Development of a chemiluminescence assay for tissue plasminogen activator inhibitor complex and its applicability to gastric cancer.

BACKGROUND: Venous thromboembolism (VTE), is a noteworthy complication in individuals with gastric cancer, but the current diagnosis and treatment methods lack accuracy. In this study, we developed a t-PAIC chemiluminescence kit and employed chemiluminescence to detect the tissue plasminogen activator inhibitor complex (t-PAIC), thrombin-antithrombin III complex (TAT), plasmin-α2-plasmin inhibitor complex (PIC) and thrombomodulin (TM), combined with D-dimer and fibrin degradation products (FDP), to investigate their diagnostic potential for venous thrombosis in gastric cancer patients. The study assessed variations in six indicators among gastric cancer patients at different stages. RESULTS: The t-PAIC reagent showed LOD is 1.2 ng/mL and a linear factor R greater than 0.99. The reagents demonstrated accurate results, with all accuracy deviations being within 5%. The intra-batch and inter-batch CVs for the t-PAIC reagent were both within 8%. The correlation coefficient R between this method and Sysmex was 0.979. Gastric cancer patients exhibited elevated levels of TAT, PIC, TM, D-D, FDP compared to the healthy population, while no significant difference was observed in t-PAIC. In the staging of gastric cancer, patients in III-IV stages exhibit higher levels of the six markers compared to those in I-II stages. The ROC curve indicates an enhancement in sensitivity and specificity of the combined diagnosis of four or six indicators. CONCLUSION: Our chemiluminescence assay performs comparably to Sysmex's method and at a reduced cost. The use of multiple markers, including t-PAIC, TM, TAT, PIC, D-D, and FDP, is superior to the use of single markers for diagnosing VTE in patients with malignant tumors. Gastric cancer patients should be screened for the six markers to facilitate proactive prophylaxis, determine the most appropriate treatment timing, ameliorate their prognosis, decrease the occurrence of venous thrombosis and mortality, and extend their survival.

Distinctive Biomarker Features in the Endotheliopathy of COVID-19 and Septic Syndromes.

BACKGROUND: Endotheliopathy is a key element in COVID-19 pathophysiology, contributing to both morbidity and mortality. Biomarkers distinguishing different COVID-19 phenotypes from sepsis syndrome remain poorly understood. OBJECTIVE: To characterize circulating biomarkers of endothelial damage in different COVID-19 clinical disease stages compared with sepsis syndrome and normal volunteers. METHODS: Patients with COVID-19 pneumonia (n = 49) were classified into moderate, severe, or critical (life-threatening) disease. Plasma samples were collected within 48 to 72 h of hospitalization to analyze endothelial activation markers, including soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1), von Willebrand Factor (VWF), A disintegrin-like and metalloprotease with thrombospondin type 1 motif no. 13 (ADAMTS-13) activity, thrombomodulin (TM), and soluble TNF receptor I (sTNFRI); heparan sulfate (HS) for endothelial glycocalyx degradation; C5b9 deposits on endothelial cells in culture and soluble C5b9 for complement activation; circulating dsDNA for neutrophil extracellular traps (NETs) presence, and α2-antiplasmin and PAI-1 as parameters of fibrinolysis. We compared the level of each biomarker in all three COVID-19 groups and healthy donors as controls (n = 45). Results in critically ill COVID-19 patients were compared with other intensive care unit (ICU) patients with septic shock (SS, n = 14), sepsis (S, n = 7), and noninfectious systemic inflammatory response syndrome (NI-SIRS, n = 7). RESULTS: All analyzed biomarkers were increased in COVID-19 patients versus controls (P < 0.001), except for ADAMTS-13 activity that was normal in both groups. The increased expression of sVCAM-1, VWF, sTNFRI, and HS was related to COVID-19 disease severity (P < 0.05). Several differences in these parameters were found between ICU groups: SS patients showed significantly higher levels of VWF, TM, sTNFRI, and NETS compared with critical COVID-19 patients and ADAMTS-13 activity was significantly lover in SS, S, and NI-SIRS versus critical COVID-19 (P < 0.001). Furthermore, α2-antiplasmin activity was higher in critical COVID-19 versus NI-SIRS (P < 0.01) and SS (P < 0.001), whereas PAI-1 levels were significantly lower in COVID-19 patients compared with NI-SIRS, S, and SS patients (P < 0.01). CONCLUSIONS: COVID-19 patients present with increased circulating endothelial stress products, complement activation, and fibrinolytic dysregulation, associated with disease severity. COVID-19 endotheliopathy differs from SS, in which endothelial damage is also a critical feature of pathobiology. These biomarkers could help to stratify the severity of COVID-19 disease and may also provide information to guide specific therapeutic strategies to mitigate endotheliopathy progression.

