RESUMO
BACKGROUND: Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. METHODS: Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. RESULTS: External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. CONCLUSIONS: The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.
RESUMO
The lipid envelope of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an essential component of the virus; however, its molecular composition is undetermined. Addressing this knowledge gap could support the design of antiviral agents as well as further our understanding of viral-host protein interactions, infectivity, pathogenicity, and innate immune system clearance. Lipidomics revealed that the virus envelope comprised mainly phospholipids (PLs), with some cholesterol and sphingolipids, and with cholesterol/phospholipid ratio similar to lysosomes. Unlike cellular membranes, procoagulant amino-PLs were present on the external side of the viral envelope at levels exceeding those on activated platelets. Accordingly, virions directly promoted blood coagulation. To investigate whether these differences could enable selective targeting of the viral envelope in vivo, we tested whether oral rinses containing lipid-disrupting chemicals could reduce infectivity. Products containing PL-disrupting surfactants (such as cetylpyridinium chloride) met European virucidal standards in vitro; however, components that altered the critical micelle concentration reduced efficacy, and products containing essential oils, povidone-iodine, or chlorhexidine were ineffective. This result was recapitulated in vivo, where a 30-s oral rinse with cetylpyridinium chloride mouthwash eliminated live virus in the oral cavity of patients with coronavirus disease 19 for at least 1 h, whereas povidone-iodine and saline mouthwashes were ineffective. We conclude that the SARS-CoV-2 lipid envelope i) is distinct from the host plasma membrane, which may enable design of selective antiviral approaches; ii) contains exposed phosphatidylethanolamine and phosphatidylserine, which may influence thrombosis, pathogenicity, and inflammation; and iii) can be selectively targeted in vivo by specific oral rinses.
Assuntos
COVID-19 , Antissépticos Bucais , Antivirais , Cetilpiridínio , Humanos , Lipídeos , Antissépticos Bucais/farmacologia , Povidona-Iodo , RNA Viral , SARS-CoV-2RESUMO
Abdominal aortic aneurysm (AAA) is an inflammatory vascular disease with high mortality and limited treatment options. How blood lipids regulate AAA development is unknown. Here lipidomics and genetic models demonstrate a central role for procoagulant enzymatically oxidized phospholipids (eoxPL) in regulating AAA. Specifically, through activating coagulation, eoxPL either promoted or inhibited AAA depending on tissue localization. Ang II administration to ApoE-/- mice increased intravascular coagulation during AAA development. Lipidomics revealed large numbers of eoxPL formed within mouse and human AAA lesions. Deletion of eoxPL-generating enzymes (Alox12 or Alox15) or administration of the factor Xa inhibitor rivaroxaban significantly reduced AAA. Alox-deficient mice displayed constitutively dysregulated hemostasis, including a consumptive coagulopathy, characterized by compensatory increase in prothrombotic aminophospholipids (aPL) in circulating cell membranes. Intravenously administered procoagulant PL caused clotting factor activation and depletion, induced a bleeding defect, and significantly reduced AAA development. These data suggest that Alox deletion reduces AAA through diverting coagulation away from the vessel wall due to eoxPL deficiency, instead activating clotting factor consumption and depletion in the circulation. In mouse whole blood, â¼44 eoxPL molecular species formed within minutes of clot initiation. These were significantly elevated with ApoE-/- deletion, and many were absent in Alox-/- mice, identifying specific eoxPL that modulate AAA. Correlation networks demonstrated eoxPL belonged to subfamilies defined by oxylipin composition. Thus, procoagulant PL regulate AAA development through complex interactions with clotting factors. Modulation of the delicate balance between bleeding and thrombosis within either the vessel wall or circulation was revealed that can either drive or prevent disease development.
