RESUMO
Protein S (PS), the critical plasma cofactor for the anticoagulants tissue factor (TF) pathway inhibitor (TFPI) and activated protein C (APC), circulates in two functionally distinct pools: free (anticoagulant) or bound to complement component 4b-binding protein (C4BP) (anti-inflammatory). Acquired free PS deficiency is detected in several viral infections, but its cause is unclear. Here, we identified a shear-dependent interaction between PS and von Willebrand Factor (VWF) by mass spectrometry. Consistently, plasma PS and VWF comigrated in both native and agarose gel electrophoresis. The PS/VWF interaction was blocked by TFPI but not APC, suggesting an interaction with the C-terminal sex hormone binding globulin (SHBG) region of PS. Microfluidic systems, mimicking arterial laminar flow or disrupted turbulent flow, demonstrated that PS stably binds VWF as VWF unfolds under turbulent flow. PS/VWF complexes also localized to platelet thrombi under laminar arterial flow. In thrombin generation-based assays, shearing plasma decreased PS activity, an effect not seen in the absence of VWF. Finally, free PS deficiency in COVID-19 patients, measured using an antibody that binds near the C4BP binding site in SHBG, correlated with changes in VWF, but not C4BP, and with thrombin generation. Our data suggest that PS binds to a shear-exposed site on VWF, thus sequestering free PS and decreasing its anticoagulant activity, which would account for the increased thrombin generation potential. As many viral infections present with free PS deficiency, elevated circulating VWF, and increased vascular shear, we propose that the PS/VWF interaction reported here is a likely contributor to virus-associated thrombotic risk.
RESUMO
Antithrombotic therapy is a delicate balance between the benefits of preventing a thrombotic event and the risks of inducing a major bleed. Traditional approaches have included antiplatelet and anticoagulant medications, require careful dosing and monitoring, and all carry some risk of bleeding. In recent years, several new targets have been identified, both in the platelet and coagulation systems, which may mitigate this bleeding risk. In this review, we briefly describe the current state of antithrombotic therapy, and then present a detailed discussion of the new generation of drugs that are being developed to target more safely existing or newly identified pathways, alongside the strategies to reverse direct oral anticoagulants, showcasing the breadth of approaches. Combined, these exciting advances in antithrombotic therapy bring us closer than we have ever been to the "holy grail" of the field, a treatment that separates the hemostatic and thrombotic systems, preventing clots without any concurrent bleeding risk.
RESUMO
Platelets contribute to COVID-19 clinical manifestations, of which microclotting in the pulmonary vasculature has been a prominent symptom. To investigate the potential diagnostic contributions of overall platelet morphology and their α-granules and mitochondria to the understanding of platelet hyperactivation and micro-clotting, we undertook a 3D ultrastructural approach. Because differences might be small, we used the high-contrast, high-resolution technique of focused ion beam scanning EM (FIB-SEM) and employed deep learning computational methods to evaluate nearly 600 individual platelets and 30 000 included organelles within three healthy controls and three severely ill COVID-19 patients. Statistical analysis reveals that the α-granule/mitochondrion-to-plateletvolume ratio is significantly greater in COVID-19 patient platelets indicating a denser packing of organelles, and a more compact platelet. The COVID-19 patient platelets were significantly smaller -by 35% in volume - with most of the difference in organelle packing density being due to decreased platelet size. There was little to no 3D ultrastructural evidence for differential activation of the platelets from COVID-19 patients. Though limited by sample size, our studies suggest that factors outside of the platelets themselves are likely responsible for COVID-19 complications. Our studies show how deep learning 3D methodology can become the gold standard for 3D ultrastructural studies of platelets.
COVID-19 patients exhibit a range of symptoms including microclotting. Clotting is a complex process involving both circulating proteins and platelets, a cell within the blood. Increased clotting is suggestive of an increased level of platelet activation. If this were true, we reasoned that parts of the platelet involved in the release of platelet contents during clotting would have lost their content and appear as expanded, empty "ghosts." To test this, we drew blood from severely ill COVID-19 patients and compared the platelets within the blood draws to those from healthy volunteers. All procedures were done under careful attention to biosafety and approved by health authorities. We looked within the platelets for empty ghosts by the high magnification technique of electron microscopy. To count the ghosts, we developed new computer software. In the end, we found little difference between the COVID patient platelets and the healthy donor platelets. The results suggest that circulating proteins outside of the platelet are more important to the strong clotting response. The software developed will be used to analyze other disease states.
