Plasmin-α2-antiplasmin Complex Research Articles

Low factor XIII levels and altered fibrinolysis in patients with multiple myeloma

BackgroundAcquired factor FXIII (FXIII) deficiency can be immune- or non-immune mediated and may cause severe bleeding symptoms. The incidence of acquired FXIII deficiency and its etiology in patients with multiple myeloma (MM) are poorly understood. ObjectivesTo assess FXIII levels and the balance of fibrinolysis in newly diagnosed, untreated MM and monoclonal gammopathy of undetermined significance (MGUS) patients. MethodsFXIII activity, mixing studies, FXIII-A2B2 antigen, total FXIII-B antigen were measured in platelet-poor plasma from 17 untreated MM patients, 33 untreated MGUS patients, and 30 age and sex-matched healthy controls. Besides routine laboratory measurements, the balance of coagulation and fibrinolysis was evaluated using quantitative fibrin monomer (FM) test, thrombin-antithrombin assay, α2-antiplasmin activity, plasmin-α2-antiplasmin (PAP) complex, D-dimer, plasmin generation assay, clot lysis assay, and ClotPro-TPA test. ResultsFXIII-A2B2 levels were significantly lower in MM patients compared to controls [median (IQR):14.6 (11.2–19.4) vs. 21.8 (17.1–26.4) mg/L, p = 0.0015], whereas total FXIII-B did not differ between groups. Decrease in FXIII activity was parallel to the decrease in FXIII-A2B2. An immune-mediated inhibitory mechanism was ruled out. Free/total FXIII-B was significantly higher in MM patients compared to MGUS and healthy controls, suggesting an etiology of FXIII-A consumption. In MM and MGUS patients, FM, D-dimer, and PAP complex were significantly elevated compared to controls, indicating hypercoagulability and ongoing fibrinolysis. ConclusionsLow FXIII levels due to consumption were observed in MM patients at diagnosis. Hypercoagulability and ongoing fibrinolysis were detected in MM and MGUS, indicating that a disturbed hemostasis balance is already present in the latter benign condition.

Fibrinogenolysis and fibrinolysis in vaccine-induced immune thrombocytopenia and thrombosis

BackgroundVaccine-induced immune thrombocytopenia and thrombosis (VITT) is a rare syndrome associated with adenoviral vector vaccines for COVID-19. The syndrome is characterized by thrombosis, anti-platelet factor 4 (PF4) antibodies, thrombocytopenia, high D-dimer, and hypofibrinogenemia. ObjectivesTo investigate abnormalities in fibrinolysis that contribute to the clinical features of VITT. MethodsPlasma samples from 18 suspected VITT cases were tested for anti-PF4 by ELISA and characterized as meeting criteria for VITT (11/18) or deemed unlikely (7/18; non-VITT). Antigen levels of PAI-1, factor XIII (FXIII), plasmin-α2antiplasmin (PAP), and inflammatory markers were quantified. Plasmin generation was quantified by chromogenic substrate. Western blotting was performed with antibodies to fibrinogen, FXIII-A, and plasminogen. ResultsVITT patients 10/11 had scores indicative of overt disseminated intravascular coagulation, while 0/7 non-VITT patients met the criteria. VITT patients had significantly higher levels of inflammatory markers, IL-1β, IL-6, IL-8, TNFα, and C-reactive protein. In VITT patients, both fibrinogen and FXIII levels were significantly lower, while PAP and tPA-mediated plasmin generation were higher compared to non-VITT patients. Evidence of fibrinogenolysis was observed in 9/11 VITT patients but not in non-VITT patients or healthy controls. Fibrinogen degradation products were apparent, with obvious cleavage of the fibrinogen α-chain. PAP complex was evident in those VITT patients with fibrinogenolysis, but not in non-VITT patients or healthy donors. ConclusionVITT patients show evidence of overt disseminated intravascular coagulation and fibrinogenolysis, mediated by dysregulated plasmin generation, as evidenced by increased PAP and plasmin generation. These observations are consistent with the clinical presentation of both thrombosis and bleeding in VITT.

Changes in thrombosis-related parameters after AstraZeneca COVID-19 vaccination in a male volunteer: a case report