Assuntos
Aorta Abdominal/fisiopatologia , Aneurisma da Aorta Abdominal , Fosfolipídeos , Angiotensinas/metabolismo , Animais , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/fisiopatologia , Fatores de Coagulação Sanguínea/genética , Fatores de Coagulação Sanguínea/metabolismo , Modelos Animais de Doenças , Feminino , Lipoxigenase/genética , Lipoxigenase/metabolismo , Masculino , Camundongos , Camundongos Knockout para ApoE , Fosfolipídeos/genética , Fosfolipídeos/metabolismoRESUMO
OBJECTIVE: Recent studies have demonstrated that galectin-1 (Gal-1) and galectin-3 (Gal-3) can bind von Willebrand factor and directly modulate von Willebrand factor-dependent early thrombus formation in vivo. Because the glycans expressed on human factor VIII (FVIII) are similar to those of von Willebrand factor, we investigated whether galectins might also bind and modulate the activity of FVIII. APPROACH AND RESULTS: Immunosorbant assays and surface plasmon resonance analysis confirmed that Gal-1 and Gal-3 bound purified FVIII with high affinity. Exoglycosidase removal of FVIII N-linked glycans significantly reduced binding to both Gal-1 and Gal-3. Moreover, combined removal of both the N- and O-glycans of FVIII further attenuated Gal-3 binding. Notably, specific digestion of FVIII high-mannose glycans at N239 and N2118 significantly impaired FVIII affinity for Gal-1. Importantly Gal-1, but not Gal-3, bound to free FVIII in the plasma milieu, and significantly inhibited FVIII functional activity. Interestingly, commercial recombinant FVIII (rFVIII) concentrates are manufactured in different cell lines and differ in their glycosylation profiles. Although the biological mechanism has not been defined, recent studies in previously untreated patients with severe hemophilia A reported significant differences in inhibitor development associated with different rFVIII products. Interestingly, Gal-1 and Gal-3 both displayed enhanced affinity for BHK-rFVIII compared with CHO-rFVIII. Furthermore, binding of Gal-1 and Gal-3 to BDD-FVIII was markedly reduced compared with full-length rFVIII. CONCLUSIONS: We have identified Gal-1 and Gal-3 as novel-binding partners for human FVIII and demonstrated that Gal-1 binding can influence the procoagulant activity of FVIII.
Assuntos
Fator VIII/metabolismo , Galectina 1/metabolismo , Galectina 3/metabolismo , Animais , Sítios de Ligação , Coagulação Sanguínea , Proteínas Sanguíneas , Células CHO , Cricetulus , Fator VIII/química , Fator VIII/genética , Galectina 1/química , Galectina 3/química , Galectinas , Glicosilação , Humanos , Tempo de Tromboplastina Parcial , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
Modern thrombophilia testing fails to identify any underlying prothrombotic tendency in a significant number of patients presenting with objectively confirmed venous thromboemboembolism (VTE). This observation has led to a search for other novel inherited or acquired human thrombophilias. Although a number of putative mechanisms have been described, the evidence behind many of these candidates remains weak. In contrast, an increasing body of work supports the hypothesis that increased plasma factor VIII (FVIII) levels may be important in this context. An association between elevated plasma FVIII levels and VTE was first described in the Leiden Thrombophilia Study (LETS). Subsequently, these conclusions have been supported by an increasing number of independent case-control studies. Cumulatively, these studies have clearly demonstrated that high FVIII levels constitute a prevalent, dose-dependent risk factor for VTE. Furthermore, more recent studies have shown that the risk of recurrent venous thrombosis is also significantly increased in patients with high FVIII levels. In this review, we present the evidence supporting the hypothesis that elevated FVIII levels constitute a clinically important thrombophilia. In addition, we examine the biological mechanisms that may underlie persistently elevated FVIII levels, and the pathways through which high FVIII may serve to increase thrombotic risk.
Assuntos
Fator VIII/metabolismo , Tromboembolia Venosa/sangue , Trombose Venosa/sangue , Estudos de Casos e Controles , Fator IXa/genética , Fator IXa/metabolismo , Fator VIII/genética , Hemofilia A/sangue , Hemofilia A/genética , Hemostasia/genética , Humanos , Fatores de Risco , Tromboembolia Venosa/genética , Trombose Venosa/genéticaRESUMO
Phospholipids (PLs) are found in all cell types and are required for structural support and cell activation signalling pathways. In resting cells, PLs are asymmetrically distributed throughout the plasma membrane with native procoagulant aminophospholipids (aPLs) being actively maintained in the inner leaflet of the membrane. Upon platelet activation, aPLs rapidly externalize to the outer leaflet and are essential for supporting the coagulation cascade by providing binding sites for factors in the cell-based model. More recent work has uncovered a role for enzymatically oxidized PLs (eoxPLs) in facilitating coagulation, working in concert with native aPLs. Despite this, the role of aPLs and eoxPLs in thrombo-inflammatory conditions, such as arterial and venous thrombosis, has not been fully elucidated. In this review, we describe the biochemical structures, distribution and regulation of aPL externalization and summarize the literature on eoxPL generation in circulating blood cells. We focus on the currently understood role of these lipids in mediating coagulation reactions in vitro, in vivo and in human thrombotic disease. Finally, we highlight gaps in our understanding in how these lipids vary in health and disease, which may place them as future therapeutic targets for the management of thrombo-inflammatory conditions.