Assuntos
COVID-19 , Aprendizado Profundo , Humanos , RNA Viral , SARS-CoV-2 , Plaquetas/ultraestrutura , OrganelasRESUMO
Background: Patients with COVID-19 have a higher risk of thrombosis and thromboembolism, but the underlying mechanism(s) remain to be fully elucidated. In patients with COVID-19, high lipoprotein(a) (Lp(a)) is positively associated with the risk of ischemic heart disease. Lp(a), composed of an apoB-containing particle and apolipoprotein(a) (apo(a)), inhibits the key fibrinolytic enzyme, tissue-type plasminogen activator (tPA). However, whether the higher Lp(a) associates with lower tPA activity, the longitudinal changes of these parameters in hospitalized patients with COVID-19, and their correlation with clinical outcomes are unknown. Objectives: To assess if Lp(a) associates with lower tPA activity in COVID-19 patients, and how in COVID-19 populations Lp(a) and tPA change post infection. Methods: Endogenous tPA enzymatic activity, tPA or Lp(a) concentration were measured in plasma from hospitalized patients with and without COVID-19. The association between plasma tPA and adverse clinical outcomes was assessed. Results: In hospitalized patients with COVID-19, we found lower tPA enzymatic activity and higher plasma Lp(a) than that in non-COVID-19 controls. During hospitalization, Lp(a) increased and tPA activity decreased, which associates with mortality. Among those who survived, Lp(a) decreased and tPA enzymatic activity increased during recovery. In patients with COVID-19, tPA activity is inversely correlated with tPA concentrations, thus, in another larger COVID-19 cohort, we utilized plasma tPA concentration as a surrogate to inversely reflect tPA activity. The tPA concentration was positively associated with death, disease severity, plasma inflammatory, and prothrombotic markers, and with length of hospitalization among those who were discharged. Conclusion: High Lp(a) concentration provides a possible explanation for low endogenous tPA enzymatic activity, and poor clinical outcomes in patients with COVID-19.
RESUMO
Systemic blood coagulation accompanies inflammation during severe infection like sepsis and COVID. We've previously established a link between pyroptosis, a vital defense mechanism against infection, and coagulopathy. During pyroptosis, the formation of gasdermin-D (GSDMD) pores on the plasma membrane leads to the release of tissue factor (TF)-positive microvesicles (MVs) that are procoagulant. Mice lacking GSDMD release fewer TF MVs. However, the specific mechanisms leading from activation of GSDMD to MV release remain unclear. Plasma membrane rupture (PMR) in pyroptosis was recently reported to be actively mediated by the transmembrane protein Ninjurin-1 (NINJ1). Here we show that NINJ1 promotes procoagulant MV release during pyroptosis. Haploinsuffciency or glycine inhibition of NINJ1 limited the release of procoagulant MVs and inflammatory cytokines and protected against blood coagulation and lethality triggered by bacterial flagellin. Our findings suggest a crucial role for NINJ1-dependent PMR in inflammasome-induced blood coagulation and inflammation.
RESUMO
INTRODUCTION: Thrombin, the enzyme which converts fibrinogen into a fibrin clot, is produced by the prothrombinase complex, composed of factor Xa (FXa) and factor Va (FVa). Down-regulation of this process is critical, as excess thrombin can lead to life-threatening thrombotic events. FXa and FVa are inhibited by the anticoagulants tissue factor pathway inhibitor alpha (TFPIα) and activated protein C (APC), respectively, and their common cofactor protein S (PS). However, prothrombinase is resistant to either of these inhibitory systems in isolation. MATERIALS AND METHODS: We hypothesized that these anticoagulants function best together, and tested this hypothesis using purified proteins and plasma-based systems. RESULTS: In plasma, TFPIα had greater anticoagulant activity in the presence of APC and PS, maximum PS activity required both TFPIα and APC, and antibodies against TFPI and APC had an additive procoagulant effect, which was mimicked by an antibody against PS alone. In purified protein systems, TFPIα dose-dependently inhibited thrombin activation by prothrombinase, but only in the presence of APC, and this activity was enhanced by PS. Conversely, FXa protected FVa from cleavage by APC, even in the presence of PS, and TFPIα reversed this protection. However, prothrombinase assembled on platelets was still protected from inhibition, even in the presence of TFPIα, APC, and PS. CONCLUSIONS: We propose a model of prothrombinase inhibition through combined targeting of both FXa and FVa, and that this mechanism enables down-regulation of thrombin activation outside of a platelet clot. Platelets protect prothrombinase from inhibition, however, supporting a procoagulant environment within the clot.
