Antithrombin Activity Research Articles

Effect of Chlorella pyrenoidosa meal and Sinotaia quadrata viscera as diets on growth, digestive enzyme activities, antithrombin activity, and gut morphology of Whitmania pigra

Effect of Chlorella pyrenoidosa meal and Sinotaia quadrata viscera as diets on growth, digestive enzyme activities, antithrombin activity, and gut morphology of Whitmania pigra

Comparison of the pharmacokinetics and pharmacodynamics of apixaban and rivaroxaban in dogs.

Comparative pharmacokinetics and pharmacodynamics (PK/PD) of apixaban and rivaroxaban have not been studied in dogs and the propensity of these drugs to cause hypercoagulability after discontinuation is unknown. Compare the PK/PD of clinical dosing regimens of PO apixaban and rivaroxaban administered repeatedly to healthy dogs and assess the effect of abrupt drug discontinuation on coagulation. Six University-owned, purpose-bred, middle-aged, mixed-breed dogs (4 male, 2 female). Dogs were given apixaban or rivaroxaban PO at 0.5 mg/kg q12h for 7 days with a 14-day washout period between drugs. Plasma drug concentrations were quantitated, and anticoagulant effects were measured using clotting times, calibrated anti-Xa bioactivity assays, and measurements of thrombin generation. The potential for rebound hypercoagulability was assessed by measuring D-dimers, thrombin-antithrombin (TAT) complexes, and antithrombin activity after drug discontinuation. Plasma drug concentrations and anti-Xa bioactivities were closely correlated for both drugs, but drug concentrations varied considerably among dogs, despite consistent dose regimens. Thrombin generation variables were significantly correlated with the anti-Xa bioactivity of both drugs and no significant differences in the effects of apixaban and rivaroxaban on thrombin generation were observed. Drug discontinuation had no effect on D-dimer concentrations. The concentration of TAT complexes decreased after apixaban discontinuation and did not change after rivaroxaban discontinuation. Repeated PO administration of apixaban or rivaroxaban to healthy dogs produced comparable anticoagulant effects measured by inhibition of thrombin formation. Rebound hypercoagulability after drug discontinuation was not observed and weaning of these drugs in clinical patients might not be necessary.

PEGylation of New Thrombin Inhibitor Peptide Ultravariegin for Prolonged In Vivo Circulation and Enhanced Antithrombotic Effects.

Anticoagulant therapy is commonly used to prevent and treat arterial and venous blood clots in patients with cardiovascular disease, cerebrovascular disease, and cancer. Venous blood clots are the third leading cause of cardiovascular death following acute coronary artery disease and stroke. There is a significant need for effective anticoagulant therapy with minimal risk of bleeding. Variegin and its variants are a new type of antithrombin peptide that has shown promising results in preclinical studies. Variegin and its best variant, ultravariegin (UV), can more effectively inhibit blood clot formation while causing less bleeding than traditional medications such as heparin and bivalirudin. However, the short lifespan of UV remains a limitation for its use in clinical settings. PEGylation, a method of conjugating poly(ethylene glycol) (PEG) chains to peptides or drugs, may help improve the effectiveness of UV by extending its circulation time in the body. In this study, UV was PEGylated using maleimide-PEG5k and 10k. The impact of PEGylation on the antithrombin activity of UV was assessed in vitro and ex vivo in rat and rabbit plasma, showing minimal effects on the efficacy. In vivo studies in rats and rabbits revealed that PEGylated UV had a longer half-life and greater anticoagulant effects than unmodified UV did, especially when it was administered subcutaneously. PEGylation significantly extended the half-life of UV in rabbits, resulting in sustained anticoagulant effects for up to 4 days. This demonstrated that increasing the size of UV and shielding it with PEG could reduce clearance by the kidneys and prolong its circulation time. The improved half-life and antithrombin activity of PEGylated UV make it a more favorable choice for anticoagulant therapy.