Tranexamic acid rapidly inhibits fibrinolysis, yet transiently enhances plasmin generation in vivo.

Tranexamic acid (TXA) is a lysine analogue that inhibits plasmin generation and has been used for decades as an antifibrinolytic agent to reduce bleeding. Recent reports have indicated that TXA can paradoxically promote plasmin generation. Blood was obtained from 41 cardiac surgical patients randomly assigned to TXA or placebo before start of surgery (preOP), at the end of surgery (EOS), then again on postoperative day 1 (POD-1) as well as POD-3. Plasma levels of tissue-type plasminogen activator (t-PA), urokinase (u-PA), the plasmin-antiplasmin (PAP) complex, as well as t-PA and u-PA-induced clot lysis assays were then determined. Clot lysis and PAP complex levels were also assessed in healthy volunteers before and at various time points after taking 1 g TXA orally. Surgery induced an increase in circulating t-PA, yet not u-PA at EOS. t-PA levels were unaffected by TXA; however, u-PA levels were significantly reduced in patients on POD-3. t-PA and u-PA-induced clot lysis were both inhibited in plasma from TXA-treated patients. In contrast, PAP complex formation, representing plasmin generation, was unexpectedly enhanced in the plasma of patients administered TXA at the EOS time point. In healthy volunteers, oral TXA effectively blocked fibrinolysis within 30 min and blockade was sustained for 8 h. However, TXA also increased PAP levels in volunteers 4 h after administration. Our findings demonstrate that TXA can actually augment PAP complex formation, consistent with an increase in plasmin generation in vivo despite the fact that it blocks fibrinolysis within 30 min. This may have unanticipated consequences in vivo.

28-day thawed plasma maintains α2 -antiplasmin levels and inhibits tPA-induced fibrinolysis.

INTRODUCTION: Evidence supports the use of plasma-first resuscitation in the treatment of trauma-induced coagulopathy (TIC). While thawed plasma (TP) has logistical benefits, the ability of plasma proteins to attenuate fibrinolysis and correct TIC remain unknown. We hypothesize that TP retains the ability to inhibit tissue plasminogen activator(tPA)-induced fibrinolysis at 28-day storage. METHODS: Healthy volunteers underwent blood draws followed by 50% dilution of whole blood (WB) with TP at 28-, 21-, 14-, 7-, 5-, and, 0-day storage, normal saline (NS), and WB control. Samples underwent citrated tPA-challenge (75 ng/ml) thromboelastography (TEG). Plasminogen activator inhibitor-1 (PAI-1) and α2 -antiplasmin (α2 -AP) concentrations in thawed or stored plasma were determined. RESULTS: In the presence of tPA, 28-day TP inhibited tPA-induced coagulopathy as effectively as WB. 28-day TP had a similar R-time, MA, and fibrinolysis (P > 0·05 for all) compared to WB, while angle was enhanced (P = 0·02) compared to WB. Significant correlations were present between storage time and clot strength (P = 0·04) and storage time and fibrinolysis (P = 0·0029). Active PAI-1 levels in thawed plasma were 1·10 ± 0·54 ng/mL while total PAI-1 levels were 4·79 ± 1·41 ng/mL. There was no difference of α2 -AP levels in FFP (40·45 ± 3·5 µg/mL) compared to plasma thawed for 14 (36·78 ± 5·39 µg/mL, P = 0·65) or 28 days (45·16 ± 5·61 µg/mL, P = 0·51). DISCUSSION: Thawed plasma retained the ability to inhibit tPA-induced fibrinolysis over 28-day storage at 1-4°C. α2 -AP levels were maintained in plasma thawed for 28 days and FFP. These in vitro results suggest consideration should be made to increasing the storage life of TP.

