In vivo-generated thrombin and plasmin do not activate the complement system in baboons.
ABSTRACT: Sepsis concurrently activates both coagulation and complement systems. Although complement activation by bacteria is well documented, work in mice and in vitro suggests that coagulation proteases can directly cleave complement proteins. We aimed to determine whether generation of coagulation proteases in vivo can activate the complement cascade in 2 highly coagulopathic models. We compared temporal changes in activation biomarkers of coagulation (thrombin-antithrombin [TAT]), fibrinolysis (plasmin-antiplasmin [PAP]), and complement (C3b, C5a, C5b-9) in baboons infused with factor Xa (FXa) and phospholipids (FXa/phosphatidylcholine-phosphatidylserine [PCPS]) vs LD100 Escherichia coli We found that, albeit with different timing, both FXa/PCPS and E coli infusion led to robust thrombin and plasmin generation. Conversely, only E coli challenge activated the complement system, reaching a maximum at 2 hours postchallenge during the peaks of lipopolysaccharide and bacteremia but not of TAT and PAP. Despite inducing a strong burst of thrombin and plasmin, FXa/PCPS infusion did not produce measurable levels of complement activation in vivo. Similarly, ex vivo incubation of baboon serum with thrombin, plasmin, or FXa did not show noticeable complement cleavage unless supraphysiologic amounts of enzymes were used. Our results suggest that in vivo-generated thrombin and plasmin do not directly activate the complement in nonhuman primates.
Project description:Colistin electrostatically interacts with lipopolysaccharides (LPS). Pre-clinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial 16 healthy volunteers received a 2 ng/kg LPS bolus after infusion of 2.5 million IU colistin or placebo. Plasma levels of F1+2 prothrombin fragments, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70?%, t-PA antigen levels by 63?% and t-PA activity by 48?%, while PAI-1 levels decreased numerically by 63?%. Two hours after the LPS bolus F1+2 levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ~50?% and the two-fold increase in vWF antigen levels by ~70?%. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected.
Project description:The generation of thrombin is a critical process in the formation of venous thrombi. In isolated plasma under static conditions, phosphatidylserine (PS)-exposing platelets support coagulation factor activation and thrombin generation; however, their role in supporting coagulation factor binding under shear conditions remains unclear. We sought to determine where activated factor X (FXa), (pro)thrombin, and fibrin(ogen) are localized in thrombi formed under venous shear.Fluorescence microscopy was used to study the accumulation of platelets, FXa, (pro)thrombin, and fibrin(ogen) in thrombi formed in vitro and in vivo. Co-perfusion of human blood with tissue factor resulted in formation of visible fibrin at low, but not at high shear rate. At low shear, platelets demonstrated increased Ca(2+) signaling and PS exposure, and supported binding of FXa and prothrombin. However, once cleaved, (pro)thrombin was observed on fibrin fibers, covering the whole thrombus. In vivo, wild-type mice were injected with fluorescently labeled coagulation factors and venous thrombus formation was monitored in mesenteric veins treated with FeCl(3). Thrombi formed in vivo consisted of platelet aggregates, focal spots of platelets binding FXa, and large areas binding (pro)thrombin and fibrin(ogen).FXa bound in a punctate manner to thrombi under shear, while thrombin and fibrin(ogen) distributed ubiquitously over platelet-fibrin thrombi. During thrombus formation under venous shear, thrombin may relocate from focal sites of formation (on FXa-binding platelets) to dispersed sites of action (on fibrin fibers).
Project description:The objective of this study was to evaluate the elimination kinetics of hemostasis-related biomarkers including the prothrombin activation fragment F1+2, thrombin-antithrombin complex (TAT), plasmin-?2-antiplasmin complex (PAP), and D-dimer in humans. Autologous serum was used as a biomarker source and infused into 15 healthy volunteers. Serum was prepared from whole blood in the presence of recombinant tissue-type plasminogen activator (final concentration 20 ?g/mL) to induce plasmin generation required for PAP and D-dimer formation. Serum transfusions (50 mL/30 min) were well tolerated by all subjects. Endogenous thrombin formation was not induced by serum infusions as measured using a highly sensitive oligonucleotide-based enzyme capture assay. Median peak levels (x-fold increase over baseline) of F1+2, TAT, PAP, and D-dimer of 3.7 nmol/L (28.9), 393 ng/mL (189.6), 3,829 ng/mL (7.0), and 13.4 mg/L (34.2) were achieved at the end of serum infusions. During a 48 h lasting follow-up period all biomarkers showed elimination kinetics of a two-compartment model. Median (interquartile range) terminal half-lives were 1.9 (1.3-3.6) h for F1+2, 0.7 (0.7-2.6) h for TAT, and 10.8 (8.8-11.4) h for PAP. With 15.8 (13.1-23.1) h the D-dimer half-life was about twice as long as previously estimated from radiolabeling studies in animals and small numbers of human subjects. The serum approach presented here allows label-free and simultaneous analysis of the elimination kinetics of various hemostasis-related biomarkers. Based on these data changes in biomarker levels could more precisely used to estimate the activity level of the hemostatic system.
