Plasminogen activator-coated nanobubbles targeting cellbound ß2-glycoprotein I as a novel thrombus-specific thrombolytic strategy.

ß2-glycoprotein I (ß2-GPI) is a serum protein widely recognized as the main target of antibodies present in patients with antiphospholipid syndrome (APS). ß2-GPI binds to activated endothelial cells, platelets and leukocytes, key players in thrombus formation. We developed a new targeted thrombolytic agent consisting of nanobubbles (NB) coated with recombinant tissue plasminogen activator (rtPA) and a recombinant antibody specific for cell-bound ß2-GPI. The therapeutic efficacy of targeted NB was evaluated in vitro, using platelet-rich blood clots, and in vivo in three different animal models: i) thrombosis developed in a rat model of APS; ii) ferric chloride-induced mesenteric thrombosis in rats, and iii) thrombotic microangiopathy in a mouse model of atypical hemolytic uremic syndrome (C3-gain-of-function mice). Targeted NB bound preferentially to platelets and leukocytes within thrombi and to endothelial cells through ß2-GPI expressed on activated cells. In vitro, rtPA-targeted NB (rtPA-tNB) induced greater lysis of platelet-rich blood clots than untargeted NB. In a rat model of APS, administration of rtPA-tNB caused rapid dissolution of thrombi and, unlike soluble rtPA that induced transient thrombolysis, prevented new thrombus formation. In a rat model of ferric chloride triggered thrombosis, rtPA-tNB, but not untargeted NB and free rtPA, induced rapid and persistent recanalization of occluded vessels. Finally, treatment of C3-gain-of-function mice with rtPA-tNB, that target ß2-GPI deposited in kidney glomeruli, decreased fibrin deposition, and improved urinalysis data with a greater efficiency than untargeted NB. Our findings suggest that targeting cell-bound ß2-GPI may represent an efficient and thrombus-specific thrombolytic strategy in both APS-related and APS-unrelated thrombotic conditions.

Differential effect of direct oral anticoagulants on thrombin generation and fibrinolysis in patients with atrial fibrillation and venous thromboembolism.

BACKGROUND: Recent reports suggest that direct oral anticoagulants (DOAC) may induce different anticoagulant and profibrinolytic responses. We performed a head-to-head comparison of the changes in thrombin generation (TG) parameters and tissue plasminogen activator (t-PA)-induced clot lysis produced by different DOAC. MATERIAL AND METHODS: We tested 137 plasma samples from patients with non-valvular atrial fibrillation (n=72) and venous thromboembolism (n=65) under treatment with apixaban (n=38), edoxaban (n=29), rivaroxaban (n=39), or dabigatran (n=31). TG was evaluated by a fluorometric assay and fibrinolysis by measuring the lysis time of clots exposed to 40 ng/mL t-PA. RESULTS: Trough-to-peak changes of TG parameters, along with correlation analysis, showed that all DOAC prolonged the lag-time in a concentration-dependent fashion. As for the other parameters, anti-factor Xa drugs markedly reduced the thrombin peak and velocity index but had little (rivaroxaban) or no effect on endogenous thrombin potential (ETP); dabigatran, instead, reduced ETP, weakly decreased thrombin peak and did not influence the velocity index, as also inferred from the changes in TG values after neutralisation of dabigatran with idarucizumab. Concerning the profibrinolytic effect of DOAC, intergroup comparison showed that the clot lysis time of dabigatran samples was significantly shorter than that of the apixaban and rivaroxaban samples, at both C-Trough and C-Peak. Moreover, a significant correlation between trough-to-peak changes in drug level and clot lysis time was only observed in the dabigatran group (rho=0.53). Finally, after DOAC removal by DOAC-stop, only dabigatran samples showed a significant increase in lysis time. DISCUSSION: Our data show that dabigatran inhibits TG in a different way than anti-Xa DOAC; moreover, under our conditions, only dabigatran displayed profibrinolytic activity, most likely because of its distinctive effect on the TG curve.

Extracellular histones promote fibrinolysis by single-chain urokinase-type plasminogen activator in a factor seven activating protease-dependent way.