BackgroundWe speculated that subclinical thrombosis may occur frequently through crosstalk between immune/inflammatory reactions and hemostasis after corona virus disease-2019 (COVID-19) vaccination. To test this hypothesis, we measured thrombosis-related parameters after COVID-19 vaccination in a volunteer for 21 days.Case presentationThe following parameters were measured in a 72-year-old Korean man at 1 day before vaccination and on days 1, 3, 7, 14, and 21 post vaccination (AstraZeneca COVID-19 vaccine: ChAdOx1-S/nCoV-19, CTMAV563): complete blood count, platelet indices, thrombin receptor-activating peptide-induced platelet aggregation, prothrombin time, activated partial thromboplastin time, D-dimer, thrombin–antithrombin III complex (TAT), plasmin-α2 antiplasmin complex (PAP), von Willebrand factor (vWF) antigen and activity, plasminogen activator inhibitor-1 (PAI-1), protein C and protein S antigen and activity, lupus anticoagulant, fibrinogen degradation product, and plasminogen. We found that the TAT had significantly increased from 0.7 ng/mL (baseline) to 21.7 ng/mL (day 1). There was a transient increase in the PAI-1 level from 7.2 ng/mL (baseline) to 10.9 ng/mL (day 3), followed by a decrease in PAP level from 0.9 ng/mL (baseline) to 0.3 μg/mL (day 7), suggesting that plasmin generation is suppressed by PAI-1.ConclusionsIncreased thrombotic factors (such as decreased protein S) and decreased fibrinolytic activity due to increased PAI-1 were potential factors causing thrombogenesis after COVID-19 vaccination. Sequential measurement of platelet indices, TAT, PAP, protein C, protein S, vWF, D-dimer, and PAI-1 following COVID-19 vaccination was informative.

Automated measurement of coagulation and fibrinolytic activation markers: Outcomes in coronavirus disease 2019 (COVID-19) patients.

BackgroundSevere coronavirus disease 2019 (COVID‐19) is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition.Patients/MethodsWe present a retrospective observational study of 104 patients admitted to hospital with COVID‐19. Plasma samples were collected within 72 h of admission. In addition to routine coagulation and haematology testing, soluble thrombomodulin (sTM), thrombin‐antithrombin (TAT), tissue plasminogen activator‐plasminogen activator inhibitor 1 complex (tPAI‐C) and plasmin‐α2 antiplasmin complex (PIC) were performed by automated chemiluminescent enzyme immunoassays.ResultsSignificantly higher levels of D‐dimer, TAT, sTM and tPAI‐C were observed in non‐survivors compared to survivors. To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D‐dimer, TAT. sTM, tPAI‐C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for each 1.0 TU/mL increment (95% CI 1.025–1.115).ConclusionsOf the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID‐19, and this suggests that endothelial dysfunction plays an important role in disease progression.

Pattern of Coagulation Activation in Patients with Myeloproliferative Neoplasms

Background: Myeloproliferative neoplasms (MPN), comprising essential thrombocythemia (ET), polycythemia vera (PV) and myelofibrosis (MF), are associated with a high incidence of venous thromboembolism (VTE), especially splanchnic vein thrombosis (SVT), but the etiology of a hypercoagulable state in MPN remains elusive. JAK2V617F-associated development of acquired protein C (APC) resistance has been reported, suggesting an alteration of the thrombin-protein C (PC) axis.Methods: To further study the impact of the thrombin-PC axis on hypercoagulability in MPN, plasma levels of thrombin and APC were measured using oligonucleotide-based enzyme capture assays (OECA) in prospectively collected samples from 21 MPN patients (10 females) with a history of VTE (thereof 17 with SVT) and 24 MPN patients (13 females) without VTE. In addition to the OECA, markers of coagulation and fibrinolysis were measured including prothrombin activation fragment F1+2, D-dimer, plasmin-α2-antiplasmin complex (PAP), and thrombin-antithrombin-complex (TAT). All but one patient with prior VTE and 21 patients (88%) without prior thrombosis were positive for the JAK2V617F or the CALR mutation. Due to repeated study visits over a period of 21 months, a total of 35 samples from patients with a history of thrombosis and 46 from patients without VTE were available for analysis. Another comparison was performed between patients under anticoagulant treatment (17 patients, 27 samples) vs no anticoagulant treatment (28 patients, 54 samples). Furthermore, the results obtained in MPN patients were compared to those in 43 healthy subjects (22 females) who acted as a control group.Results: All biomarkers were significantly increased in MPN patients compared to healthy controls (Table 1). ANOVA neither revealed a difference between the disease subgroups of MF, PV, and ET nor between MPN patients with and without a history of VTE. In both patients with and without prior VTE APC, D-Dimer, F1+2, and PAP were significantly increased: Plasma levels of APC (median, IQR) of 0.078 (0.033-0.146) ng/mL, D-Dimer of 0.45 (0.29-0.67) mg/L, F1+2 of 0.219 (0.138-0.499) nmol/L, and PAP of 195 (118-453) ng/mL were measured in MPN patients with prior VTE. In patients without a history of thrombosis APC was 0.063 (0.026-0.108) ng/mL, D-dimer 0.33 (0.27-0.54) mg/L, F1+2 0.247 (0.146-0.381) nmol/L, and PAP 321 (175-526) ng/mL. The same pattern was observed in samples from patients without anticoagulant treatment, whereas in MPN patients under anticoagulants only APC (0.103, 0.027-0.137 ng/mL, p=0.0014), D-dimer (0.35, 0.23-0.60 mg/L, p=0.0022) and PAP (202, 144-423 ng/mL, p=0.0108), but not F1+2 (0.152, 0.098-0.219 nmol/L) were increased in comparison to the healthy control group. Consistent with these findings, F1+2 was the only biomarker, that differed significantly (p=0.0021) between MPN patients with anticoagulant therapy and patients not receiving anticoagulant medication (0.296, 0.185-0.502 nmol/l).Conclusion: The obtained data indicate, that MPN-induced thrombin formation, as demonstrated by increased levels of F1+2, causes activation of the anticoagulant PC system. In addition, the increase of PAP indicates activation of fibrinolysis which might contribute to elevated plasma levels of D-dimer. Thus, F1+2 might be better suited than D-dimer to monitor the effect of anticoagulant therapy in MPN. [Display omitted] DisclosuresRühl:Grifols: Research Funding; Sobi: Consultancy.