Assuntos
Proteína C , Proteína S , Trombina , Humanos , Anticoagulantes , Coagulação Sanguínea , Fator V/metabolismo , Fator Va/metabolismo , Fator Xa/metabolismo , Proteína C/metabolismo , Proteína S/metabolismo , Trombina/metabolismo , Tromboplastina/metabolismoRESUMO
OBJECTIVES: To investigate whether COVID-19 patients with pulmonary embolism had higher mortality and assess the utility of D-dimer in predicting acute pulmonary embolism. PATIENTS AND METHODS: Using the National Collaborative COVID-19 retrospective cohort, a cohort of hospitalized COVID-19 patients was studied to compare 90-day mortality and intubation outcomes in patients with and without pulmonary embolism in a multivariable cox regression analysis. The secondary measured outcomes in 1:4 propensity score-matched analysis included length of stay, chest pain incidence, heart rate, history of pulmonary embolism or DVT, and admission laboratory parameters. RESULTS: Among 31,500 hospitalized COVID-19 patients, 1117 (3.5%) patients were diagnosed with acute pulmonary embolism. Patients with acute pulmonary embolism were noted to have higher mortality (23.6% vs.12.8%; adjusted Hazard Ratio (aHR) = 1.36, 95% CI [1.20-1.55]), and intubation rates (17.6% vs. 9.3%, aHR = 1.38[1.18-1.61]). Pulmonary embolism patients had higher admission D-dimer FEU (Odds Ratio(OR) = 1.13; 95%CI [1.1-1.15]). As the D-dimer value increased, the specificity, positive predictive value, and accuracy of the test increased; however, sensitivity decreased (AUC 0.70). At cut-off D-dimer FEU 1.8 mcg/ml, the test had clinical utility (accuracy 70%) in predicting pulmonary embolism. Patients with acute pulmonary embolism had a higher incidence of chest pain and history of pulmonary embolism or deep vein thrombosis. CONCLUSIONS: Acute pulmonary embolism is associated with worse mortality and morbidity outcomes in COVID-19. We present D-dimer as a predictive risk tool in the form of a clinical calculator for the diagnosis of acute pulmonary embolism in COVID-19.
Assuntos
COVID-19 , Embolia Pulmonar , Humanos , Estudos Retrospectivos , Embolia Pulmonar/diagnóstico , Valor Preditivo dos Testes , Dor no PeitoRESUMO
Coronavirus Disease 2019 (COVID-19) has been widely associated with increased thrombotic risk, with many different proposed mechanisms. One such mechanism is acquired deficiency of protein S (PS), a plasma protein that regulates coagulation and inflammatory processes, including complement activation and efferocytosis. Acquired PS deficiency is common in patients with severe viral infections and has been reported in multiple studies of COVID-19. This deficiency may be caused by consumption, degradation, or clearance of the protein, by decreased synthesis, or by binding of PS to other plasma proteins, which block its anticoagulant activity. Here, we review the functions of PS, the evidence of acquired PS deficiency in COVID-19 patients, the potential mechanisms of PS deficiency, and the evidence that those mechanisms may be occurring in COVID-19.
Assuntos
COVID-19 , Deficiência de Proteína S , Proteína S , Trombose , Humanos , COVID-19/complicações , COVID-19/genética , COVID-19/metabolismo , Proteína S/genética , Proteína S/metabolismo , Deficiência de Proteína S/complicações , Deficiência de Proteína S/metabolismo , Trombose/complicaçõesRESUMO
Histones are cationic nuclear proteins that are essential for the structure and functions of eukaryotic chromatin. However, extracellular histones trigger inflammatory responses and contribute to death in sepsis by unknown mechanisms. We recently reported that inflammasome activation and pyroptosis trigger coagulation activation through a tissue-factor (TF)-dependent mechanism. We used a combination of various deficient mice to elucidate the molecular mechanism of histone-induced coagulation. We showed that histones trigger coagulation activation in vivo, as evidenced by coagulation parameters and fibrin deposition in tissues. However, histone-induced coagulopathy was neither dependent on intracellular inflammasome pathways involving caspase 1/11 and gasdermin D (GSDMD), nor on cell surface receptor TLR2- and TLR4-mediated host immune response, as the deficiency of these genes in mice did not protect against histone-induced coagulopathy. The incubation of histones with macrophages induced lytic cell death and phosphatidylserine (PS) exposure, which is required for TF activity, a key initiator of coagulation. The neutralization of TF diminished the histone-induced coagulation. Our findings revealed lytic cell death as a novel mechanism of histone-induced coagulation activation and thrombosis.