INTRODUCTION: Extracellular histones inhibit tissue plasminogen activator (t-PA)-mediated fibrinolysis by modifying fibrin structure and rheological properties. However, other plasminogen activators involved in intravascular and extravascular fibrinolysis have not been considered yet. OBJECTIVES: We investigated the effect of histones on fibrinolysis driven by different plasminogen activators. METHODS: Clot lysis induced by t-PA, urokinase (u-PA) and its single chain precursor (scu-PA) was evaluated by turbidimetry. Conversion of scu-PA to u-PA and activation of factor seven activating protease (FSAP) were assessed by fluorogenic and chromogenic assays, respectively. RESULTS: Histones delayed t-PA- and u-PA-mediated fibrinolysis but strongly accelerated scu-PA-driven clot lysis through the enhancement of scu-PA to u-PA conversion. This effect required a plasma factor identified as FSAP by the following findings: 1) histones enhanced neither scu-PA activation nor scu-PA-mediated clot lysis under purified conditions; 2) in plasma, the enhancement of fibrinolytic activity by histones was abolished by a neutralizing anti-FSAP antibody; and 3) histones promoted the activation of plasma FSAP. The effect of the natural mixture of histones on scu-PA-driven fibrinolysis was differentially recapitulated by the individual recombinant histones, H4 displaying the strongest activity. When complexed to DNA, histones still accelerated scu-PA-mediated fibrinolysis but with a lesser efficiency due to a reduced FSAP activation. Finally, preincubation of histones with heparin or activated protein C, two known inhibitors of histones, further amplified histone-mediated boost of scu-PA-driven fibrinolysis. CONCLUSIONS: Enhancement of FSAP-mediated scu-PA activity by histones may play yet unforeseen roles in intravascular fibrinolysis and contribute to extravascular proteolysis and tissue damage.

FVIII/VWF complex displays a greater pro-haemostatic activity than FVIII preparations devoid of VWF: Study in plasma and cell-based models.

INTRODUCTION: Plasma-derived FVIII/VWF complex was reported to be less sensitive to inhibitors than FVIII preparations devoid of VWF. AIM: To compare the efficacy of FVIII/VWF complex (Fanhdi) and five different VWF-free FVIII preparations in restoring thrombin generation and activation of thrombin-activatable fibrinolysis inhibitor (TAFI) in haemophilic plasma, with and without inhibitor, and in cell-based models. METHODS: Experiments were performed in haemophilic plasma supplemented with inhibitory IgG or in plasma samples obtained from haemophilia A patients without (n = 11) and with inhibitor (n = 12). Thrombin generation was evaluated by calibrated automated thrombography (CAT) under standard conditions, in the presence of activated protein C (APC) or thrombomodulin (TM), and in cell-based models including endothelial cells, either alone or in combination with platelets or tissue factor-expressing blood mononuclear cells. The kinetics of TAFI activation was determined by a two-stage functional assay in the absence and in the presence of APC. RESULTS: In haemophilic plasma without inhibitor, Fanhdi enhanced thrombin generation and TAFI activation as well as recombinant (2nd-4th generation) and plasma-derived FVIII preparations devoid of VWF. On the contrary, in plasma with inhibitor, Fanhdi displayed a greater ability to restore thrombin generation and TAFI activation under all tested conditions. Notably, in cell-based models including endothelial cells, Fanhdi proved more efficient than all other preparations in improving thrombin generation even in the absence of inhibitor. CONCLUSION: The greater pro-haemostatic activity of FVIII/VWF complex, either in haemophilic plasma with inhibitor or in the presence of endothelial cells, may offer therapeutic advantages.

Randomised trial of chronic supplementation with a nutraceutical mixture in subjects with non-alcoholic fatty liver disease.