Hemostatic factor levels and cognitive decline in older adults: The Cardiovascular Health Study

BackgroundHemostasis is a key factor in cerebrovascular disease, but the association of hemostatic factors with cognitive decline is unclear. ObjectiveTo prospectively evaluate associations of 20 hemostatic factor levels with changes in cognition during ≥8 years of follow‐up in the Cardiovascular Health Study (CHS) of older adults. MethodsWe included participants of an existing CHS cross‐sectional substudy (n = 400) with hemostatic factors measured in 1989‐1990. Between 1989‐1990 and 1998‐1999, cognitive function was measured using the Modified Mini‐Mental State Examination (3MSE) and Digit Symbol Substitution Tests. Mixed‐effects linear regression models estimated change in cognitive function over time, adjusting for sociodemographic and clinical factors and APOE genotype, using Bonferroni adjustment. We also derived principal components to account for the interrelationship among factors. ResultsOf 20 factors evaluated individually, only higher levels of plasmin‐α2‐antiplasmin complex (PAP), tissue factor pathway inhibitor (TFPI), and lower factor X (FXc) levels were associated with faster cognitive decline, estimated by annual change in 3MSE points (1 standard deviation PAP β = −0.65, 95% confidence interval [CI]: −1.08 to −0.21; TFPI β = −0.55, 95% CI: −0.90 to −0.19; FXc β = 0.52, 95% CI: 0.21‐0.84). One of four principal components, loading positively on D‐dimer, prothrombin fragment 1.2 (F1.2), and PAP was significantly associated with change in 3MSE. ConclusionsLevels of PAP, TPFI, and FXc and a combination of factors driven by PAP, D‐dimer, and F1.2 were associated with cognitive decline. Whether these findings can be used to improve dementia prevention or prediction requires further study.

Trimester-specific reference intervals of TAT, TM, tPAI-C and PIC for healthy Chinese pregnant women.

Pregnancy is a hypercoagulability state, the aim of this study was to observe the changes of thrombin-antithrombin complex (TAT), thrombomodulin (TM), tissue plasminogen activator-inhibitor complex (tPAI-C) and plasmin-α2-antiplasmin complex (PIC) during pregnancy and establish trimester-specific reference intervals for Chinese healthy pregnant women. In total 190 Chinese healthy pregnant women (first trimester 59 cases, second trimester 60 cases and third trimester 71 cases) were recruited in North China. TAT, TM, tPAI-C and PIC were processed on Sysmex HISCL 5000 automated chemiluminescence immune detection system. Trimester-specific reference intervals were established with the 2.5th and 97.5th percentile of the distribution. The reference intervals for TAT, TM, tPAI-C, PIC at trimester 1 were 0.40-3.65 ng/mL, 4.85-8.80 TU/mL, 1.75-6.40 ng/mL, 0.25-1.05 μg/mL, respectively. At trimester 2, the reference intervals were 1.65-8.61 ng/mL, 5.70-9.93 TU/mL, 2.91-7.71 ng/mL, 0.33-2.02 μg/mL, respectively. At trimester 3, the reference intervals were 3.16-12.68 ng/mL, 5.50-14.24 TU/mL, 2.70-10.69 ng/mL, 0.24-1.54 μg/mL, respectively. The changes of TAT, TM, tPAI-C, PIC during pregnancy are presented, and trimester-specific reference intervals for healthy pregnant women are described. The levels of TAT, TM, tPAI-C were increased gradually from trimester 1 to trimester 3, while the PIC level remains stable during all trimesters.