Assuntos
Coagulação Intravascular Disseminada , Animais , Coagulação Intravascular Disseminada/etiologia , Histonas , Inflamassomos/metabolismo , Camundongos , Piroptose , Tromboplastina/metabolismoRESUMO
Background: Traumatic brain injury (TBI) results in neurovascular damage that initiates intrinsic mechanisms of hypercoagulation, which can contribute to the development of life-threatening complications, such as coagulopathy and delayed thrombosis. Clinical studies have hypothesized that tissue factor (TF) induces hypercoagulability after TBI; however, none have directly shown this relationship. Objectives: In the current study, we took a stepwise approach to understand what factors are driving thrombin generation following experimental TBI. Methods: We employed the contusion-producing controlled cortical impact (CCI) model and the diffuse closed head injury (CHI) model to investigate these mechanisms as a function of injury severity and modality. Whole blood was collected at 6 hours and 24 hours after injury, and platelet-poor plasma was used to measure thrombin generation and extracellular vesicle (EV) TF. Results: We found that plasma thrombin generation, dependent on TF present in the plasma, was greater in CCI-injured animals compared to sham at both 6 hours (120.4 ± 36.9 vs 0.0 ± 0.0 nM*min endogenous thrombin potential) and 24 hours (131.0 ± 34.0 vs 32.1 ± 20.6 nM*min) after injury. This was accompanied by a significant increase in EV TF at 24 hours (328.6 ± 62.1 vs 167.7 ± 20.8 fM) after CCI. Further, EV TF is also increased at 6 hours (126.6 ± 17.1 vs 63.3 ± 14.4 fM) but not 24 hours following CHI. Conclusion: TF-mediated thrombin generation is time-dependent after injury and TF increases resolve earlier following CHI as compared to CCI. Taken together, these data support a TF-mediated pathway of thrombin generation after TBI and pinpoint TF as a major player in TBI-induced coagulopathy.
RESUMO
BACKGROUND: HIV-1 infection is associated with multiple procoagulant changes and increased thrombotic risk. Possible mechanisms for this risk include heigthened expression of procoagulant tissue factor (TF) on circulating monocytes, extracellular vesicles, and viral particles and/or acquired deficiency of protein S (PS), a critical cofactor for the anticoagulant protein C (PC). PS deficiency occurs in up to 76% of people living with HIV-1 (PLWH). As increased ex vivo plasma thrombin generation is a strong predictor of mortality, we investigated whether PS and plasma TF are associated with plasma thrombin generation. METHODS: We analyzed plasma samples from 9 healthy controls, 17 PLWH on first diagnosis (naive), and 13 PLWH on antiretroviral therapy (ART). Plasma thrombin generation, total and free PS, PC, C4b-binding protein, and TF activity were measured. RESULTS: We determined that the plasma thrombin generation assay is insensitive to PS, because of a lack of PC activation, and developed a modified PS-sensitive assay. Total plasma PS was reduced in 58% of the naive and 38% of the ART-treated PLWH samples and correlated with increased thrombin generation in the modified assay. Conversely, plasma TF was not increased in our patient population, suggesting that it does not significantly contribute to ex vivo plasma thrombin generation. CONCLUSION: These data suggest that reduced total plasma PS contributes to the thrombotic risk associated with HIV-1 infection and can serve as a prothrombotic biomarker. In addition, our refined thrombin generation assay offers a more sensitive tool to assess the functional consequences of acquired PS deficiency in PLWH.
Assuntos
Infecções por HIV , Proteína S , Biomarcadores , Infecções por HIV/complicações , Infecções por HIV/tratamento farmacológico , Humanos , Trombina/metabolismo , TromboplastinaRESUMO
BACKGROUND: Serglycin (SRGN) is an intragranular, sulfated proteoglycan in hematopoietic cells that affects granule composition and function. OBJECTIVE: To understand how SRGN affects platelet granule packaging, cargo release, and extra-platelet microenvironments. METHODS: Platelets and megakaryocytes from SRGN-/- mice were assayed for secretion kinetics, cargo levels, granule morphology upon activation, and receptor shedding. RESULTS: Metabolic, 35 SO4 labeling identified SRGN as a major sulfated macromolecule in megakaryocytes. SRGN colocalized with α-granule markers (platelet factor 4 [PF4], von Willebrand factor [VWF], and P-selectin), but its deletion did not affect α-granule morphology or number. Platelet α-granule composition was altered, with a reduction in basic proteins (pI ≥8; e.g., PF4, SDF-1, angiogenin) and constitutive release of PF4 from SRGN-/- megakaryocytes. P-Selectin, VWF, and fibrinogen were unaffected. Serotonin (5-HT) uptake and ß-hexosaminidase (HEXB) were slightly elevated. Thrombin-induced exocytosis of PF4 from platelets was defective; however, release of RANTES/CCL5 was normal and osteopontin secretion was more rapid. Release of 5-HT and HEXB (from dense granules and lysosomes, respectively) were unaffected. Ultrastructural studies showed distinct morphologies in activated platelets. The α-granule lumen of SRGN-/- platelet had a grainy staining pattern, whereas that of wild-type granules had only fibrous material remaining. α-Granule swelling and decondensation were reduced in SRGN-/- platelets. Upon stimulation of platelets, a SRGN/PF4 complex was released in a time- and agonist-dependent manner. Shedding of GPVI from SRGN-/- platelets was modestly enhanced. Shedding of GP1b was unaffected. CONCLUSION: The polyanionic proteoglycan SRGN influences α-granule packaging, cargo release, and shedding of platelet membrane proteins.