A mixture of natural ingredients, namely, DHA, phosphatidylcholine, silymarin, choline, curcumin and d-α-tocopherol, was studied in subjects with non-alcoholic fatty liver disease (NAFLD). Primary endpoints were serum levels of hepatic enzymes, and other parameters of liver function, the metabolic syndrome and inflammation were the secondary endpoints. The coagulation-fibrinolysis balance was also thoroughly investigated, as NAFLD is associated with haemostatic alterations, which might contribute to increased cardiovascular risk of this condition. The present study involved a double-blind, randomised, multicentre controlled trial of two parallel groups. Subjects with NAFLD (18-80 years, either sex) received the active or control treatment for 3 months. All assays were performed on a total of 113 subjects before and at the end of supplementation. The hepatic enzymes aspartate aminotransferase (AST), alanine aminotransferase and γ-glutamyl transpeptidase decreased from 23·2 to 3·7 % after treatment, only the AST levels reaching statistical significance. However, no differences were found between control and active groups. Metabolic and inflammatory variables were unchanged, except for a slight (less than 10 %) increase in cholesterol and glucose levels after the active treatment. Coagulation-fibrinolytic parameters were unaffected by either treatment. In conclusion, chronic supplementation with the mixture of dietary compounds was well tolerated and apparently safe in NAFLD subjects. The trial failed to demonstrate any efficacy on relevant physiopathological markers, but its protocol and results may be useful to design future studies with natural compounds.

Thrombin activatable fibrinolysis inhibitor pathway alterations correlate with bleeding phenotype in patients with severe hemophilia A.

BACKGROUND: Patients with severe hemophilia A display varied bleeding phenotypes despite similar factor VIII (FVIII) activity levels. OBJECTIVE: We investigated different thrombin activatable fibrinolysis inhibitor (TAFI)-related variables in patients with severe hemophilia A and their possible correlation with bleeding tendency. PATIENTS/METHODS: Sixty-one patients with severe hemophilia A (FVIII:C <1%], treated on demand, were included. Patients were categorized as mild, moderate, and severe bleeders according to number of bleeds per year (≤2, 3-24, ≥25, respectively). Thirty healthy males served as controls. Clot lysis time was assessed by turbidimetric assay, TAFI activation by two-stage functional assay, and response to TAFIa as the prolongation of fibrinolysis time upon addition of purified TAFIa. Circulating levels of activated TAFI (TAFIa/ai) were measured by specific enzyme-linked immunosorbent assay. RESULTS: As compared to controls, hemophilic patients displayed shorter lysis time, less TAFIa generation, and reduced response to TAFIa, but similar TAFIa/ai levels. Clot lysis time was similar in mild, moderate, and severe bleeders, whereas TAFIa generation and response to TAFIa decreased with the increase in bleeding tendency; moreover, circulating TAFIa/ai levels were highest in severe bleeders. Patients with markedly impaired TAFIa generation or TAFIa response (below median) displayed 3-fold to 4-fold higher bleeding rate and factor consumption than patients whose TAFI-related values approached the control ones. CONCLUSION: The TAFI pathway impairment correlates with bleeding phenotype in severe hemophilia and may represent a promising tool to stratify the bleeding risk.

D-dimer corrected for thrombin and plasmin generation is a strong predictor of mortality in patients with sepsis.

BACKGROUND: D-dimer (DD) is the most used fibrin-related marker and has been proposed, either alone or in combination with other variables, as prognostic factor in patients with sepsis. However, DD generation depends on both coagulation and fibrinolysis, meaning that it may give false negative results in conditions associated with marked fibrinolytic inhibition such as sepsis. In this study, we tested whether correction of DD for thrombin and plasmin generation could improve its prognostic significance in septic patients. MATERIAL AND METHODS: We performed a nested study in 269 septic patients from the ALBIOS trial. DD, prothrombin fragment 1+2 (F1+2) and plasmin-antiplasmin complex (PAP) were assayed at day 1. Corrected DD (DDcorr) was calculated by the formula DD×PAP/F1+2, such that the lower the DDcorr the greater the imbalance in favour of fibrin formation over fibrin lysis, and vice-versa. Primary outcome was 90-day mortality. RESULTS: DDcorr showed a J-shaped relationship with mortality, which was highest in the first DDcorr tertile (low fibrinolysis), intermediate in the 3rd (high fibrinolysis), and lowest in the 2nd (balanced fibrinolysis), suggesting an increased risk whenever the coagulation-fibrinolysis balance is tilted (p<0.0001). Neither DD, nor PAP or F1+2 showed a comparable association with mortality. DDcorr was an independent prognostic factor in multivariable Cox models and significantly improved risk stratification (cNRI≥0.28). Finally, by combining DDcorr and SOFA tertiles, we developed a score with high discriminatory power. DISCUSSION: DDcorr is a good marker of the in vivo coagulation-fibrinolysis balance and displays a prognostic value in sepsis much higher than DD.

