Clinical value of TAT, PIC and t-PAIC as predictive markers for severe sepsis in pediatric patients.

Objective: Sepsis in pediatric patients can progress to severe sepsis, and identifying biomarkers of this progression may permit timely intervention to prevent it. This study aimed to investigate the ability of thrombin-antithrombin complex (TAT), α2-plasmininhibitor-plasmin complex (PIC) and tissue-type plasminogen activator-inhibitor complex (t-PAIC) to predict severe sepsis in pediatrics early. Methods: 148 eligible pediatric sepsis patients were enrolled in this study, and were then divided into those who progressed to severe sepsis (n = 50) or not (n = 98). Serum levels of TAT, PIC, and t-PAIC were analysed, and simplified pediatric critical illness score (PCIS) and DIC score were calculated on the day of pediatric sepsis diagnosis. Results: Compared with sepsis patients, severe sepsis patients had higher levels of TAT, PIC and t-PAIC. Correlation analysis revealed that TAT, PIC and t-PAIC were significantly correlated with simplified PCIS and DIC score. ROC curve analysis suggested that TAT, PIC and t-PAIC could serve as biomarkers for predicting severe sepsis with the AUC up to 0.862, 0.759 and 0.851, respectively. Stratified analysis demonstrated that the patients with increased levels of TAT, PIC and t-PAIC had worse illness severity and clinical outcome. Univariate logistic regression analysis revealed that TAT, PIC and t-PAIC were all risk factors for severe sepsis, yet only TAT and t-PAIC were independent risk factors in multivariate model. Conclusions: TAT, PIC and t-PAIC could serve as biomarkers for predicting severe sepsis, and correlated with illness severity in pediatrics, what's more, serum levels of TAT and t-PAIC may be independent risk factors for pediatric severe sepsis.

Antithrombin supplementation attenuates heparin resistance in plasma spiked with Gla-domainless factor Xa S195A in vitro.

BACKGROUND: Andexanet alfa is a Gla-domainless mutant (S195A) factor Xa (GDXa) approved for acute reversal of oral factor Xa inhibitors. Cardiac surgery patients exposed to andexanet before cardiopulmonary bypass often exhibit severe heparin resistance. There is a paucity of data on the effectiveness and optimal dosage of antithrombin use in this setting. The objective of this study was to evaluate the in vitro effect of increased heparin with antithrombin levels on attenuating heparin resistance induced by GDXa. METHODS: Heparinised normal pooled plasma and cardiopulmonary bypass plasma were spiked with GDXa 4 µM. Tissue factor-activated thrombin generation was used to assess heparin reversal effects of GDXa and restoration of anticoagulation with additional heparin with and without antithrombin. Serum thrombin-antithrombin complex, antithrombin activity, and tissue factor pathway inhibitor were also measured in tissue factor-activated, recalcified cardiopulmonary bypass plasma spiked with GDXa. RESULTS: In normal pooled plasma, GDXa-induced heparin reversal was mitigated by maintaining a high heparin concentration (12 U ml-1) and supplementing antithrombin (1.5-4.5 µM) based on peak and velocity of thrombin generation. Heparin reversal by GDXa was also demonstrated in cardiopulmonary bypass plasma, but supplementing both heparin (8 U ml-1) and antithrombin (3 µM) attenuated GDXa-induced changes in peak and velocity of thrombin generation by 72.5% and 72.2%, respectively. High heparin and antithrombin levels attenuated thrombin-antithrombin complex formation in tissue factor-activated, GDXa-spiked cardiopulmonary bypass plasma by 85.7%, but tissue factor pathway inhibitor remained depleted compared with control cardiopulmonary bypass plasma. CONCLUSIONS: Simultaneous supplementation of heparin and antithrombin mitigate GDXa-induced heparin resistance by compensating for the loss of tissue factor pathway inhibitor.

Performance of direct oral anticoagulant (DOAC) testing by hemostasis laboratories: The Australasian/Asia-Pacific experience.