Management of disseminated intravascular coagulation associated with aortic aneurysm and vascular malformations.

Aortic aneurysms and vascular malformations are sometimes associated with disseminated intravascular coagulation (DIC). A typical blood coagulation test shows decrease in platelet count and fibrinogen, and increases in fibrin/fibrinogen degradation products (FDP) and D-dimer. The coagulation activation marker thrombin-antithrombin complex (TAT) and the fibrinolysis activation marker plasmin-α2 plasmin inhibitor (PIC) are significantly increased. α2 plasmin inhibitor (α2PI) is significantly reduced. Since no prolongation of prothrombin time (PT) is noticeable and activated partial thromboplastin time (APTT) is shortened in some cases, DIC cannot be diagnosed or ruled out by PT and APTT alone. The cornerstone of treatment for DIC is to treat the underlying disease. However, surgery is not possible in some cases. Follow-up may be appropriate in patients with abnormal results from coagulation tests and no bleeding. However, pharmacotherapy is often required in cases with bleeding. Unfractionated heparin, low molecular weight heparin, protease inhibitors, recombinant thrombomodulin, direct oral anticoagulants, and factor XIII preparations are effective. If PIC is significantly increased and α2PI is significantly decreased, or if the bleeding is severe, tranexamic acid is used as an antifibrinolytic therapy with anticoagulant therapy. In such cases, attention should be paid not only to TAT but also changes in PIC.

Characterization of fibrin/fibrinogen degradation products reagents and their utility in critical care patients with enhanced fibrinolysis.

INTRODUCTION: Fibrin/fibrinogen degradation products (FDP) values reflect coagulation and fibrinolysis status, and FDP levels are helpful for diagnosis and classification of disseminated intravascular coagulation (DIC). FDP measurement has always played a key role in diagnosing DIC, a phenomenon that has recently gained renewed attention because of its occurrence in coronavirus disease 2019 (COVID-19) patients. Although the evaluation of FDP is crucial for the management of critical care, the variability among FDP reagents is unclear. In this study, we aimed to compare LIASAUTO P-FDP with three FDP reagents and investigate their characteristics. METHODS: In total, 172 plasmas samples were used in the correlation. The sample data were divided into three groups including negative, no and positive discrepancy based on the discrepancy percentages calculated from each correlation between LIASAUTO P-FDP and other three reagents. D-dimer, plasmin-α2 plasmin inhibitor complex (PIC), fibrin monomer complex (FMC), fibrinogen (Fbg) and Plasmin-α2 Plasmin Inhibitor (α2 PI) were measured and included in data analysis. RESULTS: The positive discrepancy groups showed higher D-dimer, PIC and FMC values than the negative discrepancy groups. The data indicated that LIASAUTO P-FDP had higher reactivity to D-dimer than other reagents and the values were elevated in the fibrinolysis-enhanced samples with various FDP fragments. CONCLUSION: LIASAUTO P-FDP displayed the reactivity towards various fibrin/fibrinogen degradation products, and it might be useful for DIC diagnosis because the fibrinolytic status differed in the DIC types and stages.

Diagnostic and Prognostic Value of TAT, PIC, TM, and t-PAIC in Malignant Tumor Patients With Venous Thrombosis.