Platelet Drop and Fibrinolytic Shutdown in Patients With Sepsis.

OBJECTIVE: Thrombocytopenia is the most common hemostatic disorder during sepsis and is associated with high mortality. We examined whether fibrinolytic changes precede incident thrombocytopenia and predict outcome in patients with severe sepsis. DESIGN: Nested study from the multicenter, randomized, controlled trial on the efficacy of albumin replacement in severe sepsis or septic shock (the Albumin Italian Outcome Sepsis trial). SETTING: Forty ICUs in Italy. PATIENTS: Three groups of patients were selected: 1) patients with platelet count less than or equal to 50 × 10/L at study entry (n = 85); 2) patients with baseline platelet count greater than or equal to 100 × 10/L who developed thrombocytopenia (≤ 50 × 10/L) within 28 days (n = 100); 3) patients with platelet count always more than or equal to 100 × 10/L (n = 95). INTERVENTIONS: Fibrinolytic variables, including fibrinolysis inhibitors and in vivo markers of plasmin generation, were measured on day 1. MEASUREMENTS AND MAIN RESULTS: Patients with early thrombocytopenia (group 1) and those who developed it later (group 2) had similar illness severity and 90-day mortality, whereas patients without thrombocytopenia (group 3) had milder disease and lower mortality. Fibrinolysis was markedly (and similarly) depressed in groups 1 and 2 as compared with group 3. Major fibrinolytic changes included increased levels of plasminogen activator inhibitor 1 and extensive activation/consumption of thrombin activatable fibrinolysis inhibitor. Most fibrinolytic variables were significantly associated with mortality in univariate models. However, only thrombin activatable fibrinolysis inhibitor level and in vivo markers of fibrinolysis activation, namely plasmin-antiplasmin complex, and D-dimer, were independently associated with mortality after adjustment for Simplified Acute Physiology Score-II score, sex, and platelet count. Furthermore, the coexistence of impaired fibrinolysis and low platelets was associated with an even greater mortality. CONCLUSIONS: Impaired fibrinolysis, mainly driven by plasminogen activator inhibitor-1 increase and thrombin activatable fibrinolysis inhibitor activation, is an early manifestation of sepsis and may precede the development of thrombocytopenia. Thrombin activatable fibrinolysis inhibitor level, in particular, proved to be an independent predictor of mortality, which may improve risk stratification of patients with severe sepsis.

Grape intake reduces thrombin generation and enhances plasma fibrinolysis. Potential role of circulating procoagulant microparticles.

Phytochemicals contained in grapes down-regulate several prothrombotic pathways in vitro. We evaluated the effect of grape consumption on coagulation and fibrinolysis in healthy volunteers. Thirty subjects were enrolled: 20 were given grape (5 g/kg body weight/day for 3 weeks), while 10 served as controls. Blood samples were taken at baseline (T0), at the end of the grape diet (T1) and after 4-week wash-out (T2). Grape intake caused a significant decrease of the procoagulant and inflammatory responses of whole blood and/or mononuclear cells to bacterial lipopolysaccharide at both T1 and T2. At plasma level, grape diet decreased thrombin generation at T1 and T2, largely through a reduction in the number and/or activity of procoagulant microparticles. This anticoagulant effect resulted in the formation of clots that were more susceptible to fibrinolysis, mainly because of a lesser activation of thrombin activatable fibrinolysis inhibitor. No difference in any variables was detected in controls at the time points considered. In conclusion, chronic grape consumption induces sustained anticoagulant and profibrinolytic effects with potential benefits for human health.

Hypercoagulability in patients with Cushing disease detected by thrombin generation assay is associated with increased levels of neutrophil extracellular trap-related factors.