Project description:Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have a lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the antihemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pathway independently of thrombin feedback loops. In a mouse model with a relatively mild thrombogenic lesion, TF-dependent FVIII activation sets the threshold for thrombus formation through contact phase-generated FIXa. In vitro, FXa stably associated with TF-FVIIa activates FVIII, but not FV. Moreover, nascent FXa product of TF-FVIIa can transiently escape the slow kinetics of Kunitz-type inhibition by TF pathway inhibitor and preferentially activates FVIII over FV. Thus, TF synergistically primes FIXa-dependent thrombin generation independently of cofactor activation by thrombin. Accordingly, FVIIa mutants deficient in direct TF-dependent thrombin generation, but preserving FVIIIa generation by nascent FXa, can support intrinsic pathway coagulation. In ex vivo flowing blood, a TF-FVIIa mutant complex with impaired free FXa generation but activating both FVIII and FIX supports efficient FVIII-dependent thrombus formation. Thus, a previously unrecognized TF-initiated pathway directly yielding FVIIIa-FIXa intrinsic tenase complex may be prohemostatic before further coagulation amplification by thrombin-dependent feedback loops enhances the risk of thrombosis.
Project description:To test the hypothesis, utilizing 2 experimental mouse models, that plasmin is an important autoantigen that drives the production of certain IgG anticardiolipin (aCL) antibodies in patients with the antiphospholipid syndrome.BALB/cJ and MRL/MpJ mice were immunized with Freund's complete adjuvant in the presence or absence of human plasmin. The mouse sera were analyzed for production of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)-glycoprotein I (anti-beta(2)GPI) antibodies. IgG monoclonal antibodies (mAb) were generated from the plasmin-immunized MRL/MpJ mice with high titers of aCL, and these 10 mAb were studied for their binding properties and functional activity in vitro.Plasmin-immunized BALB/cJ mice produced high titers of IgG antiplasmin only, while plasmin-immunized MRL/MpJ mice produced high titers of IgG antiplasmin, IgG aCL, and IgG anti-beta(2)GPI. Both strains of mice immunized with the adjuvant alone did not develop IgG antiplasmin or IgG aCL. All 10 of the IgG mAb bound to human plasmin and cardiolipin, while 4 of 10 bound to beta(2)GPI, 3 of 10 bound to thrombin, and 4 of 10 bound to the activated coagulation factor X (FXa). Functionally, 4 of the 10 IgG mAb inhibited plasmin activity, 1 of 10 hindered inactivation of thrombin by antithrombin III, and 2 of 10 inhibited inactivation of FXa by antithrombin III.Plasmin immunization leads to production of IgG antiplasmin, aCL, and anti-beta(2)GPI in MRL/MpJ mice, but leads to production of only IgG antiplasmin in BALB/cJ mice. IgG mAb generated from plasmin-immunized MRL/MpJ mice bind to various antigens and exhibit procoagulant activity in vitro. These results suggest that plasmin may drive potentially prothrombotic aCL in genetically susceptible individuals.
Project description:Activation of the blood coagulation cascade leads to fibrin deposition and platelet activation that are required for hemostasis. However, aberrant activation of coagulation can lead to thrombosis. Thrombi can cause tissue ischemia, and fibrin degradation products and activated platelets can enhance inflammation. In addition, coagulation proteases activate cells by cleavage of PARs (protease-activated receptors), including PAR1 and PAR2. Direct oral anticoagulants have recently been developed to specifically inhibit the coagulation proteases FXa (factor Xa) and thrombin. Administration of these inhibitors to wild-type mice can be used to determine the roles of FXa and thrombin in different inflammatory diseases. These results can be compared with the phenotypes of mice with deficiencies of either Par1 (F2r) or Par2 (F2rl1). However, inhibition of coagulation proteases will have effects beyond reducing PAR signaling, and a deficiency of PARs will abolish signaling from all proteases that activate these receptors. We will summarize studies that examine the roles of coagulation proteases, particularly FXa and thrombin, and PARs in different mouse models of inflammatory disease. Targeting FXa and thrombin or PARs may reduce inflammatory diseases in humans.
Project description:Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in 'global' assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5-20 ?M, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics.