Empagliflozin decreases the plasma concentration of plasminogen activator inhibitor-1 (PAI-1) in patients with type 2 diabetes: Association with improvement of fibrinolysis

AimsElevation of the plasma concentration of plasminogen activator inhibitor-1 (PAI-1), a rapid-acting inhibitor of fibrinolysis, is associated with development of vascular thrombotic diseases, including coronary artery disease and stroke. We investigated the effects of empagliflozin, a sodium-glucose co-transporter-2 (SGLT2) inhibitor, on the plasma concentration of PAI-1 and fibrinolytic activity in patients with type 2 diabetes. MethodsIn a randomized, active-controlled, open-label trial, 51 patients with type 2 diabetes were randomly allocated at a 2:1 ratio to receive empagliflozin (10 mg/day, n = 31) or standard therapy (n = 18) for 12 weeks. We measured the plasma concentrations of PAI-1 and plasmin-α2-antiplasmin complex (PAP) as indicators of fibrinolytic activity. Serum leptin and high-molecular weight (HMW) adiponectin were also measured. ResultsIn 49 patients who completed the trial, baseline plasma PAI-1 showed a positive correlation with body weight, visceral fat area (VFA), γ-glutamyltranspeptidase (GGT), leptin, and the platelet count, while it showed a negative correlation with HDL cholesterol and PAP. Body weight and VFA decreased significantly in the empagliflozin group, but not in the control group. The serum level of GGT showed a significant decrease at 12 weeks in the empagliflozin group, while it was unchanged in the control group. Serum HMW adiponectin increased significantly in the empagliflozin group. Plasma PAI-1 decreased significantly by 25% in the empagliflozin group, but not in the control group. In the empagliflozin group, the change of plasma PAI-1 was positively correlated with the changes of body weight and leptin, but was negatively correlated with the change of PAP. ConclusionsEmpagliflozin reduced the plasma PAI-1 concentration through its synergistic actions of a glucose-lowering effect, VFA loss, and restoring the adipokine balance. (Clinical trial registry: UMIN000025418).

1150-P: Empagliflozin Decreases the Plasma Concentration of Plasminogen Activator Inhibitor-1 (PAI-1) in Patients with Type 2 Diabetes: Association with Improvement of Fibrinolysis

Objectives: Elevation of the plasma concentration of plasminogen activator inhibitor-1 (PAI-1), a rapid-acting inhibitor of fibrinolysis, is associated with development of cardiovascular diseases. We investigate the effects of empagliflozin, an SGLT2 inhibitor, on the plasma concentration of PAI-1 and fibrinolytic activity in patients with type 2 diabetes. Methods: In a randomized, active-controlled, open-label trial, 51 patients with type 2 diabetes were randomly allocated at a 2:1 ratio to receive empagliflozin (10 mg/day, n=31) or standard therapy (n=18) for 12 weeks. We measured the plasma concentrations of PAI-1 and plasmin-α2-antiplasmin complex (PAP) as indicators of fibrinolytic activity. Serum leptin and HMW adiponectin were also measured. Results: Body weight and visceral fat decreased in the empagliflozin group, but not in the control group. The serum level of γ-GTP showed a significant decrease at 12 weeks in the empagliflozin group, while it was unchanged in the control group. Serum HMW adiponectin increased significantly in the empagliflozin group. Plasma PAI-1 decreased significantly by 25% in the empagliflozin group, but not in the control group. In the empagliflozin group, the change of plasma PAI-1 was positively correlated with the changes of body weight and leptin, but was negatively correlated with the change of PAP. Conclusions: Empagliflozin reduced the plasma PAI-1 concentration through its synergistic actions of a glucose-lowering effect, weight loss, and restoring the adipokine balance. The beneficial effects of empagliflozin on fibrinolysis by decreasing circulating PAI-1 may be associated with improvement of vascular thrombotic outcomes in patients with type 2 diabetes. (Clinical trial registry: UMIN000025418). Disclosure T. Niitani: None. S. Sakurai: None. T. Jojima: None. T. Iijima: None. T. Tomaru: Research Support; Self; Merck & Co., Inc., Teijin Pharma Limited. I. Usui: None. Y. Aso: None.

Combined effect of a direct oral anticoagulant edoxaban and an inhibitor of activated thrombin-activatable fibrinolysis inhibitor on clot lysis.