INTRODUCTION: Direct oral anticoagulants (DOACs) reflect anticoagulation agents given to treat or prevent thrombosis, having largely replaced vitamin K antagonists (VKAs) such as warfarin. DOACs are given in fixed daily doses and generally do not need monitoring. However, there may be a variety of reasons that justify measurement of plasma DOAC levels in individual patients. METHODS: We report updated findings for DOAC testing in our geographic region, using recent data from the RCPAQAP, an international external quality assessment (EQA) program, currently with some 40-60 participants in each of the different DOAC (rivaroxaban, apixaban, dabigatran) modules, to assess laboratory performance in this area. Data has been assessed for the past 5 years (2019-2023 inclusive), with 20 samples each per DOAC. RESULTS: Data shows a limited repertoire of assays in use, and mostly consistency in reported numerical values when assessing proficiency samples. Available assays mostly comprised reagents from four manufacturing suppliers. There was good consistency across what participants identified as 'DOAC detected', but some variability when participants attempted to grade DOAC levels as low vs moderate vs high. Inter-laboratory/method coefficient of variation (CVs) were generally <15% for each DOAC, when present at >100 ng/mL. CONCLUSION: We hope our findings, reflecting on mostly consistent reporting of DOAC levels and interpretation provides reassurance for clinicians requesting these measurements, and helps support their implementation in regions where there is a paucity of test availability.

Perioperative management of venous recanalization in a patient with inherited antithrombin deficiency: case report.

Background: Inherited antithrombin (AT) deficiency (ATD) is a severe thrombophilia causing venous thromboembolism, which can be complicated by postthrombotic syndrome (PTS). Venous recanalization, used to treat PTS, often requires a temporary withdrawal of anticoagulant therapy. In ATD patients, there is a risk of insufficient perioperative anticoagulation due to altered heparin response. Key Clinical Question: There is no consensus on how to manage perioperative anticoagulation in ATD patients. Clinical Approach: Warfarin-unfractionated heparin transition could be a more reliable strategy than low-molecular-weight heparin transition because unfractionated heparin anti-Xa activity not only reflects heparin-bound AT but also AT's activity, which correlates strongly with therapeutic anticoagulation. Biological monitoring could thus decrease the number of plasma-derived AT supplementation. Conclusion: This study describes a successful perioperative management of anticoagulation for venous recanalization that could be suggested to type 1 ATD patients with PTS.

Natural Anticoagulant Protein Levels in Patients With Beta-Thalassemia Major: A Case-Control Study.

Background: ß-thalassemia is a group of inherited blood disorders that affect the production of ß-globin chains, leading to the reduction or absence of these chains. One of the complications observed in patients with ß-thalassemia major (ß-TM) is thrombosis, especially in those who receive frequent blood transfusions. This may be due to a decrease in the levels of the natural anticoagulants: protein C (PC), total protein S (PS), and antithrombin (AT). Methods: In this case-control study, patients with ß-TM, who had received at least 20 packed cell transfusions during their lifetime, were included. Patients with other underlying diseases like bleeding or thrombotic disorders were excluded. Totally, 118 patients with ß-TM and 120 healthy individuals were included. Results: The mean level of PC and AT was significantly lower in patients with ß-TM (48.2 ± 65.4 and 57.42 ± 13.6, respectively) compared to the control group (97.1 ± 21.46 and 81.79 ± 14.3, respectively), with P value of 0.001 and 0.01, respectively. Although the difference was not statistically significant (P = 0.1), a similar trend was observed for total PS (61.12 ± 21.12 for patients versus 72.2 ± 35.2 for the control group). Of note, the decrease in PC, AT, and total PS levels compared to the control group was 50.36%, 27.5%, and 15.34%, respectively. Conclusions: It seems that ß-TM patients who receive prolonged blood transfusions frequently are at an increased risk of decreased in natural anticoagulants levels and therefore potentially are at risk of thrombosis.

A systematic review and meta-analysis of the effectiveness of primary thromboprophylaxis in acute lymphoblastic leukemia during early-phase therapy including asparaginase or its prolonged form.