Assuntos
Fosfolipídeos , Trombose , Células Sanguíneas/metabolismo , Coagulação Sanguínea , Hemostasia , Humanos , Fosfolipídeos/química , Fosfolipídeos/metabolismoRESUMO
Plasmodium falciparum infection results in adhesion of infected erythrocytes to blood vessel endothelium, and acute endothelial cell activation, together with sequestration of platelets and leucocytes. We have previously shown that patients with severe infection or fulminant cerebral malaria have significantly increased circulatory levels of the adhesive glycoprotein von Willebrand factor (VWF) and its propeptide, both of which are indices of endothelial cell activation. In this prospective study of patients from Ghana with severe (n = 20) and cerebral (n = 13) P. falciparum malaria, we demonstrate that increased plasma VWF antigen (VWF:Ag) level is associated with disproportionately increased VWF function. VWF collagen binding (VWF:CB) was significantly increased in patients with cerebral malaria and severe malaria (medians 7.6 and 7.0 IU/ml versus 1.9 IU/ml; p<0.005). This increased VWF:CB correlated with the presence of abnormal ultra-large VWF multimers in patient rather than control plasmas. Concomitant with the increase in VWF:Ag and VWF:CB was a significant persistent reduction in the activity of the VWF-specific cleaving protease ADAMTS13 (approximately 55% of normal; p<0.005). Mixing studies were performed using P. falciparum patient plasma and normal pooled plasma, in the presence or absence of exogenous recombinant ADAMTS13. These studies demonstrated that in malarial plasma, ADAMTS13 function was persistently inhibited in a time-dependent manner. Furthermore, this inhibitory effect was not associated with the presence of known inhibitors of ADAMTS13 enzymatic function (interleukin-6, free haemoglobin, factor VIII or thrombospondin-1). These novel findings suggest that severe P. falciparum infection is associated with acute endothelial cell activation, abnormal circulating ULVWF multimers, and a significant reduction in plasma ADAMTS13 function which is mediated at least in part by an unidentified inhibitor.
Assuntos
Proteínas ADAM/metabolismo , Células Endoteliais/metabolismo , Malária Falciparum/metabolismo , Fator de von Willebrand/metabolismo , Proteína ADAMTS13 , Criança , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Humanos , Lactente , Multimerização ProteicaRESUMO
Previous studies have suggested that development of inhibitors in previously treated patients (PTPs) may be attributable to a switch in factor VIII (FVIII) therapeutic product. Consequently, it is widely recognized that inhibitor development must be assessed in PTPs following the introduction of any new FVIII product. Following a national tender process in 2006, all patients with haemophilia A in Ireland changed their FVIII treatment product en masse to a plasma and albumin-free recombinant full-length FVIII product (ADVATE(®)). In this study, we retrospectively reviewed the case records of Irish PTPs to evaluate risk of inhibitor formation following this treatment switch. One hundred and thirteen patients participated in the study. Most patients (89%) had severe haemophilia. Only one of 96 patients with no inhibitor history developed an inhibitor. Prior to the switch in his recombinant FVIII (rFVIII) treatment of choice, this child had only experienced three exposure days (EDs). Consequently, in total he had only received 6 EDs when his inhibitor was first diagnosed. In keeping with this lack of de novo inhibitor development, we observed no evidence of any recurrent inhibitor formation in any of 16 patients with previously documented inhibitors. Similarly, following a previous en masse switch, we have previously reported that changing from a Chinese hamster ovary cell-produced to a baby hamster kidney cell-produced rFVIII was also associated with a low risk of inhibitor formation in PTPs. Our cumulative findings from these two studies clearly emphasizes that the risk of inhibitor development for PTPs following changes in commercial rFVIII product is low, at least in the Irish population.