Fibrinolysis is regulated by the thrombin/thrombin-activatable fibrinolysis inhibitor (TAFI) system. Thus, anticoagulants and inhibitors of TAFI are expected to accelerate fibrinolysis. The combined effects of an anticoagulant and a TAFIa inhibitor on fibrinolysis remain unknown. The aim of this study was to evaluate the combined effect of edoxaban, an oral direct factor Xa (FXa) inhibitor, and a TAFIa inhibitor, potato tuber carboxypeptidase inhibitor (PCI) on tissue-type plasminogen activator (t-PA)-induced clot lysis in human plasma in vitro. Pooled human plasma (containing 180ng/mL t-PA and 0.1nM thrombomodulin) was mixed with edoxaban and/or PCI. Clot formation was induced by 2.5pM tissue factor and 4µM phospholipids and clot lysis time was examined. Plasma plasmin-α2 antiplasmin complex (PAP) concentration was measured as a marker of plasmin generation. Edoxaban or PCI alone significantly shortened the t-PA-induced clot lysis time and plasma PAP concentration. The combination of these compounds significantly accelerated the clot lysis compared with the inhibitors alone. Addition of PCI (0.3, 1, and 3μg/mL) to 75ng/mL edoxaban increased plasma PAP concentration compared with edoxaban alone; however, compared with PCI alone only the combination of 0.3μg/mL PCI + 75ng/mL edoxaban showed the significant increase in PAP concentration. Concomitant use of an oral direct FXa inhibitor, edoxaban, and a TAFIa inhibitor, PCI, significantly accelerate fibrinolysis via enhancement of plasmin generation. These results suggest that the combination of edoxaban and a TAFIa inhibitor might be beneficial for the treatment of thromboembolic diseases.

A direct oral anticoagulant edoxaban accelerated fibrinolysis via enhancement of plasmin generation in human plasma: dependent on thrombin-activatable fibrinolysis inhibitor

A direct oral anticoagulant, edoxaban, is as effective as vitamin K antagonists for the treatment of venous thromboembolism (VTE). However, the mechanism underlying the treatment effect on VTE remains to be determined. The aims of this study were to evaluate the effect of edoxaban on tissue plasminogen activator (t-PA)-induced clot lysis in human plasma and to determine the roles of plasmin and thrombin-activatable fibrinolysis inhibitor (TAFI) in the profibrinolytic effect by edoxaban. Pooled human normal plasma or TAFI-deficient plasma (containing 180ng/mL t-PA and 0.1nM thrombomodulin) was mixed with edoxaban or an activated TAFI inhibitor, potato tuber carboxypeptidase inhibitor (PCI). Clot was induced by adding tissue factor and phospholipids. Clot lysis time and plasma plasmin-α2 antiplasmin complex (PAP) concentration were determined. Clot structure was imaged with a scanning electron microscope. In normal plasma, edoxaban at clinically relevant concentrations (75, 150, and 300ng/mL) and PCI significantly shortened clot lysis time. PCI increased PAP concentration and a correlation between PAP concentration and percent of clot lysis was observed. Edoxaban also dose-dependently elevated PAP concentration. In TAFI-deficient plasma, the effects of edoxaban and PCI on clot lysis and PAP concentration were markedly diminished as compared with normal plasma. Fibrin fibers were thinner in clots formed in the presence of edoxaban. In conclusion, edoxaban at clinically relevant concentrations accelerates t-PA-induced fibrinolysis via increasing plasmin generation in human plasma. The effects of edoxaban is mainly dependent on TAFI. The profibrinolytic effect of edoxaban might contribute to the efficacy for the treatment of VTE.

Clinical Impact of Coagulation and Fibrinolysis Markers for Predicting Postoperative Venous Thromboembolism in Total Joint Arthroplasty Patients.

One common complication after joint arthroplasty is venous thromboembolism (VTE). Therefore, it is essential to measure the changes in coagulation and fibrinolysis in order to predict VTE among patients who underwent joint arthroplasty. This study aimed to identify potential useful biomarkers for prognosing to VTE. This was a prospective cohort study enrolling 83 patients who underwent joint arthroplasty. The levels of d-dimer, thrombin–antithrombin complex (TAT), plasmin–α2-antiplasmin complex (PIC), soluble thrombomodulin, and tissue plasminogen activator inhibitor complex were measured on day 0 (before surgery) and days 1, 3, and 6 after surgery. Ultrasound examination was used to diagnose VTE on preoperative day 0 and postoperative day 6. A total of 35 patients developed VTE after surgery. Patients with VTE exhibited significantly higher levels of d-dimer and TAT on postoperative days 3 and 6 (all P < .05). The area under curves (AUC) of receiver operating characteristic (ROC) were 0.65 and 0.68 and 0.68 and 0.74 for d-dimer and TAT levels on postoperative days 3 and 6, respectively. The level of TAT/PIC ratio on postoperative day 6 was significantly increased among patients with VTE compared to non-VTE patients (P < .0001). In addition, the AUC of ROC, cutoff level, sensitivity, specificity, positive-predictive value, and negative-predictive value of TAT/PIC ratio were 0.78, 4.03 ng/TU, 97.14%, 33.33%, 51.52%, and 94.12%, respectively. The high sensitivity and negative predictive value of TAT/PIC ratio make it a potential prognostic index for diagnosing VTE during the early phase of postoperative joint arthroplasty.