Asparaginase is essential in the initial management of acute lymphoblastic leukemia (ALL) but frequently leads to venous thromboembolism (VTE). Using anticoagulants for primary VTE prevention has been studied with no consensus. We conducted a systematic literature search in PubMed, Scopus, and Web of science and performed random-effect meta-analysis using Mantel-Haenszel method in RevMan 5.4 to analyze primary pharmacological thromboprophylaxis during asparaginase treatment in early-phase (induction, consolidation, or intensification phase) therapy in patients with ALL with all ages and followed with subgroup analysis by age. Meta-analysis of 13 articles describing the effect of antithrombin supplementation in 1375 patients showed that antithrombin prophylaxis decreases the risk of VTE by 43% (RR, 0.57; 95% CI, 0.38 - 0.83; p=0.004), with mild heterogeneity (I2=35%, p=0.10) and moderate certainty by GRADE. 8 articles included for meta-analysis of low-molecular weight heparin (LMWH) treatment in 612 patients showed that it decreased the risk of VTE by nearly 40% (RR, 0.61; 95% CI, 0.45 - 0.81; p=0.00081), with minimal heterogeneity (I2=14%, p=0.31) but low certainty. Subgroup analysis showed that only prophylaxis with antithrombin supplementation significantly decreased the VTE rate in adult patients with moderate certainty. In pediatric patients, one nonrandomized prospective study showed that LMWH combined with antithrombin has a better thromboprophylaxis effect than antithrombin alone. In the PREVAPIX-ALL trial, prophylaxis with direct factor Xa inhibitor Apixaban did not benefit children younger than 18 years except for cases of obesity. We concluded that thromboprophylaxis with antithrombin is effective in ALL patients older than 18 years during the early phase of therapy, and LMWH combined with antithrombin supplementation might be effective for pediatric patients with ALL. Apixaban is effective in pediatric ALL patients with obesity and needs further study in other high-risk patients.

Efficacy of antithrombin administration for patients with sepsis: A systematic review, meta-analysis, and meta-regression.

Aims: There have been inconsistent reports regarding the effect of antithrombin on sepsis; furthermore, there are limited reports on how dosage affects therapeutic efficacy. Thus, we aimed to perform a systematic review and meta-analysis of the use of antithrombin for sepsis and a meta-regression analysis of antithrombin dosage. Methods: We included randomized controlled trials (RCTs) and observational studies of adult patients with sepsis who received antithrombin. Outcomes included all-cause mortality and serious bleeding complications. Statistical analyses and data synthesis were performed using a random-effects model; further, meta-regression and funnel plots were used to explore heterogeneity and biases. Results: Seven RCTs and six observational studies were included. Most patients in the RCTs and observational studies had severe sepsis and septic-disseminated intravascular coagulation (DIC), respectively. A meta-analysis using RCTs showed no significant differences in mortality between the antithrombin and control groups. However, the meta-analysis of observational studies indicated a trend of decreasing mortality rates with antithrombin administration (odds ratio [OR], 0.79; 95% confidence interval [CI], 0.68-0.92; p = 0.002). Bleeding complications were significantly higher in the antithrombin group than in the control group in both study types (OR, 1.90; 95% CI, 1.52-2.37; p < 0.01). The meta-regression analysis showed no correlation between antithrombin dosage and mortality. Conclusion: A meta-analysis of RCTs confirmed no survival benefit of antithrombin, whereas that of observational studies, which mostly focused on septic DIC, showed a significant beneficial effect on improving outcomes. Indications of antithrombin should be considered based on its beneficial and harmful effects.

Protein disulfide isomerase uses thrombin-antithrombin complex as a template to bind its target protein and alter the blood coagulation rates.

During inflammation and situations of cellular stress Protein Disulfide Isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) in addition to the purified recombinant PDI (175nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (Quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and western blot analysis showed that the PDI does not form a stable complex with either thrombin or Antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.

The role of inflammation and antithrombin supplementation on thromboelastographic parameters during veno-venous ECMO for respiratory failure.