BACKGROUND: Venous thromboembolism (VTE) is an important complication in patients with malignant tumors. Its exact diagnosis and treatment are still lacking. We used a high-sensitive chemiluminescence method to detect thrombin-antithrombin III complex (TAT), plasmin-α2-plasmininhibitor complex (PIC), thrombomodulin (TM), and tissue plasminogen activator-inhibitor complex(t-PAIC) in combination with D-dimer and fibrin degradation product (FDP) to analyze their diagnostic and prognostic value in patients with malignant tumors. METHODS: In total, 870 patients with confirmed malignant tumors were included, 82 of whom had diagnosed VTE; 200 healthy individuals were classified as the control group. The TAT, PIC, TM, and t-PAIC were detected using Sysmex HISCL5000 automated analyzers, whereas FDP and D-dimer were detected using Sysmex CS5100 coagulation analyzer. Receiver operating characteristic (ROC) curves were used to evaluate the diagnostic efficiency. Survival probabilities were determined using Kaplan-Meier analysis, and multivariate analyses were performed using a Cox regression model. RESULTS: Compared with healthy controls, patients with malignant tumors showed significantly elevated TAT, PIC, TM, t-PAIC, D-dimer, and FDP. Similarly, compared with patients in the non-thrombosis group, those in the thrombosis group showed significantly elevated levels of the above mentioned markers. Logistic regression analysis showed that TAT, PIC, TM, t-PAIC, D-Dimer, and FDP were all associated with VTE. ROC analysis showed that "TAT+PIC+TM+t-PAIC+D-dimer+FDP"showed the highest sensitivity and specificity. Patients with elevated TAT, PIC, TM, and t-PAIC had a significantly shorter survival. Multivariate Cox survival analysis showed that TM and t-PAIC were significantly associated with poor prognosis. In addition, the incidence of VTE was significantly lower in patients with malignant tumors who were treated with low-molecular-weight heparin (LMWH), and their survival period was significantly longer than that of patients with malignant tumors who were not treated with LMWH. CONCLUSION: TAT, PIC, TM, and t-PAIC combined with D-dimer and FDP were better than the application of a single marker in the diagnosis of VTE in patients with malignant tumors. TAT and PIC can be used as sensitive markers in the diagnosis of VTE but not as prognostic markers. TM and t-PAIC might be independent prognostic indicators in patients with malignant tumors, regardless of the state of thrombus.

Tranexamic acid administration in the field does not affect admission thromboelastography after traumatic brain injury.

BACKGROUND: No Food and Drug Administration-approved medication improves outcomes following traumatic brain injury (TBI). A forthcoming clinical trial that evaluated the effects of two prehospital tranexamic acid (TXA) dosing strategies compared with placebo demonstrated no differences in thromboelastography (TEG) values. We proposed to explore the impact of TXA on markers of coagulation and fibrinolysis in patients with moderate to severe TBI. METHODS: Data were extracted from a placebo-controlled clinical trial in which patients 15 years or older with TBI (Glasgow Coma Scale, 3-12) and systolic blood pressure of ≥90 mm Hg were randomized prehospital to receive placebo bolus/placebo infusion (placebo), 1 g of TXA bolus/1 g of TXA infusion (bolus maintenance), or 2 g of TXA bolus/placebo infusion (bolus only). Thromboelastography was performed, and coagulation measures including prothrombin time, activated partial thromboplastin time, international ratio, fibrinogen, D-dimer, plasmin-antiplasmin (PAP), thrombin antithrombin, tissue plasminogen activator, and plasminogen activator inhibitor 1 were quantified at admission and 6 hours later. RESULTS: Of 966 patients receiving study drug, 700 had laboratory tests drawn at admission and 6 hours later. There were no statistically significant differences in TEG values, including LY30, between groups (p > 0.05). No differences between prothrombin time, activated partial thromboplastin time, international ratio, fibrinogen, thrombin antithrombin, tissue plasminogen activator, and plasminogen activator inhibitor 1 were demonstrated across treatment groups. Concentrations of D-dimer in TXA treatment groups were less than placebo at 6 hours (p < 0.001). Concentrations of PAP in TXA treatment groups were less than placebo on admission (p < 0.001) and 6 hours (p = 0.02). No differences in D-dimer and PAP were observed between bolus maintenance and bolus only. CONCLUSION: While D-dimer and PAP levels reflect a lower degree of fibrinolysis following prehospital administration of TXA when compared with placebo in a large prehospital trial of patients with TBI, TEG obtained on admission and 6 hours later did not demonstrate any differences in fibrinolysis between the two TXA dosing regimens and placebo. LEVEL OF EVIDENCE: Diagnostic test, level III.