Novel SERPINC1 Mutation in a Brazilian Patient with Sudden-onset Sagittal Sinus Thrombosis Caused by Congenital Antithrombin Deficiency

A 19-year-old Brazilian man visited the emergency department of our hospital complaining of nausea and fever. He presented with left hemispatial neglect. Magnetic resonance imaging led to a diagnosis of sagittal sinus thrombosis. Blood tests showed decreased antithrombin (ATIII) activity, and ATIII deficiency was diagnosed. He was treated with heparin sodium and warfarin. Genetic counseling through medical interpreters was recommended, and the patient and his mother consented to genetic testing, which revealed a novel mutation variant of the SERPINC1 gene. Genetic counseling is important to connect patients to genetic testing, even if language barriers are present.

A comprehensive strategy integrating chemical profiling with deep learning for screening the antithrombin bioactive-chemical quality markers of functional food: Pueraria radix as an example

Pueraria lobata radix (PL) and Puerariae tbatae radix (PT) are nutritious medicinal food homology plants that are widely used in the food and health products industry. Both of them are used as Pueraria radix (PR) for medicine. In this study, the function-related active markers were discovered by chemical profiling coupled with deep learning for assessment of PR. Firstly, IR spectral fingerprints were established. And the IR spectra of PL and PT were different in fingerprint region. HCA, PCA and PLS-DA can distinguish the samples of PL and PT. Furtherly, UPLC-Q-TOF-MS was further applied to identify the main components of PL and PT, and 30 chemical components in PL and 15 in PT were identified. In vitro antithrombin assay was performed to indicate the different batches of PL and PT extracts and they could be distinguished according to the strength of activity. The potential active markers related to antithrombin activity were screened by PCA and PLS-DA. Finally, 3′-hydroxy puerarin, puerarin, 3′-methoxy puerarin, daidzin, and daidzein were screened-out as functional markers applying molecular docking combined with BP-ANN, CP-ANN, SIMCA, and SVMDA. The thrombin-based discovery strategy of bioactive-chemical marker combination was a powerful tool for screening the quality markers for evaluation of PR.

Reduced Plasma Selenoprotein P Is Associated With Type I Antithrombin Deficiency and a Prothrombotic State.

A positive association between antithrombin activity and selenium level was reported. Selenoprotein P, the most important selenium carrier, was identified within human plasma fibrin clots. To investigate the relationship between selenoprotein P and antithrombin and its role in modulation of fibrin clot properties in antithrombin-deficient patients. Proteomic analysis of plasma fibrin clots was performed with mass spectrometry. In 108 patients with genetically confirmed type I (57%) or type II (43%) antithrombin deficiency and in healthy controls (n = 50), we assessed plasma selenoprotein P levels and thiobarbituric acid-reactive substances by enzyme-linked immunosorbent assay, along with fibrin clot permeability, clot lysis time, and thrombin generation. Clot-bound antithrombin concentration was 0.46 ± 0.32 mg/g protein, while selenoprotein P level was 30-fold lower (0.015 ± 0.012 mg/g). Type I compared to type II antithrombin-deficient patients had higher clot-bound antithrombin and selenoprotein P levels (both P < .001), associated together (ρ = 0.93, P < .001). Individuals with type I compared to type II antithrombin deficiency or controls had about 40% lower plasma selenoprotein P levels (P < .001). In antithrombin-deficient patients, plasma selenoprotein P was associated with antithrombin antigen (ρ = 0.35, P < .001) and thiobarbituric acid-reactive substances (ρ = 0.42, P < .001). Plasma selenoprotein P correlated also with endogenous thrombin potential (r = -0.33, P < .001), fibrin clot permeability (r = 0.43, P < .001), and clot lysis time (r = -0.40, P < .001) in antithrombin-deficient patients but not in controls. Patients with type I antithrombin deficiency had higher clot-bound selenoprotein P and reduced plasma selenoprotein P levels. Plasma selenoprotein P was associated with prothrombotic fibrin clot phenotype and enhanced thrombin generation.