Patients with Cushing disease (CD) are at increased risk of venous thromboembolism (VTE). It was surmised, but not conclusively shown that the risk is related to plasma hypercoagulability secondary to the glucocorticoids effect. This study is aimed at detecting hypercoagulability in patients with CD. Case-control study of 48 CD patients and controls enrolled at two Italian clinics for whom we assessed the thrombin-forming-potential in the presence of optimal activation of protein C obtained by adding into the assay system its main endothelial activator, thrombomodulin. These experimental conditions mimic more closely than any other test the in vivo situation. We observed enhanced thrombin-generation in CD patients, as shown by the modification of thrombin-generation parameters [i.e., shortened lag-time and time-to-peak, increased thrombin peak and endogenous thrombin potential (ETP)]. Moreover, the ETP ratio (with/without thrombomodulin), recognized as an index of hypercoagulability, was increased in patients as compared to controls. We attempted to explain such hypercoagulability by measuring both procoagulant and anticoagulant factors, and some other non-coagulation parameters (i.e., neutrophil extracellular traps (NET), recently associated with the VTE risk and/or increased hypercoagulability. We showed that the hypercoagulability in patients with CD is associated with increased levels of factor VIII and NET-related variables. We detected plasma hypercoagulability in patients with CD and found experimental explanation for its occurrence. Whether this hypercoagulability can entirely explain the occurrence of VTE in patients with CD should be investigated by ad-hoc clinical trials. However, until these studies will be available the evidence supports the concept that patients with CD are candidates for antithrombotic prophylaxis.

Dabigatran but not rivaroxaban or apixaban treatment decreases fibrinolytic resistance in patients with atrial fibrillation.

INTRODUCTION: Most anticoagulants stimulate fibrinolysis in vitro through mechanisms dependent on and independent of thrombin activatable fibrinolysis inhibitor (TAFI). We evaluated the effect of dabigatran, rivaroxaban and apixaban treatment on plasma fibrinolysis in patients with non-valvular atrial fibrillation. METHODS AND RESULTS: Patients treated with dabigatran etexilate (n=22), rivaroxaban (n=24) or apixaban (n=22) were studied. Plasma was obtained before (trough) and 2h after drug intake (peak). Fibrinolytic resistance of clots exposed to exogenous tissue plasminogen activator was significantly lower in peak than in trough samples and correlated with drug concentration only in dabigatran group. Moreover, fibrinolytic resistance at peak was lower in dabigatran than in rivaroxaban and apixaban groups. This difference disappeared if the TAFI pathway was inhibited. Thrombin generation and TAFI activation were markedly lower in peak than in trough samples in all three groups. However, TAFIa levels in trough and peak samples were significantly lower in dabigatran group than in rivaroxaban and apixaban groups. Circulating levels of prothrombin fragment F1+2 (reflecting in vivo thrombin generation) and plasmin-antiplasmin complex (reflecting plasmin generation) were not or barely influenced by drug levels in all groups. CONCLUSIONS: Our data suggest that dabigatran, contrary to rivaroxaban and apixaban, reduces fibrinolytic resistance by virtue of its greater impact on TAFI activation. The profibrinolytic effect of dabigatran may play a role locally, at sites of fibrin formation, by making the nascent thrombus more susceptible to plasminogen-dependent degradation.

Coagulopathy of Acute Sepsis.

Coagulopathy is common in acute sepsis and may range from subclinical activation of blood coagulation (hypercoagulability), which may contribute to venous thromboembolism, to acute disseminated intravascular coagulation, characterized by widespread microvascular thrombosis and consumption of platelets and coagulation proteins, eventually causing bleeding. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the pathogen leading to the overexpression of inflammatory mediators. The latter, along with the microorganism and its derivatives drive the major changes responsible for massive thrombin formation and fibrin deposition: (1) aberrant expression of tissue factor mainly by monocytes-macrophages, (2) impairment of anticoagulant pathways, orchestrated by dysfunctional endothelial cells (ECs), and (3) suppression of fibrinolysis because of the overproduction of plasminogen activator inhibitor-1 by ECs and thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor. Neutrophils and other cells, upon activation or death, release nuclear materials (neutrophil extracellular traps and/or their components such as histones, DNA, lysosomal enzymes, and High Mobility Group Box-1), which have toxic, proinflammatory and prothrombotic properties thus contributing to clotting dysregulation. The ensuing microvascular thrombosis-ischemia significantly contributes to tissue injury and multiple organ dysfunction syndromes. These insights into the pathogenesis of sepsis-associated coagulopathy may have implications for the development of new diagnostic and therapeutic tools.