A rat model of orthopedic injury-induced hypercoagulability and fibrinolytic shutdown.

BACKGROUND: Postinjury hypercoagulability occurs in >25% of injured patients, increasing risk of thromboembolic complications despite chemoprophylaxis. However, few clinically relevant animal models of posttraumatic hypercoagulability exist. We aimed to evaluate a rodent model of bilateral hindlimb injury as a preclinical model of postinjury hypercoagulability. METHODS: Forty Wistar rats were anesthetized with isoflurane: 20 underwent bilateral hindlimb fibula fracture, soft tissue and muscular crush injury, and bone homogenate injection intended to mimic the physiological severity of bilateral femur fracture. Twenty sham rats underwent anesthesia only. Terminal citrated blood samples were drawn at 0, 6, 12, and 24 hours (n = 5 per timed group) for analysis by native thromboelastography in the presence and absence of taurocholic acid to augment fibrinolysis. Plasminogen activator inhibitor 1 and α-2 antiplasmin levels in plasma were assessed via enzyme-linked immunosorbent assay. RESULTS: Injured rats became hypercoagulable relative to baseline by 6 hours based on thromboelastography maximal amplitude (MA) and G (p < 0.005); sham rats became hypercoagulable to a lesser degree by 24 hours (p < 0.005). Compared with sham animals, injured rats were hypercoagulable by MA and G within 6 hours of injury, remained hypercoagulable by MA and G through at least 24 hours (all p < 0.01), and showed impaired fibrinolysis by taurocholic acid LY30 at 12 hours (p = 0.019) and native LY30 at 24 hours (p = 0.045). In terms of antifibrinolytic mediators, α-2 antiplasmin was elevated in trauma animals at 24 hours (p = 0.009), and plasminogen activator inhibitor 1 was elevated in trauma animals at 6 hours (p = 0.004) and 12 hours (p < 0.001) when compared with sham. CONCLUSIONS: Orthopedic injury in rodents induced platelet and overall hypercoagulability within 6 hours and fibrinolytic impairment by 12 to 24 hours, mimicking postinjury hypercoagulability in injured patients. This rodent model of orthopedic injury may serve as a preclinical testing ground for potential therapies to mitigate hypercoagulability, maintain normal fibrinolysis, and prevent thromboembolic complications.

Administration of the extra virgin olive oil (EVOO) in mild cognitive impairment (MCI) patients as a therapy for preventing the progress to AD.