A sorbent modified with melatonin and lithium: in vitro investigation of the effect on hemostatic reactions

Aim of the study was to evaluate the effect of lithium- and melatonin-containing sorbent based on aluminum oxide and polydimethylsiloxane on changes in the number of platelets during hemosorption modeling and on the features of the hemostatic response during dosed contact of the sorbent with blood in an in vitro experiment. Material and methods. An analysis of the effect of the porous sorbent modified with melatonin (MT, 0.15 %) and lithium (0.5 %) based on aluminum oxide and polydimethylsiloxane (Al2O3@PDMS/MT-Li) was carried out in comparison with sorbent without modifiers (Al2O3@PDMS) and modified with MT (Al2O3@PDMS/MT) on a number of donor blood clotting parameters under in vitro hemosorption conditions. Studies of the hemostatic system included assessment of platelet count, chronometric parameters, fibrinogen concentration, antithrombin activity and plasminogen content. For integral assessment, calibrated thrombography and computer thromboelastometry were used. Results and discussion. Contact of all studied sorbents with blood causes a moderate decrease in the number of platelets (by 5.3–10.1 % from initial). Comparison sorbents reduce fibrinogen concentration by 7.1–7.7 %, Al2O3@PDMS/MT-Li – by 2.6 times, which is likely due to the methodology for determining this protein against the background of the independent anticoagulant activity of lithium ions. Al2O3@PDMS and Al2O3@PDMS/MT cause the development of a hypercoagulable shift, as evidenced by a shortening of kaolin time (by 27.5 and 22.1 %, respectively) and of activated partial thromboplastin time (APTT) by 7.1 % for both sorbents. At the same time, when lithium was included in the sorbent, not only did the hypercoagulation shift not occur, but blood clotting was also inhibited, as evidenced by an increase in kaolin time and APTT by 1.2 and 1.6 times, respectively, as well as in silicone time. Conclusions. Modifying sorbents with biologically active substances, lithium and MT, makes it possible to obtain an original hemosorbent with new properties. The presented results demonstrated the absence of a hypercoagulable shift in donor blood after contact with a lithium-, MTcontaining sorbent in vitro and indicate the potential for its using as a basis for the development of safe drugs.

Acquired antithrombin deficiency in adult patients with postcardiotomy extracorporeal membrane oxygenation.

This study aimed to investigate the relationship between acquired antithrombin deficiency in patients undergoing postcardiotomy extracorporeal membrane oxygenation (PC-ECMO) and thromboembolic or haemorrhagic events such as bleeding, peripheral arterial thromboembolism, and ischemic cerebrovascular events. The study was designed as a single-center, prospective study and conducted at our hospital between November 2019 and June 2021. 50 patients who underwent ECMO due to postcardiotomy cardiogenic shock were included in the study. Antithrombin (AT) activity testing was performed immediately after ECMO placement and continued for 5 days. The total of haemorrhagic or thromboembolic events was defined as morbidity. The entire patient population was assessed daily for AT measurements according to morbidity status, and ROC analysis was applied to determine the cut-off point. The correlation between clinical outcomes and morbidities with antithrombin levels was analysed. In our study, we identified a cut-off for AT levels on the first postoperative day. The risk of both bleeding (p = .006) and thromboembolism (p = .012) was significantly higher in patients below the 48.9% cut-off value. AT levels were compared with data on separation from PC-ECMO. The rate of separation from ECMO was 7.969 times higher in cases with AT levels above 51.8 on the third postoperative day and 5.6 times higher in cases with AT levels above 47.5 on the fourth postoperative day. Acquired antithrombin deficiency may develop in adults undergoing PC-ECMO. In our study, we demonstrated that in patients with low antithrombin levels, the risk of bleeding and thromboembolism increased. Additionally, since AT levels were higher in survivors, this can be considered an indicator of severity. This study is the first prospective study related to determining target antithrombin levels in adult patients undergoing PC-ECMO.