Assuntos
Autoanticorpos/análise , Inibidores dos Fatores de Coagulação Sanguínea/sangue , Fator VIII/imunologia , Fator VIII/uso terapêutico , Hemofilia A/tratamento farmacológico , Adolescente , Adulto , Criança , Pré-Escolar , Hemofilia A/sangue , Humanos , Proteínas Recombinantes/sangue , Estudos Retrospectivos , Albumina Sérica , Adulto JovemRESUMO
INTRODUCTION: Low molecular weight heparins (LMWHs) are used worldwide for the treatment and prophylaxis of thromboembolic disorders. Routine laboratory tests are not required due to the predictable pharmacokinetics of LMWHs, with the exception of pregnant patients, children, patients with renal failure, morbid obesity, or advanced age. Anti-Factor Xa (anti-FXa) plasma levels are most often employed in the assessment and guidance of accurate dosing in these patient cohorts. MATERIALS AND METHODS: A LMWH calibration curve was generated using citrated human pooled plasma spiked with pharmacologically relevant concentrations (0-1.2U/ml) of two low molecular weight heparins; enoxaparin and tinzaparin. Least squares analysis determined the best curve fit for this set of data which returned low sum of squares (SS) values for the log linear fit with an R(2) value of 0.98. 30 patient samples were tested in the fluorogenic assay and concentrations were determined using the log linear regression equation and correlated with a standard chromogenic assay used for heparin monitoring. RESULTS: A statistically significant correlation was found between the fluorogenic and the chromogenic anti-FXa assays for 30 patient samples, with a slope of 0.829, offset of 0.258 and an R(2) value of 0.72 (p<0.0001). CONCLUSIONS: In the study presented here, a fluorogenic anti-FXa assay was correlated with a standard laboratory chromogenic anti-FXa assay using samples from patients on LMWH therapy. Significant correlations between the values derived by the fluorogenic and chromogenic anti-FXa assays were found for the patient cohort tested in this study.
Assuntos
Anticoagulantes/sangue , Enoxaparina/química , Inibidores do Fator Xa , Heparina de Baixo Peso Molecular/sangue , Técnicas de Química Analítica , Relação Dose-Resposta a Droga , Fator Xa/química , Fator Xa/isolamento & purificação , Corantes Fluorescentes , HumanosRESUMO
Platelet factor 4 (PF4) is an abundant platelet alpha-granule chemokine released following platelet activation. PF4 interacts with thrombomodulin and the gamma-carboxyglutamic acid (Gla) domain of protein C, thereby enhancing activated protein C (APC) generation by the thrombin-thrombomodulin complex. However, the protein C Gla domain not only mediates protein C activation in vivo, but also plays a critical role in modulating the diverse functional properties of APC once generated. In this study we demonstrate that PF4 significantly inhibits APC anti-coagulant activity. PF4 inhibited both protein S-dependent APC anticoagulant function in plasma and protein S-dependent factor Va (FVa) proteolysis 3- to 5-fold, demonstrating that PF4 impairs protein S cofactor enhancement of APC anticoagulant function. Using recombinant factor Va variants FVa-R506Q/R679Q and FVa-R306Q/R679Q, PF4 was shown to impair APC proteolysis of FVa at position Arg(306) by 3-fold both in the presence and absence of protein S. These data suggest that PF4 contributes to the poorly understood APC resistance phenotype associated with activated platelets. Finally, despite PF4 binding to the APC Gla domain, we show that APC in the presence of PF4 retains its ability to initiate PAR-1-mediated cytoprotective signaling. In summary, we propose that PF4 acts as a critical regulator of APC generation, but also differentially targets APC toward cytoprotective, rather than anticoagulant function at sites of vascular injury with concurrent platelet activation.
Assuntos
Anticoagulantes/farmacologia , Fator Plaquetário 4/metabolismo , Proteína C/metabolismo , Apoptose/fisiologia , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Fator Va/metabolismo , Brometo de Hexadimetrina/farmacologia , Humanos , Fator Plaquetário 4/genética , Protaminas/farmacologia , Proteína C/antagonistas & inibidores , Proteína S/metabolismo , Trombina/metabolismoRESUMO
For many years, an association between ABO histo-blood group and risk of thrombosis has been recognized. Blood group non-O (A, B, and AB) individuals have consistently been found to demonstrate increased incidence of both arterial and venous thrombotic disease, compared to group O individuals. This increased risk is attributable to the fact that ABO blood group influences plasma levels of a coagulation glycoprotein named von Willebrand factor (VWF). VWF levels are 25 percent higher in non-O compared to group O individuals. The mechanism by which ABO group determines plasma VWF levels has not been determined. ABO(H) carbohydrate antigenic determinants, however, are expressed on the N-linked glycan chains of circulating plasma VWF. This review will focus on the carbohydrate structures of VWF and recent studies suggesting that subtle variations in these structures (particularly differences in ABO blood group antigen expression) may have clinically significant effects on VWF proteolysis and clearance.