OBJECTIVE: Although Mediterranean diet is connected with longevity and lower rate of many disorders including Alzheimer's disease (AD), the effect of olive oil, which is the principal component of the Mediterranean diet, on fibrinolytic system related to AD and especially on plasminogen activator inhibitor-1 (PAI-1) and a2-antiplasmin in aged participants are not yet examined. This study was performed on 108 aged participants allocated into 5 groups: Mild Cognitive Impairment (MCI) (36) patients subjected to 1-year therapy with extra virgin olive oil (EVOO), MCI without therapy patients (26), MCI without therapy 1-year later patients (11), AD patients (30) and healthy individuals (16). Hypothesis/Purpose: To examine the effect of EVOO therapy on the fibrinolytic factors PAI-1 and a2-antiplasmin, on hallmarks of AD, tau and Aß amyloid fragments and on an oxidative stress biomarker, MDA in the serum of MCI patients aiming to be exploited as a future preventive therapy. RESULTS: Using ELISA method, the levels of both fibrinolytic factors PAI-1 and a2- antiplasmin in the serum of MCI patients were reduced notably in the EVOO treated patients versus the control group and were lower than those of all other groups. For better determination of AD from other pathological conditions the ratio Aß1-42/Aß1-40 was measured in serum of all participants. The more lessened the ratio is, the more cognitive impairment is observed in patients. The MCI group with one-year EVOO therapy displayed a ratio similar to this of healthy individuals. Moreover, patients with EVOO therapy showed decreased tau protein levels in comparison with all the other groups. The levels of the oxidative stress's biomarker, malondialdehyde (MDA) showed a significant decrease in MCI patients subjected to EVOO therapy revealing the involvement of the beneficial antioxidative properties of EVOO in the progression of AD. CONCLUSION: We demonstrated that EVOO therapy may prevent the risk of patients with MCI to progress to AD via decreasing fibrinolytic factors PAI-1 and a2 antiplasmin that reflecting in the diminution of the hallmarks proteins of AD, tau and Aß amyloid as well and in a biomarker of oxidative stress, MDA.

Effect of storage of plasma in the presence of red blood cells and platelets: re-evaluating the shelf life of whole blood.

BACKGROUND: There is renewed interest in administering whole blood (WB) for the resuscitation of patients with bleeding trauma. The shelf life of WB was established decades ago based on the viability of red blood cells. However, plasma quality during WB storage is not established. STUDY DESIGN AND METHODS: White blood cell- and platelet-reduced WB (WB-PLT) was prepared using standard processes and compared to WB processed using a platelet-sparing WBC reduction (WB + PLT) filter. WB (± PLT) was held at 2 to 6°C for 35 days alongside control units of red blood cells (RBCs) in saline, adenine, glucose, and mannitol and liquid plasma. A series of assays explored the coagulation potential and RBC quality. RESULTS: While fibrinogen and α2-antiplasmin remained unaffected by storage, other factors varied between components or over time at 2 to 6°C. At 14 days factor V, factor VII, α2 -antiplasmin and free protein S antigen remained on average greater than 0.50 IU/mL or 50%, as appropriate, in WB ± PLT. Factor VIII was on average 0.49 IU/mL in WB+PLT, and 0.56 IU/mL for WB-PLT. Free protein S activity decreased significantly in all arms but remained on average greater than 40% at Day 14. Contact activation was not demonstrated before Day 14. Thrombin generation in plasma remained relatively stable to Day 35 in all arms. CONCLUSIONS: Clotting factor activity remained at or above a mean of 0.5 IU/mL, or 50%, at Day 14 for factor V, factor VII, factor VIII, free protein S, fibrinogen, and α2-antiplasmin in all arms. Further data on platelet function in WB+PLT is needed to inform its shelf life.

Prothrombotic clot properties can predict venous ulcers in patients following deep vein thrombosis: a cohort study.

Venous ulcers are the most severe manifestation of post-thrombotic syndrome (PTS). We have previously demonstrated that formation of compact fibrin clots resistant to lysis is observed in patients following deep-vein thrombosis (DVT) who developed PTS. The current study investigated whether unfavourable fibrin clot properties can predict post-thrombotic venous ulcers. In a cohort study on 186 consecutive patients following DVT, we determined plasma fibrin clot characteristics, including clot permeability and lysability, inflammatory markers, thrombin generation, fibrinolysis proteins at 3 months since the index event. Occurrence of PTS and venous ulcers was recorded during follow-up (median, 53; range 24 to 76 months). Fifty-seven DVT patients (30.6%) developed PTS, including 12 subjects (6.45%) with a venous ulcer (4 individuals with recurrent ulcers). Patients who developed ulcers compared with the remainder had at enrolment 13.0% lower clot permeability (Ks), 17.4% longer clot lysis time (CLT), 13.1% longer lag phase of clot formation, and 5.0% higher maximum absorbance, with no difference in fibrinogen, C-reactive protein, and thrombin generation. The baseline prothrombotic fibrin clot phenotype (Ks ≤ 6.5 × 10-9 cm2 and CLT > 100 min) was associated with a higher risk of ulcers [hazard ratio (HR), 5.37; 95% confidence interval (CI), 1.3-21.5]. A multivariate model adjusted for age, sex, and fibrinogen showed that independent predictors of the ulcer occurrence were body mass index (HR 1.53; 95% CI 1.30-1.86), CLT (HR 1.43; 95% CI 1.04-2.05), and α2-antiplasmin (HR 0.95; 95% CI 0.90-0.99). This study suggests that formation of denser fibrin clots with impaired fibrinolysis predisposes to post-thrombotic venous ulcers.