Structural characteristics of sulfated xylogalactomannan isolated from Caulerpa okamurae and its anticoagulant activity

Due to wonderful taste, rich nutrition and biological functions, many marine green algae in the genus Caulerpa have been recently developed as candidates for green caviar. A novel water-soluble sulfated xylogalactomannan CO-0-1 was obtained from the green algae Caulerpa okamurae. CO-0-1 was mainly composed of mannose (Man), galactose (Gal), and xylose (Xyl) at the ratio of 4.4:4.0:1.4 with the molecular weight at 470 kDa and the sulfate content at 12.78 %. The sulfated xylogalactomannan had Man at the backbone with →4)-β-D-Manp-(1→ and →2)-β-D-Manp-(1→ as the main chain and branches at O-3 position. The side chains contained →3)-β-D-Galp-(1→ and minor →2)-β-D-Xylp(1→. The sulfate groups only distributed at the side chains and at O-6 position of →3)-β-D-Galp-(1→ and O-4 position of (1→2)-β-D-Xylp. The anticoagulant activity indicated that CO-0-1 displayed intrinsic anticoagulant and specific anti-thrombin activities. The investigation expanded the utilization and development scene and scope of the green algae Caulerpa okamurae.

Toward the Establishment of a Harmonized Physicochemical Profiling Platform for Therapeutic Oligonucleotides: A Case Study for Aptamers Where the Higher-Order Structure Influences Physical Properties.

Oligonucleotides are short nucleic acids that serve as one of the most promising classes of drug modality. However, attempts to establish a physicochemical evaluation platform of oligonucleotides for acquiring a comprehensive view of their properties have been limited. As the chemical stability and the efficacy as well as the solution properties at a high concentration should be related to their higher-order structure and intra-/intermolecular interactions, their detailed understanding enables effective formulation development. Here, the higher-order structure and the thermodynamic stability of the thrombin-binding aptamer (TBA) and four modified TBAs, which have similar sequences but were expected to have different higher-order structures, were evaluated using ultraviolet spectroscopy (UV), circular dichroism (CD), differential scanning calorimetry (DSC), and nuclear magnetic resonance (NMR). Then, the relationship between the higher-order structure and the solution properties including solubility, viscosity, and stability was investigated. The impact of the higher-order structure on the antithrombin activity was also confirmed. The higher-order structure and intra-/intermolecular interactions of the oligonucleotides were affected by types of buffers because of different potassium concentrations, which are crucial for the formation of the G-quadruplex structure. Consequently, solution properties, such as solubility and viscosity, chemical stability, and antithrombin activity, were also influenced. Each instrumental analysis had a complemental role in investigating the higher-order structure of TBA and modified TBAs. The utility of each physicochemical characterization method during the preclinical developmental stages is also discussed.

Altered fibrin clot phenotype in young adults with intracerebral hemorrhage of unknown cause: A case-control study

BackgroundIntracerebral hemorrhage (ICH) of undetermined etiology occurs infrequently in young and middle-aged adults. We hypothesized that slight decreases in coagulation factors and formation of less compact fibrin clots prone to faster lysis predispose to this type of ICH. MethodsWe recruited 44 consecutive patients aged <50 years following ICH of unknown cause at least 3 months since the event. Subjects free of ICH (n = 47) matched for age, sex, BMI, and hypertension served as the control group. We assessed plasma fibrin clot permeability, turbidity and fibrinolytic capacity, along with thrombin generation, coagulation factors (F) II, FV, FVII, FVIII, FIX, FX, FXI, antithrombin, and fibrinolysis proteins. ResultsICH patients (median age 41 years, 45.5 % women) had 8.4 % lower FII (p = 0.0001) and 10.1 % lower FVII activity (p = 0.0003), 9.4 % higher antithrombin activity (p = 0.0004) and 13.5 % lower platelet count (p = 0.02). Other factors and thrombin generation did not differ between the two groups. The ICH survivors were characterized by impaired fibrin polymerization reflected by 10.1 % longer lag phase of the turbidimetry curve (p = 0.0002), decreased fiber density indicated by 11.8 % lower maximum absorbance (p = 0.004), as well as 11.1 % shorter clot lysis time (p = 0.014) and 10.0 % faster increase of maximal D-Dimer levels (p = 0.000001). ConclusionsWe demonstrated a prohemorrhagic fibrin clot phenotype, along with lower FII, FVII and higher antithrombin activity in adults below 50 years of age who suffered from ICH of unknown cause, which might indicate novel mechanisms contributing to ICH in younger individuals.