Assuntos
Megacariócitos , Proteoglicanas , Animais , Plaquetas , Grânulos Citoplasmáticos , Camundongos , Proteínas de Transporte Vesicular/genéticaAssuntos
Coagulação Sanguínea , Plaquetas/metabolismo , Ativação Plaquetária , Trombina/metabolismo , Tromboplastina/metabolismo , Trombose/sangue , Animais , Anticoagulantes/uso terapêutico , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/efeitos dos fármacos , Fibrinolíticos/uso terapêutico , Humanos , Ativação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/uso terapêutico , Transdução de Sinais , Trombose/tratamento farmacológicoAssuntos
Malus , Pré-Calicreína , Fator XI , Fator XII , Fibrinolíticos , Calicreínas , Calicreína PlasmáticaRESUMO
Dyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction-induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI-mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet-fed conditions comparable to that seen in chow diet-fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.
Assuntos
Plaquetas/metabolismo , Antígenos CD36/metabolismo , Caspases/metabolismo , Fibrina/metabolismo , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Fosfatidilserinas/metabolismo , Animais , Plaquetas/citologia , Antígenos CD36/antagonistas & inibidores , Venenos de Crotalídeos/farmacologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Humanos , Hiperlipidemias/complicações , Hiperlipidemias/patologia , Lectinas Tipo C , Lipoproteínas LDL/farmacologia , Camundongos , Camundongos Knockout , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Ativação Plaquetária/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Trombose/etiologia , Trombose/patologia , Quinases da Família src/metabolismoRESUMO
This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would become NovoSeven, they sponsored a series of meetings to understand the basic biology and clinical applications of factor VIIa. The first meeting in Chapel Hill was held April 4-6, 2002 with Dr. Roberts as the organizer. Over the years, the conference emphasis has expanded from discussions of factor VIIa and tissue factor to additional topics in hemostasis and thrombosis. This year's meeting includes presentations by internationally renowned speakers that discuss the state-of-the-art on an array of important topics, including von Willebrand factor, engineering advances, coagulation and disease, tissue factor biology, therapeutic advances, and basic clotting factor biology. Included in this review article are illustrated abstracts provided by our speakers, which highlight the main conclusions of each invited talk. This will be the first meeting without Dr. Roberts in attendance, yet his commitment to excellent science and his focus on turning science to patient care are pervasively reflected in the presentations by our speakers.
RESUMO
Activated factor V (FVa) and factor X (FXa) form prothrombinase, which converts prothrombin to thrombin. The α isoform of tissue factor (TF) pathway inhibitor (TFPI) dampens early procoagulant events, partly by interacting with FV. FV Leiden (FVL) is the most common genetic thrombophilia in Caucasians. Thrombosis risk is particularly elevated in women with FVL taking oral contraceptives, which produce acquired TFPIα deficiency. In mice, FVL combined with 50% reduction in TFPI causes severe thrombosis and perinatal lethality. However, a possible interaction between FVL and TFPIα has not been defined in humans. Here, we examined this interaction using samples from patients with FVL in thrombin generation and fibrin formation assays. In dilute TF- or FXa-initiated reactions, these studies exposed a TFPI-dependent activation threshold for coagulation initiation that was greatly reduced by FVL. The reduced threshold was progressively overcome with higher concentrations of TF or FXa. Plasma assays using anti-TFPI antibodies or a TFPI peptide that binds and inhibits FVa demonstrated that the decreased activation threshold resulted from reduced TFPIα inhibition of prothrombinase. In assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL than with FV. Thus, FVL reduces the threshold for initiating coagulation, and this threshold is further reduced in situations of low TFPIα concentration. Individuals with FVL are likely prone to thrombosis in response to weak procoagulant stimuli that would not initiate blood clot formation in individuals with FV.