A new ELISA method for the measurement of total α2-plasmin inhibitor level in human body fluids.

The ever-increasing research efforts to develop new antithrombotic therapies have led to the reassessment of the role of alpha-2-plasmin inhibitor (α2-PI) in pathological conditions. In particular, experimental stroke studies have suggested correlation between increased free α2-PI level and mortality. However there are only a small number of well-characterized and specific assays available for the measurements of free α2-PI. In plasma α2-PI undergoes both N- and/or C-terminal cleavages resulting four isoforms with modified susceptibility to FXIII catalyzed cross-linking to fibrin and/or loss of plasmin(ogen) binding. Present paper describes a new sandwich ELISA method for the determination of free total α2-PI in plasma and other body fluids. A newly generated biotinylated monoclonal antibody recognizes and captures all the four N- and/or C-terminally modified isoforms of α2-PI while HRPO-labeled polyclonal anti-α2-PI antibody detects the captured antigen. Performing the 2-step assay in streptavidin-coated microplate can be completed within three hours. The assay is well reproducible, total (within laboratory) imprecision in the normal, pathological and very low ranges were 7.4%, 9.1% and < 19%, respectively. When examining the plasma samples of 197 healthy volunteers, 100 acute ischemic stroke patients and 102 patients with venous thrombosis, strong correlation was observed between total α2-PI antigen levels and α2-PI activity for each group. Using the assay a reference interval of 45-86 mg/L was established for total α2-PI mass concentration in the plasma. α2-PI levels were also measured in cerebrospinal fluid samples of 47 individuals the median value and range was 132 (36-379) µg/L. In conclusion, our ELISA enables accurate and fast measurement of total free α2-PI in human body fluids.

Performance evaluation of thrombomodulin, thrombin-antithrombin complex, plasmin-α2-antiplasmin complex, and t-PA: PAI-1 complex.

BACKGROUND: To conduct a comprehensive performance evaluation of a fully automated analyzer for measuring thrombomodulin (TM), thrombin-antithrombin complex (TAT), plasmin-α2-antiplasmin complex (PAP), and t-PA: PAI-1 complex (tPAI-C). METHODS: According to the Clinical and Laboratory Standards Institute (CLSI) EP05-A2, EP06-A specifications, TM, TAT, PAP, and tPAI-C were analyzed to evaluate intraassay variability and interassay variability, linear range, carryover rate, reference range, sample stability, and interferences. RESULTS: The intraassay variability and interassay variability of the four factors were all below 5%. The carryover rates were below 1%. Linear verification analysis revealed correlation coefficients of 0.998-0.999. The recommended reference ranges of TM, TAT, and PAP were appropriate for our laboratory, whereas the reference of tPAI-C should be established by each laboratory. Stability assessment revealed that TM is stable for 2 days at room temperature but lacks stability at colder temperatures. In contrast, TAT is stable for 5 days at 4°C and -20°C but has poor stability at room temperature. PAP and tPAI-C are stable for 3 days at all three temperatures. The measurement of TM, TAT, PAP, and tPAI-C is not altered by the presence of 510 mg/dL hemoglobin, 1490 FTU triglycerides, or 21.1 mg/dL conjugated and free bilirubin. CONCLUSION: The determination of TM, TAT, PAP, and tPAI-C using a high-sensitivity chemiluminescence analyzer performs well in terms of precision, carryover rate, linear range, and interference. Thus, this method is suitable for the detection of these substances in clinical specimens.