Evaluation of coagulation profile in dogs with ACTH-dependent hyperadrenocorticism compared to healthy dogs by rotational thromboelastometry (ROTEM)

Despite their low morbidity, thromboembolic events in hyperadrenocorticism are associated with high mortality. Identifying the main hemostatic abnormalities will improve the prophylactic approach of these canine patients. The aim of this study was to evaluate hemostatic alterations related with ACTH-dependent HAC and its association with hypercoagulable state. For this purpose, 25 dogs diagnosed with ACTH-dependent HAC were compared with 28 healthy dogs as a control group. The hemostatic variables included platelet count, antithrombin, fibrinogen, D-dimer, PT, aPTT, rotational thromboelastometry (ROTEM) and platelet aggregation. Results showed a hypercoagulable state in 32% (8/25) dogs by ROTEM, which had at least 2 of the next features: decreased coagulation time (CT) or clot formation time (CFT) on INTEM (5/25) or EXTEM (4/25); increased maximum clot firmness (MCF) on INTEM (9/25), EXTEM (6/25) and FIBTEM (9/25). These same variables had a significant difference (P≤ 0.05) compared with the control group, as well as the parameters of α-angle and CT. Median fibrinogen levels (310 vs.178 mg/dL), mean platelet aggregation (11.1 vs. 7.9 Ohms), median platelet count (360 vs. 225 ×103/µL) and mean antithrombin activity (140 vs. 119%) were increased in ACTH-dependent HAC dogs compared to control group. PT (7.1 vs. 8.0 seconds) and aPTT (11.6 vs. 15.2 seconds) were also shortened in ACTH-dependent HAC dogs. Our findings confirm the presence of a hypercoagulable tendency in dogs with HAC. Although multifactorial, fibrinogen concentration and MCF FIBTEM showed the relevance of this protein for hypercoagulability in HAC.

Clinical outcomes of antithrombin III supplementation in an overt disseminated intravascular coagulation: a longitudinal single-institutional experience and retrospective analysis.

Antithrombin is a small plasma glycoprotein synthesized in the liver that belongs to the serpin family of serine protease inhibitors and inactivates several enzymes in the coagulation pathway. It plays a leading major factor on coagulation pathway, therefore administration of antithrombin is essential to treat serious clinical conditions such as disseminated intravascular coagulation (DIC). Despite the theoretical benefits of antithrombin supplementation, the optimal antithrombin activity for heparin efficacy and the benefits of antithrombin supplementation in various disease entities are not yet fully understood. The strict administration guidelines on antithrombin III in cases of DIC by the National Health Insurance Service and the Ministry of Food and Drug Safety complied as follows: antithrombin levels below 20 mg/dL in adults; antithrombin activity below 70% of normal in adults; total administration period of antithrombin must be carefully limited to within maximum 3 days, and the total administration dose must be below 7,000 international unit (IU), (loading dose, 1,000 IU in 1 hour: maintenance dose, 500 IU every 6 hours for 3 days). We identified 76 eligible for analysis according to the above-mentioned criteria in our institution (male/female, 59/17). Forty-four were identified to the non-survivor group and 32 patients were recognized as the survivor group. The baseline parameters in the non-survivor and survivor groups were comparable with no significant differences in age (66.5±18.1 vs. 66.0±16.2 years, P=0.90), sex (32/12 vs. 27/5, P=0.35), hospital length of stay (31.1±34.5 vs. 31.2±26.1 days, P=0.99), sequential organ failure assessment (SOFA) (7.3±2.5 vs. 6.6±2.0, P=0.22), simplified acute physiology score II (SAPS II) (46.0±8.8 vs. 43.5±9.2, P=0.23), cause for DIC (P=0.95), and underlying disease (P=0.38). The levels of antithrombin III on the day just before the administration significantly lower in the non-survivor groups than in the survivor groups (50.1%±13.6% vs. 57.6%±12.5%, P=0.01). The hemoglobin level in the 2nd day and 7th day after antithrombin III administration was significantly different between the non-survivor and survivor groups (9.9±1.9 vs. 11.0±2.0 g/dL, P=0.01, and 9.4±1.8 vs. 10.5±1.6 g/dL, P=0.006). The antithrombin III levels on the day of administration [area under the curve (AUC) =0.672] demonstrated significantly better prediction of mortality than the A antithrombin III levels on 1st day (AUC =0.552), the 2nd day (AUC =0.624), and 7th day (AUC =0.593). Our study suggests that the antithrombin administration may be effective tools for DIC treatment, and may be more positively considered, especially in the cases of DIC, which is a frequent complication of septic shock, sepsis, and other critical disease entities and which is associated with a high level of mortality. Furthermore, our study also suggests that the total doses and periods of antithrombin administration, which recommended by national guidelines, may be insufficient, therefore prolongation of period and increase of total dose of antithrombin supplement might be necessary.