A Direct Oral Anticoagulant Edoxaban Enhanced Fibrinolysis Via Inhibition of Thrombin-Activatable Fibrinolysis Inhibitor Activation and Enhancement of Plasmin Generation in Human Plasma

Introduction: Direct oral anticoagulants are as effective as vitamin K antagonists for the treatment of venous thromboembolism and are associated with less bleeding. We have reported that a direct oral factor Xa inhibitor edoxaban exerts a thrombus resolution effect in a rat model of venous thrombosis and enhances tissue plasminogen activator (t-PA)-induced clot lysis in human plasma. However, the mechanism underlying the thrombus resolution effect and fibrinolysis enhancement by edoxaban remains to be determined.Purposes: To evaluate the effect of edoxaban on plasmin generation during clot lysis in human plasma in vitro. To determine the role of thrombin-activatable fibrinolysis inhibitor (TAFI) in the enhancement of fibrinolysis by edoxaban.Methods: Pooled human normal plasma or TAFI-deficient plasma (both containing 180 ng/mL t-PA and 0.1 nM thrombomodulin) was mixed with edoxaban, an activated TAFI (TAFIa) inhibitor (a fibrinolysis enhancer) potato tuber carboxypeptidase inhibitor (PCI), or vehicle. Clot was induced by adding 2.5 pM tissue factor and 4 µM phospholipids. To monitor the clot formation and lysis, the absorbance of plasma at 405 nm was measured every 30 sec. Clot lysis time was defined as the interval between the time of the midpoint of the clear to maximum turbidity transition and the midpoint of the maximum turbidity to clear transition. Plasmin-α2 antiplasmin complex (PAP) concentration was measured by ELISA as an indicator of plasmin generation.Results: In normal plasma, PCI accelerated clot lysis and increased plasma PAP concentration. There was a correlation between plasma PAP concentration and percent of clot lysis, indicating that plasma PAP concentration is an appropriate marker of fibrinolysis. Edoxaban at clinically relevant concentrations (75, 150, and 300 ng/mL) significantly shortened clot lysis time and elevated plasma PAP concentration. The additive combined effect of edoxaban and PCI was observed. In TAFI-deficient plasma, clot lysis time was shortened and plasma PAP concentration increased compared with normal plasma. In these samples, the effects of edoxaban and PCI on clot lysis and plasma PAP concentration were markedly diminished as compared with normal plasma.Conclusions: Edoxaban at clinically relevant concentrations enhanced t-PA-induced clot lysis with increasing plasma PAP concentration in human plasma. The effects of edoxaban on clot lysis and plasmin generation were diminished in TAFI-deficient plasma. A TAFIa inhibitor PCI exerted similar effects. These data suggest that edoxaban enhanced fibrinolysis via inhibition of TAFI activation and enhancement of plasmin generation. DisclosuresMorishima:Daiichi Sankyo Co., Ltd.: Employment. Honda:Daiichi Sankyo Co., Ltd.: Employment. Furugohri:Daiichi Sankyo Co., Ltd.: Employment.

Role of fibrinolysis and α2-macroglobulin in growth of primary adenocarcinoma and rectal polyps.

e15093 Background: An important role in tumor angiogenesis induction is played by hydrolytic systems, in particular plasmin one which provides degradation of the extracellular matrix, activates growth factors and metalloproteinases. Our purpose was to analyze the tissue fibrinolytic system and α2-macroglobulin (α2M) in primary adenocarcinoma (PA) and polyps (P) of the rectum (R). Methods: Tissues of tumors (РА, G2, T2-3N0M0, 42-73 years) and their perifocal zone (PZ) in R (n = 24) and Р (n = 27) were studied by ELISA; the result was calculated per 1 mg of tissue. Results: Resection line (RL) tissues contained levels of tРА antigen and its active form (tРА-Ag and tРА-act) 6.7 and 33.8 times higher than prourokinase and urokinase levels (uPA-Ag and uPA-act). RL tissues in P did not differ significantly from R tissues at PA resection. uPA-act in PA was higher than in RL by 3.2 times, uPA-Ag - 8 times higher, tРА-act - 2.3 times lower; tPA-Ag was similar to RL tissue values. Plasmin-α2-antiplasmin complex (PAP) in PA was 1.4 times higher than in RL. Plasminogen (PG) in PA was 1.5 times lower than in RL (p &lt; 0.01). Activity of α2M in PA and RL did not differ. PAP and α2М in PA PZ were similar to RL, and PG did not differ from PA. Other indices were between tumor and RL levels. P tissue did not show changes in uPA; tPA-act activity was 1.3 times lower than in RL, and tPA-Ag 1.3 times higher (p &lt; 0.01). PAP in tissues of 88.2% P was 1.5 times higher than in RL and 1.7 times lower than in PA. PG and α2М were similar in P and RL. PAP in PZ of P was 2.3 times higher than in RL and 1.5 times higher than in PA PZ. PG in P PZ was 1.4 times lower than in RL (p &lt; 0.01). α2М activity in P PZ was higher than in RL and P by 5.4 times on average. uPA in P PZ did not differ from RL; tРА-Ag and tРА-act were higher than in RL by 1.5 and 1.9 times and exceeded P by 2.1 and 1.5 times (p &lt; 0.01). Prevalence of uPA, activation of PG in PA and its PZ indicated degradation of the extracellular matrix compared with the corresponding P tissues. Conclusions: The role of R tumor PZ as a "metabolic" tumor field in neoplasm progression was confirmed. Activation of fibrinolysis in PA and its PZ with a deficiency of inhibitors stimulates the migration and proliferation of cells. Increasing tРА, РАР and α2М in PZ of polyps has a protective effect.