INTRODUCTION: Extracorporeal membrane oxygenation (ECMO) may act as a driver or propagator of systemic inflammation. In turn, cytokine release can modify thromboelastographic (TEG) tests which are commonly used for anticoagulation monitoring. In this context, antithrombin (AT) supplementation might further modify TEG. METHODS: This is a pre-specified sub-study of the "Randomized Controlled Trial of Antithrombin Supplementation During Extracorporeal Membrane Oxygenation" study (investigator-initiated, randomized, single-blind, two-arm trial) conducted in two Italian ECMO referral ICUs. Adult patients requiring vv-ECMO for respiratory failure and undergoing unfractioned heparin (UFH) administration were enrolled and randomized whether to receive AT supplementation. Plasma samples for cytokine assay (IL-8, IL-10, IL-6, IL-1ß, TNF-α and Pro-ADM) and heparinase TEG were collected from every patient before ECMO start, 24 h and 72 h after ECMO start, before ECMO removal, and 7 days after ECMO removal or upon ICU discharge whichever happened first. AT concentration, coagulation and clinical data were collected before ECMO start and at pre-fixed time points. RESULTS: Thirty-nine patients were enrolled (21 treatments, 18 controls). TEG-R had a weak-to-moderate positive correlation with IL-8, IL-6, IL-10 and TNF-α and a moderate positive correlation with Pro-ADM. TEG-ANG showed a weak negative correlation with IL-8, IL-6 and TNF-α, while TEG-MA negatively correlated with IL-8, TNF-α and Pro-ADM. AT supplementation seemed to modify the association between TEG-MA and IL-8, IL-10 and Pro-ADM; conversely, AT did not affect the relationship among TEG-R or TEG-ANG and the studied cytokines. CONCLUSIONS: High concentrations of systemic cytokines correlated with longer reaction times and decreased angle and amplitude at TEG, suggesting that an increase in inflammation is related with hypocoagulability as revealed by thromboelastography.

Normalizing Anti-Thrombin III for heparin management during routine cardiopulmonary bypass for congenital cardiothoracic surgery: A single institution practice review.

INTRODUCTION: Over the past decade, there has been an increase in the use of recombinant Anti-Thrombin III (AT-III) administration during neonatal and pediatric short- and long-term mechanical support for the replacement of acquired deficiencies. Recombinant AT-III (Thrombate) administration is an FDA licensed drug indicated primarily for patients with hereditary deficiency to treat and prevent thromboembolism and secondarily to prevent peri-operative and peri-partum thromboembolism. Herein we propose further use of Thrombate for primary AT-III deficiency of the newborn as well as for acquired dilution and consumption secondary to cardiopulmonary bypass (CPB). METHODOLOGY: All patients undergoing CPB obtain a preoperative AT-III level. Patients with identified deficiencies are normalized in the OR using recombinant AT-III as a patient load, in the CPB prime, or both. Patient baseline Heparin Dose Response (HDR) is assessed using the Heparin Management System (HMS) before being exposed to AT-III. If a patient load of AT-III is given, a second HDR is obtained and this AT-III Corrected HDR is used as the primary goal during CPB. Once CPB is initiated, an AT-III level is obtained with the first patient blood analysis. A subtherapeutic level results in an additional dose of AT-III. During the rewarm period, a final AT-III level is obtained and AT-III treated once again if subtherapeutic. A retrospective, matched analysis review of practice analyzing two groups, a Study Group (Repeat HDR, May 2022 onward) and Matched Group (Without Repeat HDR, July 2019 to April 2022), for age (D), weight (Kg) and operation was conducted. The focus of the study was to determine any change in heparin sensitivity identified post AT-III patient bolus load in the HDR (U/mL), Slope (U/mL/s), ACT (s), and total amount of heparin on CPB (U) and protamine (mg) used in each group. RESULTS: No significance was seen in Baseline AT-III (%), post heparin load HDR (U/mL), first CPB ACT (s), first CPB HDR (U/mL), or total CPB heparin (u/Kg) between the two groups. Statistical significance was seen in Baseline ACT (s), Baseline HDR (U/mL), Baseline Slope (U/mL/s), Post Heparin Load ACT (s), first CPB AT-III (%), and Protamine (mg/Kg) (p < .05). No statistical significance was seen in the Study Intragroup between pre versus post AT-III patient load baseline sample in ACT (s), however significance was seen in HDR (U/mL) and Slope (U/mL/s) (p < .05). CONCLUSION: Implementation of AT-III monitoring and therapy before and during CPB in conjunction with the HMS allows patients to maintain a steady state of anticoagulation with overall less need for excessive heparin replacement and potentially thrombin activation. The result is obtaining a steady state of anticoagulation, a reduced fluctuation in the heparin and ACT levels and a potential for lower co-morbidities associated with prolonged CPB times.

A novel factor IXa-specific enzyme-linked immunosorbent assay detects factor IXa in human plasma.