Factor IX-Padua enhances the fibrinolytic resistance of plasma clots.

Hypercoagulable conditions may determine a hypofibrinolytic state by increasing the activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Factor (F)IX-Padua is a mutated FIX with an eight-fold increased clotting activity and associates with a higher venous thrombotic risk. We evaluated the influence of FIX-Padua on TAFI-mediated regulation of fibrinolysis. A subject hemizygous for FIX-Padua, two family members (heterozygous and normal) and six healthy controls were studied. Clot lysis, TAFI activation and thrombin generation were evaluated in contact-inhibited plasma challenged with low concentrations of tissue factor. Fibrinolysis times were significantly longer in FIX-Padua carriers than controls. The difference disappeared when activated TAFI (TAFIa) was inhibited, when TAFI activation was avoided or when clotting was made independent of FIX. TAFIa generation was markedly enhanced in FIX-Padua carriers as compared to controls, and this could be explained by a greater thrombin generation in the former. Hyperactive FIX, but not wild-type FIX, enhanced fibrinolytic resistance also when the FXI-dependent positive feedback was blocked by a neutralising anti-FXI antibody. This thrombin-mediated, TAFI-dependent down-regulation of fibrinolysis provides new clues for explaining the heightened thrombotic risk in subjects carrying the FIX-Padua mutation.

Sepsis, thrombosis and organ dysfunction.

Sepsis is often associated with haemostatic changes ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by widespread microvascular thrombosis and subsequent consumption of platelets and coagulation proteins, eventually causing bleeding manifestations. The key event underlying this life-threatening complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. The latter, along with the micro-organism and its derivatives are now believed to drive the major changes responsible for massive thrombin formation and fibrin deposition, namely 1) the aberrant expression of the TF by different cells (especially monocytes-macrophages), 2) the impairment of physiological anticoagulant pathways, orchestrated mainly by dysfunctional endothelial cells (ECs) and 3) the suppression of fibrinolysis due to overproduction of plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). The ensuing microvascular thrombosis and ischemia are thought to contribute to tissue injury and multiple organ dysfunction syndrome (MODS). Recent evidence indicates that extracellular nuclear materials released from activated and especially apoptotic or necrotic cells, e.g. High Mobility Group Box-1 (HMGB-1) and histones, are endowed with cell toxicity, proinflammatory and clot-promoting properties and thus, during sepsis, they may represent late mediators that propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenesis of DIC and MODS may have implications for the development of new therapeutic agents potentially useful for the management of severe sepsis.

Extracellular histones promote thrombin generation through platelet-dependent mechanisms: involvement of platelet TLR2 and TLR4.

The release of histones from dying cells is associated with microvascular thrombosis and, because histones activate platelets, this could represent a possible pathogenic mechanism. In the present study, we assessed the influence of histones on the procoagulant potential of human platelets in platelet-rich plasma (PRP) and in purified systems. Histones dose-dependently enhanced thrombin generation in PRP in the absence of any trigger, as evaluated by calibrated automated thrombinography regardless of whether the contact phase was inhibited. Activation of coagulation required the presence of fully activatable platelets and was not ascribable to platelet tissue factor, whereas targeting polyphosphate with phosphatase reduced thrombin generation even when factor XII (FXII) was blocked or absent. In the presence of histones, purified polyphosphate was able to induce thrombin generation in plasma independently of FXII. In purified systems, histones induced platelet aggregation; P-selectin, phosphatidylserine, and FV/Va expression; and prothrombinase activity. Blocking platelet TLR2 and TLR4 with mAbs reduced the percentage of activated platelets and lowered the amount of thrombin generated in PRP. These data show that histone-activated platelets possess a procoagulant phenotype that drives plasma thrombin generation and suggest that TLR2 and TLR4 mediate the activation process.