Project description:OBJECTIVE:To investigate the blood coagulation function in COVID-19 patients, and the correlation between coagulopathy and disease severity. METHODS:We retrospectively collected 147 clinically diagnosed COVID-19 patients at Wuhan Leishenshan Hospital of Hubei, China. We analyzed the coagulation function in COVID-19 patients through the data including thrombin-antithrombin complex (TAT), ?2-plasmininhibitor-plasmin Complex (PIC), thrombomodulin (TM), t-PA/PAI-1 Complex (t-PAIC), prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (APTT), fibrinogen (FIB), thrombin time (TT), D-Dimer (DD), and platelet (PLT). RESULT:The levels of TAT, PIC, TM, t-PAIC, PT, INR, FIB, and DD in COVID-19 patients were higher than health controls (p<0.05), and also higher in the patients with thrombotic disease than without thrombotic disease (p<0.05). What's more, the patients with thrombotic disease had a higher case-fatality (p<0.05). TAT, PIC, TM, t-PAIC, PT, INR, APTT, FIB, DD, and PLT were also found correlated with disease severity. Meanwhile, we found that there were significant difference in TAT, TM, t-PAIC, PT, INR, APTT, DD, and PLT in the death and survival group. Further using univariate and multivariate logistic regression analysis also found that t-PAIC and DD were independent risk factors for death in patients and are excellent predicting the mortality risk of COVID-19. CONCLUSION:Most COVID-19 patients with inordinate coagulation systems, dynamic monitoring of coagulation parameters might be a key in the control of COVID-19 death.
Project description:INTRODUCTION: Current criteria for early diagnosis of coagulopathy in sepsis are limited. We postulated that coagulopathy is already complicated with sepsis in the initial phase, and severe coagulopathy or disseminated intravascular coagulation (DIC) becomes overt after progressive consumption of platelet and coagulation factors. To determine early diagnostic markers for severe coagulopathy, we evaluated plasma biomarkers for association with subsequent development of overt DIC in patients with sepsis. METHODS: A single-center, prospective observational study was conducted in an adult ICU at a university hospital. Plasma samples were obtained from patients with sepsis at ICU admission. Fourteen biomarkers including global markers (platelet count, prothrombin time, activated partial thromboplastin time, fibrinogen and fibrin degradation product (FDP)); markers of thrombin generation (thrombin-antithrombin complex (TAT) and soluble fibrin); markers of anticoagulants (protein C (PC) and antithrombin); markers of fibrinolysis (plasminogen, ?2-plasmin inhibitor (PI), plasmin-?2-PI complex, and plasminogen activator inhibitor (PAI)-1); and a marker of endothelial activation (soluble E-selectin) were assayed. Patients who had overt DIC at baseline were excluded, and the remaining patients were followed for development of overt DIC in 5 days, and for mortality in 28 days. RESULTS: A total of 77 patients were enrolled, and 37 developed overt DIC within the following 5 days. Most patients demonstrated hemostatic abnormalities at baseline with 98.7% TAT, 97.4% FDP and 88.3% PC. Most hemostatic biomarkers at baseline were significantly associated with subsequent development of overt DIC. Notably, TAT, PAI-1 and PC discriminated well between patients with and without developing overt DIC (area under the receiver operating characteristic curve (AUROC), 0.77 (95% confidence interval, 0.64 to 0.86); 0.87 (0.78 to 0.92); 0.85 (0.76 to 0.91), respectively), and using the three together, significantly improved the AUROC up to 0.95 (vs. TAT, PAI-1, and PC). Among the significant diagnostic markers for overt DIC, TAT and PAI-1 were also good predictors of 28-day mortality (AUROC, 0.77 and 0.81, respectively). CONCLUSIONS: Severe coagulation and fibrinolytic abnormalities on ICU admission were associated with subsequent development of overt DIC. A single measurement of TAT, PAI-1, and PC activity could identify patients with ongoing severe coagulopathy, early in the course of sepsis.
Project description:Activation of coagulation and vascular inflammation are prominent features of sickle cell disease (SCD). Previously, we have shown that inhibition of tissue factor (TF) attenuates activation of coagulation and vascular inflammation in mouse models of SCD. In this study, we examined the mechanism by which coagulation proteases enhance vascular inflammation in sickle BERK mice. To specifically investigate the contribution of FXa and thrombin, mice were fed chow containing either rivaroxaban or dabigatran, respectively. In addition, we used bone marrow transplantation to generate sickle mice deficient in either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) on nonhematopoietic cells. FXa inhibition and PAR-2 deficiency in nonhematopoietic cells attenuated systemic inflammation, measured by plasma levels of interleukin-6 (IL-6). In contrast, neither thrombin inhibition nor PAR-1 deficiency in nonhematopoietic cells affected plasma levels of IL-6 in sickle mice. However, thrombin did contribute to neutrophil infiltration in the lung, independently of PAR-1 expressed by nonhematopoietic cells. Furthermore, the TF-dependent increase in plasma levels of soluble vascular cell adhesion molecule-1 in sickle mice was not mediated by FXa or thrombin. Our data indicate that TF, FXa, and thrombin differentially contribute to vascular inflammation in a mouse model of SCD.