Background: Factor (F)IXa activity has been detected in human plasma and may impact thrombotic risk. Current FIXa activity assays are complex and cumbersome. Objectives: To develop a reproducible enzyme-linked immunosorbent assay (ELISA) using a novel monoclonal antibody that detects total FIXa in human plasma. Methods: A monoclonal antibody was raised against the new N-terminus exposed upon activation of FIX to FIXa by cleavage after R226. This antibody is specific for FIXa protease and does not recognize FIX zymogen or FIXα. The antibody was used to develop a FIXa-specific ELISA capable of quantifying total FIXa (free FIXa and FIXa-antithrombin complex) in human plasma. Total FIXa quantified using the ELISA was compared to that of FIXa-antithrombin quantified using modifications of a previously described ELISA. Results: The FIXa-specific ELISA was reproducible and quantified total FIXa in human plasma. Total FIXa levels correlated with FIXa-antithrombin levels. Conclusion: A monoclonal antibody was developed that specifically detects human FIXa protease. A FIXa-specific ELISA using the new antibody is capable of reproducibly measuring total FIXa in human plasma (both free FIXa and FIXa-antithrombin). This assay should facilitate the evaluation of total FIXa levels in a variety of clinical circumstances.

Prophylaxis with enoxaparin and antithrombin III in drug-induced coagulation alterations in childhood leukemia: a retrospective experience of 20 years.

BACKGROUND: Thromboembolic complications are well known in the treatment of childhood acute lymphoblastic leukemia. Over the years it has not been possible to reach a consensus on a possible prophylaxis of thromboembolic events during intensive therapy. Only the administration of enoxaparin was able to achieve evidence in the literature to date. METHODS: In this retrospective study, 173 childhood leukemia patients were treated over 20 years with a thromboembolic prophylaxis including enoxaparin and AT III during induction therapy with L-asparaginase and cortisone. RESULTS: We here report the effectiveness of administration of enoxaparin and AT III in childhood leukemia, showing a strikingly low prevalence of deep vein thrombosis (2.9%). Especially in adolescent patients, a particularly great need for AT III was demonstrated. CONCLUSIONS: We recommend thromboembolic prophylaxis with enoxaparin and AT III substitution during induction/reinduction therapy with L-asparaginase and glucocorticosteroids, especially from adolescence onwards.

Efficacy and safety of antithrombin supplementation in neonates and infants on a continuous heparin infusion.

Background: Antithrombin (AT) is a natural anticoagulant and potent inhibitor of several coagulation proteins, including activated factor X (FXa) and FIIa. The therapeutic activity of heparin depends on the presence of AT. Levels of plasma AT are low in neonates and young infants compared to those in adults. Exogenous AT supplementation is postulated to enhance the activity of heparin and facilitate attainment of therapeutic anticoagulation in infants. Objectives: To describe the efficacy and safety of AT administration in infants on a continuous heparin infusion. Methods: Retrospective cohort study of 50 infants who received AT while on a heparin infusion. The primary efficacy outcome was attainment of therapeutic anticoagulation within 48 hours after AT administration. Secondary outcomes included the percent of partial thromboplastin time (PTT) levels and/or antifactor Xa (anti-FXa) activity within the therapeutic window, attainment of the target AT activity level, the incidence and severity of bleeding, and all-cause in-hospital mortality. A secondary analysis investigated the relationship between simultaneously measured PTT levels and anti-FXa activity used for heparin monitoring. Results: AT supplementation resulted in achievement of at least one therapeutic PTT level or anti-FXa activity in 90% of AT courses, though not sustained. PTT was within the therapeutic window more often than anti-FXa activity. When measured simultaneously, therapeutic anti-FXa levels were associated with supratherapeutic PTT levels. Conclusion: AT supplementation in infants on a continuous heparin infusion may transiently improve the therapeutic effect of heparin, but this is largely dependent on the laboratory parameters used for monitoring.

Clinical and Molecular Characterization of Nine Novel Antithrombin Mutations.