Fitusiran reduces bleeding in factor X–deficient mice

AbstractFactor X (FX) deficiency is a rare bleeding disorder manifesting a bleeding tendency caused by low FX activity levels. We aim to explore the use of fitusiran (an investigational small interfering RNA that silences antithrombin expression) to increase thrombin generation and the in vivo hemostatic potential under conditions of FX deficiency. We therefore developed a novel model of inducible FX deficiency, generating mice expressing <1% FX activity and antigen (f10low mice). Compared with control f10WT mice, f10low mice had sixfold and fourfold prolonged clotting times in prothrombin time and activated partial prothrombin time assays, respectively (P < .001). Thrombin generation was severely reduced, irrespective of whether tissue factor or factor XIa was used as an initiator. In vivo analysis revealed near-absent thrombus formation in a laser-induced vessel injury model. Furthermore, in 2 distinct bleeding models, f10low mice displayed an increased bleeding tendency compared with f10WT mice. In the tail-clip assay, blood loss was increased from 12 ± 16 μL to 590 ± 335 μL (P < .0001). In the saphenous vein puncture (SVP) model, the number of clots generated was reduced from 19 ± 5 clots every 30 minutes for f10WT mice to 2 ± 2 clots every 30 minutes (P < .0001) for f10low mice. In both models, bleeding was corrected upon infusion of purified FX. Treatment of f10low mice with fitusiran (2 × 10 mg/kg at 1 week interval) resulted in 17 ± 6% residual antithrombin activity and increased thrombin generation (fourfold and twofold to threefold increase in endogenous thrombin potential and thrombin peak, respectively). In the SVP model, the number of clots was increased to 8 ± 6 clots every 30 minutes (P = .0029). Altogether, we demonstrate that reduction in antithrombin levels is associated with improved hemostatic activity under conditions of FX deficiency.

Implications of Natural 3′–5′– Linkages in the Loop Region of Isomeric 2′–5′–Linked Thrombin–Binding Aptamer

AbstractThe backbone modification of the thrombin‐binding aptamer (TBA) in the TT and TGT loop regions by isomeric 2′–5′–linkages was found to impose additive destabilizing effects on the thermal stability of the G‐quadruplex structure. In contrast, the thermal stability of isomeric 2′–5′–linked TBA, i. e., isoTBA, was significantly improved by isomeric 3′–5′–phosphodiester linkages. The isoTBA, when modified with 3′–5′–linkages in both lateral TT loops (isoTBA202), exhibited higher thermal stability and enzymatic stability in comparison to other oligomers in the present study, and TBA202 showed higher antithrombin activity than other loop‐modified TBA oligomers.

Effect of Xe/O2 Inhalation on Hemostasis in Experimental Thromboplastin Pneumonitis.

