombofilia/etiología , Trombofilia/metabolismo , Ticlopidina/análogos & derivados , Ticlopidina/uso terapéuticoRESUMEN
BACKGROUND & AIMS: The mechanism of thrombogenicity in cirrhosis is largely unknown. Our objective was to study the relationship between phosphatidylserine on blood cells and endothelial cells and the hypercoagulable state in cirrhotic patients. METHODS: Patients with cirrhosis and healthy controls were studied. Lactadherin was used to quantify phosphatidylserine exposure on blood cells and endothelial cells. Procoagulant activity of cells was evaluated using clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. Phosphatidylserine exposure, fibrin strands and FVa/Xa binding on cells were observed using confocal microscopy. RESULTS: Our study showed that phosphatidylserine exposure on erythrocytes, platelets and leucocytes in cirrhotic patients increased progressively with Child-Pugh categories. In addition, we found that endothelial cells treated with cirrhotic serum in vitro exposed more phosphatidylserine than those exposed to healthy serum. The exposed phosphatidylserine supported a shorter coagulation time and increased FXa, thrombin and fibrin formation. Notably, phosphatidylserine+ erythrocytes also promoted shorter coagulation times and more fibrin generation in cirrhotic microparticle-depleted plasma, regardless of Child-Pugh categories. Confocal microscopy data showed that the FVa/FXa complex and fibrin fibrils colocalized with phosphatidylserine on endothelial cells. Lactadherin significantly inhibited FXa and thrombin generation and consequently decreased fibrin production in normal or cirrhotic plasma. CONCLUSIONS: These results lead us to believe that exposed phosphatidylserine on activated or injured erythrocytes, platelets, leucocytes and endothelial cells plays an important role in the hypercoagulable state in cirrhotic patients. Thus, blocking phosphatidylserine binding sites might be a new therapeutic target for preventing thrombosis.
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Células Sanguíneas/metabolismo , Células Endoteliales/metabolismo , Cirrosis Hepática/sangre , Fosfatidilserinas/metabolismo , Trombofilia/etiología , Adulto , Pruebas de Coagulación Sanguínea , Plaquetas/metabolismo , Estudios de Casos y Controles , Micropartículas Derivadas de Células , China , Femenino , Fibrina/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Trombina/metabolismoRESUMEN
OBJECTIVE: We evaluated cellular origin, numbers, and procoagulant activity of phosphatidylserine-positive microparticles (MPs) among subgroups in acute coronary syndromes (ACS). MATERIALS AND METHODS: Parameters were measured on admission, days 1 (within 24 h of admission), 2, 3, and 7. All ST-elevated myocardial infarction (STEMI) patients presented more than 3 h from symptom onset and received fibrinolysis treatment; controls included unstable angina and non-STEMI patients as well as healthy controls. Phosphatidylserine-positive MPs were detected by flow cytometry, whereas procoagulant activity was assessed by coagulation time, purified coagulation complex assays, and fibrin formation. MP-induced fibrins were visualized by confocal microscopy. RESULTS: On admission, the total MP count was â¼2.5-fold higher in the ACS groups compared with the healthy controls (P<0.05), primarily originating from platelets and endothelial cells, and there were no significant differences among ACS subgroups. Specifically, leukocyte-derived and erythrocyte-derived MPs were higher in the STEMI group compared with unstable angina and non-STEMI groups (both P<0.05). Further, MPs from the ACS groups reduced coagulation time by 27.5% and induced intrinsic and extrinsic FXase, prothrombinase, and fibrin formation by 2.8-, 2.3-, 2.5-, and 1.7-fold, respectively (P<0.05 for all), whereas blocking phosphatidylserine with lactadherin inhibited â¼70% of procoagulant activity. MP number and concomitant coagulation decreased significantly by day 2 and continued to decrease gradually during the recovery period. CONCLUSION: This study shows that MP characteristics from circulating blood may be used as prognostic indicators to reflect the origin cell of activation and thrombophilic states found in ACS subgroups.
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Síndrome Coronario Agudo/sangre , Angina Inestable/sangre , Coagulación Sanguínea , Micropartículas Derivadas de Células/metabolismo , Infarto del Miocardio sin Elevación del ST/sangre , Fosfatidilserinas/sangre , Infarto del Miocardio con Elevación del ST/sangre , Síndrome Coronario Agudo/diagnóstico , Síndrome Coronario Agudo/tratamiento farmacológico , Adulto , Anciano , Angina Inestable/diagnóstico , Angina Inestable/tratamiento farmacológico , Biomarcadores/sangre , Coagulación Sanguínea/efectos de los fármacos , Pruebas de Coagulación Sanguínea , Estudios de Casos y Controles , Femenino , Citometría de Flujo , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Infarto del Miocardio sin Elevación del ST/diagnóstico , Infarto del Miocardio sin Elevación del ST/tratamiento farmacológico , Valor Predictivo de las Pruebas , Infarto del Miocardio con Elevación del ST/diagnóstico , Infarto del Miocardio con Elevación del ST/tratamiento farmacológico , Terapia Trombolítica , Factores de Tiempo , Tiempo de TratamientoRESUMEN
BACKGROUND: Colon cancer is invariably accompanied by altered coagulation activity; however, the precise role of phosphatidylserine (PS) in the hypercoagulable state of colon cancer patients remains unclear. We explored the exposure of PS on platelets and microparticles (MPs), and evaluate its role in procoagulant activity in colon cancer patients. METHODS: PS-positive platelets and MPs, mainly from platelets and endothelial cells, were detected by flow cytometry and confocal microscopy, and their procoagulant activity was assessed with purified coagulation complex assays, clotting time, and fibrin turbidity. RESULTS: Plasma levels of PS-positive platelets increased gradually from stage I to IV and were higher in all stages of the patients than in the healthy control, while PS-positive platelet-derived MPs only increased significantly in stage III/IV patients. Meanwhile, PS-positive MPs and endothelial-derived MPs in stage II/III/IV patients were markedly higher than ones in controls but no difference with stage I. Tissue factor positive MPs were higher in all 4 stages of colon cancer patients than in the healthy control. Platelets and MPs from the patients demonstrated significantly enhanced intrinsic/extrinsic FXa and thrombin generation, greatly shortened coagulation time, and increased fibrin formation. Combined treatment with PS antagonist lactadherin, strongly prolonged the coagulation time and reduced fibrin formation by inhibiting factor tenase and prothrombinase complex activity. In contrast, pretreatment with anti tissue factor antibody played a lesser role in suppression of procoagulant activity. CONCLUSION: Our results suggest that PS-positive platelets and MPs contribute to hypercoagulability and represent a potential therapeutic target to prevent coagulation in patients with colon cancer.