Differences in plasminogen activation in primary rectal adenocarcinoma with and without liver metastasis.

e15111 Background: Plasminogen activators play a key role in the cascade fibrinolytic system as they catalyze plasmin formation from plasminogen (PG). Plasminogen activators together with plasmin are directly or indirectly involved in tumor growth. The purpose of the study was to compare the plasminogen activation system in primary adenocarcinomas (РА) of the rectum (R) with and without liver metastases (LM). Methods: Tissues of tumors (РА, st. III, G2, 38-74 years) and their perifocal zone (PZ) with LM (T2-3NхM1, n = 24) and without them (T2-3N0M0, n = 32) were studied by ELISA. Results: The studied parameters did not differ in apparently intact R tissues in the resection line (RL) with and without LM. Plasmin-α2-antiplasmin complex (PAP) was 1.4 times higher in PA with LM than without LM (p &lt; 0.05). Prourokinase and urokinase levels (uPA-Ag and uPA-act) were 12.4 and 3.7 higher than in RL. uPA-Ag and uPA-act in PA+LM were higher than without LM by 1.4 and 1.6 times. PG levels and α2-macroglobulin (α2M) activity in PA+LM were lower by 1.5 and 2.7 times than in PA without LM and lower than in RL (p &lt; 0.01). All studied parameters, except α2M, in PA+LM were activated more than in PA without LM. Low α2M content supposed realization of effects of uPA and plasmin in insufficient inhibitory control. Most parameters in PZ+LM were between the levels in PA and RL being significantly different from both and significantly exceeding the values in PZ of PA without LM. α2М was an exception being 1.8 times lower in PZ of PA+LM than in PZ of PA without LM. The results demonstrated higher uPA and РАР levels in PA and its PZ with LM than without them, with increased PG consumption and decreased α2M activity in these parts of the rectum. Activation of plasminogen system in tissues of PA+LM and in PA without LM was unidirectional. Conclusions: Levels of uPA and PAP, as well as low α2M content in PA and PZ with LM can be used as an indicator of tumor metastatic activity.

Intracardiac Hemostasis and Fibrinolysis Parameters in Patients with Atrial Fibrillation.

Aims To identify intracardiac hemostasis or fibrinolysis abnormalities, which are associated with atrial fibrillation (AF) and increase the risk of thromboembolism. Patients and Methods Patient group consisted of 24 patients with AF and control group included 14 individuals with other supraventricular tachycardia undergoing transcatheter radiofrequency ablation. Blood samples were drawn from the femoral vein (FV), left atrium (LA), and left atrial appendage (LAA) before the ablation procedure. Fibrinogen, factor VIII (FVIII) and factor XIII activity, von Willebrand factor (VWF) antigen, thrombin-antithrombin (TAT) complex, quantitative fibrin monomer (FM), plasminogen, α2-plasmin inhibitor, plasmin-α2-antiplasmin (PAP) complex, PAI-1 activity, and D-dimer were measured from all samples. Results Levels of FVIII and VWF were significantly elevated in the FV and LA of AF patients as compared to controls. TAT complex, FM, PAP complex, and D-dimer levels were significantly elevated in the LA as compared to FV samples in case of both groups, indicating a temporary thrombotic risk associated with the catheterization procedure. Conclusions None of the investigated hemostasis or fibrinolysis parameters showed significant intracardiac alterations in AF patients as compared to non-AF controls. AF patients have elevated FVIII and VWF levels, most likely due to endothelial damage, presenting at both intracardiac and systemic level.