We studied the effectiveness of Xe/O2 mixture inhalation (30% Xe and 70% O2, 20 min for 5 days) in a model of experimental thromboplastin pneumonitis. Inhalation of the studied mixture decreased the intensity of the inflammatory process in the lung tissue assessed by the temperature response of animals, changed lung weight and lung weight coefficient. At acute stage of pneumonitis, an increase in xenon consumption was recorded due to its retention in the gas exchange zone and a natural decrease in oxygen consumption due to partial alveolar/capillary block. The formation of pneumonitis was accompanied by a pronounced procoagulant shift in the regulation system of the aggregate state of blood. The Xe/O2 inhalations ensured physiologically optimal levels of prothrombin and activated partial thromboplastin time against the background of a moderate decrease in fibrinogen level throughout the experiment. At the same time, the activity of the natural anticoagulant antithrombin III increased from day 5 to day 14.

Phenotypic and genetic analysis of a Chinese pedigree affected with Hereditary antithrombin deficiency due to a novel variant of SERPINC1 gene

To analyze the clinical phenotype and genetic characteristics of a Chinese pedigree affected with Hereditary antithrombin deficiency. A pedigree diagnosed at the the Second Affiliated Hospital of Wenzhou Medical University, Yuying Children's Hospital in June, 2020 was selected as the study subject. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and thrombin time (TT) of the probands and their pedigree members were determined using a STA-R automatic coagulation analyzer. Antithrombin activity (AT: A) and antithrombin antigen (AT: Ag) in plasma were determined with chromogenic substrate and immunonephelometry assays. All exons and flanking sequences of the anticoagulant protein gene SERPINC1 were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with bioinformatic tools (PolyPhen-2, SIFT, Mutation Taster and PYMOL) to explore their effect on the function and structural conformation of the protein. The probands (II-2, II-10), their brother (II-5) and sons (III-1, III-8) had shown normal PT, APTT, FIB, and TT, but significantly decreased AT: A and AT: Ag, with their levels being 34%, 57%, 56%, 48%, 53% and 13.51 mg/dL, 13.44 mg/dL, 18.39 mg/dL, 17.36 mg/dL, 17.71 mg/dL, respectively. The remaining pedigree members had normal values. Sanger sequencing revealed that the probands and all affected pedigree members had harbored a heterozygous c.851T>C (p.Met284Thr) missense variant in exon 5 of the SERPINC1 gene. Bioinformatic analysis and simulation suggested that the variant has resulted in alteration of hydrogen bonds at the c.851 position, which may affect the structure of the protein. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS1+PM1+PM5+PP1+PP4). The probands and other affected members were all diagnosed with type I hereditary AT deficiency, for which the c.851T>C (p.Met284Thr) variant of the SERPINC1 gene may be accountable.

Blood coagulation system in experimental acute lung injury and its treatment with dexamethasone

The effects of short- and long-term administrations of dexamethasone on survival, severity of pulmonary edema, and hemostasis on experimental lipopolysaccharide-induced acute lung injury in rats were analyzed. Acute lung injury in rats was modeled by the intratracheal injection of lipopolysaccharide from the Salmonella enterica cell wall. White male rats were randomly divided into nine groups: the intact group consisted of 10 animals; two control groups of 20 animals each, in which acute lung injury was simulated without further treatment and removed from the experiment on day 3 or 7; six comparison groups of 20 animals each, in which, 3 h after modeling of acute lung injury and then once a day for 3 days (short mode of administration) or 7 days (long mode of administration), dexamethasone solution was administered intraperitoneally in the following doses: 0. 52 (equivalent to 6 mg/day for humans), 1. 71 (20 mg/day for humans), and 8 mg/kg/day (94 mg/day, pulse therapy for humans). On days 3 and 7, the survival rate, coagulogram values (active partial thromboplastin time, prothrombin time, activity of antithrombin, and soluble fibrin monomer complexes), and low-frequency piezotromboelastography data were assessed in the surviving animals. The results revealed that dexamethasone reduces mortality in acute lung injury and has a dose-dependent effect on the hemostasis system: with an increase in the dose administered, blood clotting processes increase and fibrinolysis is inhibited. Low-frequency piezothromboelastography with a conventional coagulogram allows for a comprehensive assessment of the hemostasis system, identifying violations, and timely drug correction.