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Plaquetas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Neoplasias del Colon/patología , Fosfatidilserinas/farmacología , Adulto , Anciano , Anciano de 80 o más Años , Plaquetas/patología , Micropartículas Derivadas de Células/patología , Neoplasias del Colon/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Trombina/metabolismo , Tromboplastina/metabolismoRESUMEN
Sepsis is invariably accompanied by altered coagulation cascade; however, the precise role of phosphatidylserine (PS) in inflammation-associated coagulopathy in sepsis has not been well elucidated. We explored the possibility of exposed PS on microparticles (MPs), blood cells, as well as on endothelium, and defined its role in procoagulant activity (PCA) in sepsis. PS-positive MPs and cells were detected by flow cytometry, while PCA was assessed with clotting time, purified coagulation complex, and fibrin formation assays. Plasma levels of PS MPs derived from platelets, leukocytes (including neutrophils, monocytes, and lymphocytes), erythrocytes, and endothelial cells were elevated by 1.49-, 1.60-, 2.93-, and 1.53-fold, respectively, in septic patients. Meanwhile, PS exposure on blood cells was markedly higher in septic patients than in controls. Additionally, we found that the endothelial cells (ECs) treated with septic serum in vitro exposed more PS than with healthy serum. Isolated MPs/blood cells from septic patients and cultured ECs treated with septic serum in vitro demonstrated significantly shortened coagulation time, greatly enhanced intrinsic/extrinsic FXa generation, and increased thrombin formation. Importantly, confocal imaging of MPs or septic serum-treated ECs identified binding sites for FVa and FXa to form prothrombinase, and further supported fibrin formation in the area where PS exposure was abundant. Pretreatment with lactadherin blocked PS on MPs/blood cells/ECs, prolonged coagulation time by at least 25%, reduced FXa/thrombin generation, and inhibited fibrin formation by approximately 85%. Our findings suggest a key role for PS exposed on MPs, blood cells, and endothelium in augmenting coagulation in sepsis. Therefore, therapies targeting PS may be of particular importance.
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Células Sanguíneas/metabolismo , Micropartículas Derivadas de Células/metabolismo , Endotelio Vascular/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Fosfatidilserinas/farmacología , Sepsis/metabolismo , Adulto , Anciano , Células Sanguíneas/patología , Micropartículas Derivadas de Células/patología , Endotelio Vascular/patología , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Masculino , Persona de Mediana Edad , Sepsis/patologíaRESUMEN
BACKGROUND: Relatively little information is available about phosphatidylserine positive (PS(+)) microparticles (MPs) and their originating cells in IgA nephropathy (IgAN) despite well-established intraglomerular coagulation. Our objectives were to detect PS exposure on MP membranes and MP-origin cells and to evaluate its role in procoagulant activity (PCA) and fibrin formation and their association with pathological lesions in the disease. METHODS: Patients with IgAN and healthy controls were studied. Lactadherin was used to quantify PS exposure on MPs and MP-origin cells. PCA of MPs and MP-origin cells was evaluated by clotting time and purified coagulation complex assays. Fibrin production was determined by turbidity. PS exposure, fibrin strands and FVa/Xa binding were observed on MPs/cells using confocal microscopy. RESULTS: Using flow cytometry, we found that IgAN patients had high levels of PS(+) MPs derived from lymphocytes, monocytes, neutrophils, platelets, erythrocytes and endothelial cells (ECs). The PS exposure on MP-origin cells also increased in these patients. MPs and MP-origin cells (leukocytes, platelets and erythrocytes) isolated from IgAN patients and ECs cultured with IgAN serum had a significantly shorter median coagulation time (P < 0.001), higher median intrinsic FXa (P < 0.001) and higher thrombin (P < 0.001) generation than controls. These coagulation functional assays were associated with the glomerular lesions. The lesions were also correlated with glomerular fibrin deposition (all P < 0.05). In the presence of patient MPs or their related cells, fibrin formation peaked faster with a higher maximum turbidity when compared with healthy controls. Blocking PS with lactadherin in the IgAN group prolonged coagulation time to control levels, inhibited the PCA up to 80% and markedly reduced fibrin formation. More importantly, we observed that fibrin strands formed on MPs and ECs in the same regions that bound lactadherin, similar to the FVa/Xa costaining. CONCLUSIONS: We find that high levels of PS(+) MPs and the MP-origin cells are associated with the coagulation process in IgAN, and this may provide a previously unrecognized contribution to intraglomerular coagulation.