Coagulation-fibrinolysis changes during off-pump bypass: effect of two heparin doses.

BACKGROUND: To date, no study has tested the effect of different heparin dosages on the hemostatic changes during off-pump coronary artery bypass graft (OPCABG) surgery, and a wide variety of empirical anticoagulation protocols are being applied. We tested the effect of two different heparin dosages on the activation of the hemostatic system in patients undergoing OPCABG procedures. METHODS: Forty-two patients eligible for OPCABG procedures were assigned in a randomized fashion to low-dose heparin (150 IU/kg) or high-dose heparin (300 IU/kg). Prothrombin fragment 1+2, plasmin/alpha(2)-plasmin inhibitor complex, D-dimer, soluble tissue factor, tissue factor pathway inhibitor, total thrombin activatable fibrinolysis inhibitor (TAFI), and activated TAFIa were assayed by specific enzyme-linked immunosorbent assays at six different timepoints, before, during, and after surgery. Platelet function was evaluated by means of an in vitro bleeding time test, platelet function analyzer-100. RESULTS: The OPCABG surgery was accompanied by significant changes of all plasma biomarkers, indicative of systemic activation of coagulation and fibrinolysis. A significant increase in circulating TAFIa was detected perioperatively and postoperatively, and multiple regression analysis indicated that prothrombin F1+2 but not plasmin/alpha(2)-antiplasmin complex was independently associated with TAFIa level. Platelet function analyzer-100 values did not change significantly after OPCABG. All hemostatic changes were similar in the two heparin groups, even perioperatively, when the difference in anticoagulation was maximal. CONCLUSIONS: Both early and late hemostatic changes, including TAFI activation, are similarly affected in the low-dose and high-dose heparin groups, suggesting that the increase in heparin dosage is not accompanied by a better control of clotting activation during OPCABG surgery.

Sepsis-associated disseminated intravascular coagulation and thromboembolic disease.

Sepsis is almost invariably associated with haemostatic abnormalities ranging from subclinical activation of blood coagulation (hypercoagulability), which may contribute to localized venous thromboembolism, to acute disseminated intravascular coagulation (DIC), characterized by massive thrombin formation and widespread microvascular thrombosis, partly responsible of the multiple organ dysfunction syndrome (MODS), and subsequent consumption of platelets and coagulation proteins causing, in most severe cases, bleeding manifestations. There is general agreement that the key event underlying this life-threatening sepsis complication is the overwhelming inflammatory host response to the infectious agent leading to the overexpression of inflammatory mediators. Mechanistically, the latter, together with the micro-organism and its derivatives, causes DIC by 1) up-regulation of procoagulant molecules, primarily tissue factor (TF), which is produced mainly by stimulated monocytes-macrophages and by specific cells in target tissues; 2) impairment of physiological anticoagulant pathways (antithrombin, protein C pathway, tissue factor pathway inhibitor), which is orchestrated mainly by dysfunctional endothelial cells (ECs); and 3) suppression of fibrinolysis due to increased plasminogen activator inhibitor-1 (PAI-1) by ECs and likely also to thrombin-mediated activation of thrombin-activatable fibrinolysis inhibitor (TAFI). Notably, clotting enzymes non only lead to microvascular thrombosis but can also elicit cellular responses that amplify the inflammatory reactions. Inflammatory mediators can also cause, directly or indirectly, cell apoptosis or necrosis and recent evidence indicates that products released from dead cells, such as nuclear proteins (particularly extracellular histones), are able to propagate further inflammation, coagulation, cell death and MODS. These insights into the pathogenetic mechanisms of DIC and MODS may have important implications for the development of new therapeutic agents that could be potentially useful particularly for the management of severe sepsis.

Extracellular histones are major mediators of death in sepsis.

Hyperinflammatory responses can lead to a variety of diseases, including sepsis. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra-alveolar hemorrhage and macro- and microvascular thrombosis. We detected histone in the circulation of baboons challenged with Escherichia coli, and the increase in histone levels was accompanied by the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co-infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality, which was reversed by treatment with antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.