A Multicentric Verification of Hereditary and Acquired Thrombophilia Coagulation Assays

Aims: With an ever-growing number of patients being admitted to our University Hospital Sveti Duh (Zagreb, Croatia), there was a need to positively respond to clinicians' demands about hereditary and acquired thrombophilia testing.&#x0D; Study Design: An extensive and multicentric verification of coagulation assays included in thrombophilia testing was performed on the BCS XP coagulation analyzer (Siemens Healthineers, Marburg, Germany): antithrombin activity (AT) (Innovance Antithrombin), protein C activity (PC) (Berichrom Protein C), protein S activity (PS) (Protein S Ac), free protein S antigen (free PS:Ag) (Innovance Free PS Ag), activated protein C resistance (APCR) (ProC Ac R and ProC Global + Coagulation Factor V Deficient Plasma), lupus anticoagulant (LA) screening (LA1 and activated partial thromboplastin time by using Dade Actin FSL as reagent) and confirmation test (LA2), factor VIII activity (FVIII) (Dade Actin FS and coagulation FVIII Deficient Plasma).&#x0D; Place and Duration of Study: Department of Medical Laboratory Diagnostics, University Hospital Sveti Duh, Zagreb, Croatia, between January 2020 and December 2021.&#x0D; Methodology: Verification protocol included: the precision study (CLSI EP15-A3 protocol), trueness estimation by comparison of seventy remnant plasma samples with two large Croatian hospital laboratories with established thrombophilia testing, and verification of reference intervals and cut-off values (CLSI EP28-A3C and CLSI HA-60 guidelines).&#x0D; Results: All of the obtained imprecision CVs were within the manufacturer's claims (&lt;5/10/15%). While the observed bias for PC (+1.9%) was within the EFLM performance specifications (6.7%), the average bias for AT was higher than acceptance criteria (+10.8% vs 3.2% allowed). P&amp;B regression revealed a significant positive proportional difference (slope=1.14). Comparison of PS activity yielded a high negative bias (-33.4%) that exceeded the acceptance criteria of 8.1%. Regarding LA testing, the diagnostic accuracy was 99% when compared to Sestre milosrdnice UHC (N=70) and 95% when compared to University Hospital Centre Zagreb (N=19). All manufacturer's reference intervals and cut-offs were verified.&#x0D; Conclusion: The verification study confirmed all manufacturer's claims, except for PS which has been replaced with free PS:Ag determinations.

Risk stratification utilizing sequential organ failure assessment (SOFA) score, antithrombin activity, and demographic data in sepsis-associated disseminated intravascular coagulation (DIC)

Disseminated intravascular coagulation (DIC) is a frequent complication in patients with sepsis and is associated with increased mortality. Anticoagulant therapy may be appropriate for certain patients with DIC, particularly those with increased disease severity and deficiency in the physiologic anticoagulant antithrombin. We retrospectively analyzed post-marketing survey data from 1562 patients with sepsis-associated DIC and antithrombin activity of 70% or less. All the patients were treated with antithrombin concentrates. Baseline sequential organ failure assessment (SOFA) score, DIC score, and antithrombin activity were assessed. Cox multivariate regression analysis, Kaplan–Meier curve analysis, and receiver operating characteristic (ROC) curve analysis were performed to evaluate the performance of variables used to assess mortality. Furthermore, a decision tree was constructed to classify the risk of 28-day mortality. COX multivariate regression analysis demonstrated a significant association of age, sex, baseline SOFA score, baseline antithrombin activity, and the presence of pneumonia or skin/soft tissue infection with increased mortality. The area under the curve of SOFA score or antithrombin activity for mortality was 0.700 and 0.614, respectively. Kaplan–Meier analysis demonstrated that mortality was significantly higher in patients with SOFA score ≥ 12 and antithrombin activity < 47%. The decision tree analysis accurately classified the risk of death into high (> 40%), medium (40%–20%), and low (< 20%) categories in 86.1% of the cohort. Twenty eight-day mortality can be strongly predicted using baseline SOFA score, antithrombin activity, infection site, age, and sex as variables in the clinical decision tree for patients with sepsis-associated disseminated intravascular coagulation (DIC).