Antithrombin (AT) is the major plasma inhibitor of thrombin (FIIa) and activated factor X (FXa), and antithrombin deficiency (ATD) is one of the most severe thrombophilic disorders. In this study, we identified nine novel AT mutations and investigated their genotype-phenotype correlations. Clinical and laboratory data from patients were collected, and the nine mutant AT proteins (p.Arg14Lys, p.Cys32Tyr, p.Arg78Gly, p.Met121Arg, p.Leu245Pro, p.Leu270Argfs*14, p.Asn450Ile, p.Gly456delins_Ala_Thr and p.Pro461Thr) were expressed in HEK293 cells; then, Western blotting, N-Glycosidase F digestion, and ELISA were used to detect wild-type and mutant AT. RT-qPCR was performed to determine the expression of AT mRNA from the transfected cells. Functional studies (AT activity in the presence and in the absence of heparin and heparin-binding studies with the surface plasmon resonance method) were carried out. Mutations were also investigated by in silico methods. Type I ATD caused by altered protein synthesis (p.Cys32Tyr, p.Leu270Argfs*14, p.Asn450Ile) or secretion disorder (p.Met121Arg, p.Leu245Pro, p.Gly456delins_Ala_Thr) was proved in six mutants, while type II heparin-binding-site ATD (p.Arg78Gly) and pleiotropic-effect ATD (p.Pro461Thr) were suggested in two mutants. Finally, the pathogenic role of p.Arg14Lys was equivocal. We provided evidence to understand the pathogenic nature of novel SERPINC1 mutations through in vitro expression studies.

SERPINC1 c.1247dupC: a novel SERPINC1 gene mutation associated with familial thrombosis results in a secretion defect and quantitative antithrombin deficiency.

BACKGROUND: Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. METHODS: Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein (GFP) or hemagglutinin (HA) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. RESULTS: The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. CONCLUSION: The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

The Inhibition of Serine Proteases by Serpins Is Augmented by Negatively Charged Heparin: A Concise Review of Some Clinically Relevant Interactions.

Serine proteases are members of a large family of hydrolytic enzymes in which a particular serine residue in the active site performs an essential role as a nucleophile, which is required for their proteolytic cleavage function. The array of functions performed by serine proteases is vast and includes, among others, the following: (i) the ability to fight infections; (ii) the activation of blood coagulation or blood clot lysis systems; (iii) the activation of digestive enzymes; and (iv) reproduction. Serine protease activity is highly regulated by multiple families of protease inhibitors, known collectively as the SERine Protease INhibitor (SERPIN). The serpins use a conformational change mechanism to inhibit proteases in an irreversible way. The unusual conformational change required for serpin function provides an elegant opportunity for allosteric regulation by the binding of cofactors, of which the most well-studied is heparin. The goal of this review is to discuss some of the clinically relevant serine protease-serpin interactions that may be enhanced by heparin or other negatively charged polysaccharides. The paired serine protease-serpin in the framework of heparin that we review includes the following: thrombin-antithrombin III, plasmin-anti-plasmin, C1 esterase/kallikrein-C1 esterase inhibitor, and furin/TMPRSS2 (serine protease Transmembrane Protease 2)-alpha-1-antitrypsin, with the latter in the context of COVID-19 and prostate cancer.

Prothrombin conversion and thrombin decay in patients with cirrhosis-role of prothrombin and antithrombin deficiencies.

BACKGROUND: Thrombin generation (TG) in the presence of thrombomodulin (TG-TM) in the plasma of patients with cirrhosis (PWC) is tilted toward a hypercoagulable phenotype. Low protein C and elevated factor VIII levels play a role, but other determinants, such as the prothrombin/antithrombin pair, must also be studied. OBJECTIVES: The objectives were (i) to quantitatively assess the subprocesses (prothrombin conversion and thrombin decay) and (ii) to understand the underlying mechanism by studying TG dynamics after prothrombin and antithrombin plasma level correction in PWC. METHODS: We studied TG-TM in plasma samples of 36 healthy controls (HCs) and 41 PWC with prothrombin and antithrombin levels of <70% and after their correction. We initiated coagulation with an intermediate picomolar concentration of tissue factor. We determined the overall thrombin potential, prothrombin conversion, and thrombin decay. RESULTS: TG-TM was increased in PWC compared with HC due to impaired thrombin inhibition. Indeed, thrombin decay capacity (min-1) decreased from 0.37 (0.35-0.40) in HC to 0.33 (0.30-0.37) in the Child-Turcotte-Pugh A (CTP-A; P = .09), 0.27 (0.26-0.30) in the CTP-B (P < .001), and 0.20 (0.19-0.20) in the CTP-C (P < .001) group. Concomitant correction of prothrombin and antithrombin increased endogenous thrombin potential with prothrombin conversion surpassing thrombin decay. By contrast, when we corrected only antithrombin, TG-TM was normalized and even consistent with a hypocoagulable phenotype in the CTP-C group. CONCLUSION: Our results highlight that in PWC, hypercoagulability (evidenced in the presence of TM) is due to impaired thrombin decay, whereas low prothrombin levels do not translate into decreased prothrombin conversion, likely due to altered TM-activated protein C negative feedback.