Coagulation during elective neurosurgery with hydroxyethyl starch fluid therapy: an observational study with thromboelastometry, fibrinogen and factor XIII

BackgroundSeveral studies have described hypercoagulability in neurosurgery with craniotomy for brain tumor resection. In this study, hydroxyethyl starch (HES) 130/0.42 was used for hemodynamic stabilization and initial blood loss replacement. HES can induce coagulopathy with thromboelastographic signs of decreased clot strength. The aim of this study was to prospectively describe perioperative changes in coagulation during elective craniotomy for brain tumor resection with the present fluid regimen.MethodsForty patients were included. Perioperative whole-blood samples were collected for EXTEM and FIBTEM assays on rotational thromboelastometry (ROTEM) and plasma fibrinogen analysis immediately before surgery, after 1 L of HES infusion, at the end of surgery and in the morning after surgery. Factor (F)XIII activity, thrombin-antithrombin complex (TAT) and plasmin-α2-antiplasmin complex (PAP) were analysed in the 25 patients receiving ≥1 L of HES.ResultsMost patients (37 of 40) received HES infusion (0.5–2 L) during surgery. Preoperative ROTEM clot formation/structure, plasma fibrinogen and FXIII levels were generally within normal range but approached a hypocoagulant state during and at end of surgery. ROTEM variables and fibrinogen levels, but not FXIII, returned to baseline levels in the morning after surgery. Low perioperative fibrinogen levels were common. TAT levels were increased during and after surgery. PAP levels mostly remained within the reference ranges, not indicating excessive fibrinolysis. There were no differences in ROTEM results and fibrinogen levels in patients receiving <1 L HES and ≥1 L HES.ConclusionsOnly the increased TAT levels indicated an intra- and postoperative activation of coagulation. On the contrary, all other variables deteriorated towards hypocoagulation but were mainly normalized in the morning after surgery. Although this might be an effect of colloid-induced coagulopathy, we found no dose-dependent effect of HES. The unactivated fibrinolysis indicates that prophylactic use of tranexamic acid does not seem warranted under normal circumstances in elective neurosurgery. Individualized fluid therapy and coagulation factor substitution is of interest for future studies.

TAVI induces an elevation of hemostasis-related biomarkers, which is not causative for post-TAVI thrombocytopenia

BackgroundThrombocytopenia after transcatheter aortic valve implantation (TAVI) has been evaluated in several studies, but the pathomechanisms behind this phenomenon are not well described. Here, we assess the influence of TAVI on hemostasis-related biomarkers and their potential role in post-TAVI thrombocytopenia. Methods and ResultsWe assessed perioperative platelet counts in 307 patients undergoing transfemoral TAVI. Additionally, hemostasis-related biomarkers including thrombin–anti-thrombin complex (TAT), prothrombin activation fragment 1+2 (F1+2), plasmin–α₂-antiplasmin complex (PAP), and d-dimer were assessed in a subcohort of 35 patients. The course of these parameters was correlated with platelet counts and compared in patients with and without a relevant thrombocytopenia, i.e., a decline in platelet count of ≥50% compared to baseline.Levels of TAT (8.6±8.7μg/l), PAP (992±597μg/l), and d-dimer (1.43±1.66mg/l) were elevated above normal range before TAVI. TAT, F1+2, and d-dimer significantly increased after TAVI, and d-dimer as well as PAP remained elevated until day 7, indicating TAVI-induced increased thrombin formation associated with secondary fibrinolysis. Post-TAVI thrombocytopenia occurred in 17.9% of patients and was associated with a significantly higher incidence of post-TAVI complications, e.g. acute kidney injury and vascular complications. No impact of activated coagulation on thrombocytopenia was observed. ConclusionsTAVI induces an increase in hemostasis-related biomarkers, which persists until 1week after the procedure. The frequent finding of post-TAVI thrombocytopenia appears not to be linked to increased thrombin formation.

Label-Free Kinetic Studies of Hemostasis-Related Biomarkers Including D-Dimer Using Autologous Serum Transfusion.

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-α2-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 μg/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.

Biomarkers of Coagulation and Fibrinolysis during Cemented Total Hip Arthroplasty with Pre- versus Postoperative Start of Thromboprophylaxis

Venous thrombosis is common in elective hip surgery, and prophylaxis is recommended. Clinical trials suggest that the drug dose and timing of initiating prophylaxis significantly influence antithrombotic effectiveness and safety. We studied the time course and gradient of plasma coagulation and fibrinolysis during total hip arthroplasty (THA) in twenty patients that were randomly assigned to have the first dose of 5000 IU dalteparin subcutaneously (sc) injected 12 hours before or 6 hours after surgery. Baseline characteristics were similar in both groups. Specific biomarkers on coagulation (prothrombin fragment 1+2 (F1+2)) and fibrinolytic activity (plasmin/α2-antiplasmin complex (PAP) and D-dimer) were collected at six events during hospitalization and analysed. There were no significant group differences in the biomarkers at any time point. The highest concentrations were measured 6 hours after surgery and before the first postoperative injection. A marked decrease followed at the first postoperative day, and then a second increase in plasma concentrations was observed 6 days after surgery. This study showed that activation of coagulation and fibrinolysis by the operative trauma was the same when the first dose of dalteparin was injected 12 hours before or 6 hours after surgery.