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 180 ng/mL t-PA and 0.1 nM 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 300 ng/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.

Association of imbalanced sex hormone production with excessive procoagulation factor SerpinF2 in preeclampsia.

BACKGROUND: Preeclampsia, a serious pregnancy-associated syndrome, is the leading cause of maternal and perinatal morbidity and mortality. Significant exacerbation of the hypercoagulation status as well as imbalanced steroid hormones have been reported in developed preeclampsia. However, it remains unclear whether the two pathological changes are directly associated. METHOD AND RESULTS: Our proteomic analysis revealed a significantly elevated SerpinF2/α2-antiplasmin level in preeclampsia plasma. Measurement of the longitudinally gestational change of plasmin-α2-antiplasmin (PAP) complex, testosterone, estradiol in preeclampsia patients and normal pregnant women demonstrated that the circulating PAP and testosterone levels in the early-onset preeclampsia (E-PE) patients were substantially higher, whereas estradiol concentration was significantly lower than that in normal pregnant controls from early pregnancy throughout gestation. Correlation analysis revealed that circulating PAP is in positive correlation with the concentration of testosterone, and in negative correlation with estradiol in E-PE patients. In E-PE placenta, the productions and activities of 17ß-hydroxysteroid dehydrogenases 3 and aromatase, the essential enzymes for testosterone and estradiol synthesis, were compromised. In human renal and trophoblastic cells, testosterone and estradiol could regulate SerpinF2 expression in opposite ways. In addition, obvious fibrin deposition was colocalized with SerpinF2 in intervillous spaces and the area surrounding syncytiotrophoblasts in E-PE placenta. CONCLUSION: The findings reveal a tight correlation between the imbalanced steroid hormone production and the procoagulation factor in E-PE patients, which provide potential biomarkers to predict preeclampsia, and bring new insight into the pathogenesis of preeclampsia.

Evaluation the combined diagnostic value of TAT, PIC, tPAIC, and sTM in disseminated intravascular coagulation: A multi-center prospective observational study.

BACKGROUND: Accurate and early diagnosis is important in the management of disseminated intravascular coagulation (DIC). We employed new automation technology to detect plasma biomarkers, including thrombin-antithrombin complex (TAT), α2-plasmininhibitor-plasmin complex (PIC), soluble thrombomodulin (sTM), and tissue plasminogen activator-inhibitor complex (tPAIC), and evaluated their diagnostic performance and prognostic value for DIC in Chinese population. METHODS: This prospective observational study included 444 patients with suspected DIC and 137 healthy people. The molecular markers were measured by qualitative chemiluminescence enzyme immunoassay performed on HISCL automated analyzers. All patients with suspected DIC were followed for 7 days to screen for the development of overt-DIC and 28 days for mortality. RESULTS: According to the International Society of Thrombosis and Haemostasis (ISTH) scoring system, 157 patients were diagnosed as overt-DIC and 36 were diagnosed as pre-DIC. All four biomarkers were significantly higher in DIC patients than in non-overt DIC patients; TAT, tPAIC, and sTM were significantly higher in pre-DIC patients than in non-overt DIC patients. Four molecular markers behaved differently among various underlying diseases. TAT, tPAIC, and sTM were also good predictors of 28-day mortality, high levels were associated with poor outcomes. CONCLUSIONS: TAT, PIC, tPAIC, and sTM demonstrated good diagnostic performance and prognostic value in DIC patients with different underlying diseases. Besides, TAT, tPAIC and sTM have certain implications in pre-DIC stage. Combination of four makers was demonstrated better behavior than single one.