Daboialipase, a phospholipase A2 from Vipera russelli russelli venom posesses anti-platelet, anti-thrombin and anti-cancer properties.

Snake venoms are known to contain toxins capable of interfering with normal physiological processes of victims. Specificity of toxins from snake venoms give scope to identify new molecules with therapeutic action and/or help to understand different cellular mechanisms. Russell's viper venom (RVV) is a mixture of many bioactive molecules with enzymatic and non-enzymatic proteins. The present article describes Daboialipase (DLP), an enzymatic phospholipase A2 with molecular mass of 14.3 kDa isolated from RVV. DLP was obtained after cation exchange chromatography followed by size-exclusion high performance liquid chromatography (SE-HPLC). The isolated DLP presented strong inhibition of adenosine di-phosphate (ADP) and collagen induced platelet aggregation. It also showed anti-thrombin properties by significantly extending thrombin time in human blood samples. Trypan blue and resazurin cell viability assays confirmed time-dependent cytotoxic and cytostatic activities of DLP on MCF7 breast cancer cells, in vitro. DLP caused morphological changes and nuclear damage in MCF7 cells. However, DLP did not cause cytotoxic effects on non-cancer HaCaT cells. Peptide sequences of DLP obtained by O-HRLCMS analysis showed similarity with a previously reported PLA2 (Uniprot ID: PA2B_DABRR/PDB ID: 1VIP_A). An active Asp at 49th position, calcium ion binding site and anticoagulant activity sites were identified in 1 VIP_A. These findings are expected to contribute to designing new anti-platelet, anticoagulant and anti-cancer molecules.

Thrombosis-related characteristics of pregnant women with antithrombin deficiency, protein C deficiency and protein S deficiency in Japan.

BACKGROUND:  We previously conducted a primary survey of pregnant women with hereditary thrombophilia based on national surveillance in Japan, but did not examine their thrombosis-related characteristics. Antithrombin (AT) deficiency, protein C (PC) deficiency and protein S (PS) deficiency are the major types of hereditary thrombophilia in Japan. METHODS: We examined their detailed information related to thrombosis, and evaluated peripartum outcomes in comparison with control data obtained from the Japan Society of Obstetrics and Gynecology. RESULTS: Definite or probable AT deficiency, PC deficiency and PS deficiency were observed in 80, 50, and 317 pregnancies, respectively, from 2014 to 2018 in Japan, with prevalence rates among total deliveries of 0.011%, 0.007%, 0.044%. The number of pregnancies with AT, PC and PS deficiency might have been as many as 27, 17 and 108 every year if complete answers had been provided. In the peripartum period of current pregnancies, 27.5% of women with AT deficiency, 28.0% with PC deficiency and 13.2% with PS deficiency developed thrombosis (p < 0.001 vs. control). Pregnant women with AT and PC deficiency were more susceptible to thrombosis than those with PS deficiency (P < 0.01). Of the thromboses, 92.3% occurred during pregnancy, 62.8% at less than 15 gestational weeks. The earliest onset of thrombosis was 5 gestational weeks. Prophylactic anticoagulation significantly prevented the onset of both antepartum and postpartum thrombosis (p < 0.0001). The rate of recurrent pregnancy loss in women with low PC or PS activities was significantly higher than in controls (p < 0.0001); however, it is unknown whether recurrent pregnancy loss is related to hereditary PS deficiency. There seem to have been few serious maternal or fetal/neonatal complications due to placental insufficiency related to a hypercoagulable state other than growth restriction. CONCLUSIONS: This survey revealed the thrombosis-related characteristics of pregnant women with hereditary thrombophilia in Japan. We suggest prophylactic anticoagulation to prevent maternal or fetal/neonatal complications.