No abstract available

The thromboembolic ischemia model is one of the most applicable for studying ischemic stroke in humans. The aim of this study was to develop a novel thromboembolic stroke model, allowing, by affordable tools, to reproduce cerebral infarction in rats. In the experimental group, the left common carotid artery, external carotid artery, and pterygopalatine branch of maxillary artery were ligated. A blood clot that was previously formed (during a 20 min period, in a catheter and syringe, by mixing with a thromboplastin solution and CaCl2) was injected into the left internal carotid artery. After 10 min, the catheter was removed, and the incision was sutured. The neurological status of the animals was evaluated using a 20-point scale. Histological examination of brain tissue was performed 6, 24, 72 h, and 6 days post-stroke. All groups showed motor and behavioral disturbances 24 h after surgery, which persisted throughout the study period. A histological examination revealed necrotic foci of varying severity in the cortex and subcortical regions of the ipsilateral hemisphere, for all experimental groups. A decrease in the density of hippocampal pyramidal neurons was revealed. Compared with existing models, the proposed ischemic stroke model significantly reduces surgical time, does not require an expensive operating microscope, and consistently reproduces brain infarction in the area of the middle cerebral artery supply.

Activated partial thromboplastin time (aPTT) is determined and reported as clotting time in seconds aPTT(s), but it is presumed that reporting results as patient-to-normal clotting time ratio, aPTT(r), could minimize within-laboratory variability. The aim of study was to investigate differences in reporting aPTT results that can affect comparability of the results among Croatian laboratories and suggest further steps for its harmonization.

Anticoagulation therapy with heparin is a frequent treatment in intensive care units and is monitored by activated partial thromboplastin clotting time (aPTT). It has been demonstrated that reaching an established anticoagulation target within 24 hours is associated with favorable outcomes. However, patients respond to heparin differently and reaching the anticoagulation target can be challenging. Machine learning algorithms may potentially support clinicians with improved dosing recommendations.

Activated partial thromboplastin time (aPTT) is associated with risk of thrombosis and coagulation disorders. We conducted a genome-wide association study for aPTT and identified significant associations with SNPs in three coagulation cascade genes, F12 (rs2731672, combined p = 2.16 x 10(-30)), KNG1 (rs710446, combined p = 9.52 x 10(-22)), and HRG (rs9898, combined p = 1.34 x 10(-11)). These three SNPs explain approximately 18% of phenotypic variance in aPTT in the Lothian Birth Cohorts.

Emicizumab is a bispecific humanized monoclonal antibody that shortens the activated partial thromboplastin time (aPTT), making aPTT-based tests unreliable.

Current guidelines recommend monitoring the anticoagulant effect of unfractionated heparin (UFH) by measuring anti-Xa activity rather than activated partial thromboplastin time (aPTT) in intensive care unit (ICU) patients. The primary objective of this study was to evaluate the correlation of aPTT, anti-Xa activity, and thrombin generation in UFH-treated ICU patients. A prospective observational pilot study was conducted in adult surgical ICU patients treated with UFH. aPTT and anti-Xa activity were monitored daily. The therapeutic target was aPTT between 50 s and 84 s, and/or anti-Xa between 0.3 and 0.7 U/mL. Correlation among aPTT, anti-Xa activity, and thrombin generation was determined by measuring endogenous thrombin potential (ETP), with the inflammatory response evaluated. C-reactive protein (CRP) was used as a marker of inflammatory response. The plasma of 107 samples from 30 ICU patients was analyzed. The correlation between aPTT and anti-Xa activity was 0.66, CI95% [0.54;0.76] (p < 0.0001). Although thrombin generation, aPTT, and anti-Xa were correlated with inflammatory responses, the correlation was higher with thrombin generation and anti-Xa activity compared to aPTT. When aPTT was in a therapeutic range, a low thrombin generation was observed but was 50% inhibited when anti-Xa was in a therapeutic range. Coagulation testing with aPTT, anti-Xa correlated with thrombin generation. A 50% decrease in thrombin generation was observed when anti-Xa was within a therapeutic range. Further work is needed to evaluate coagulation biomarker responses and clinical outcomes in specific ICU populations.

Activated partial thromboplastin time (aPTT) and prothrombin time (PT) are clinical tests commonly used to screen for coagulation-factor deficiencies. One genome-wide association study (GWAS) has been reported previously for aPTT, but no GWAS has been reported for PT. We conducted a GWAS and meta-analysis to identify genetic loci for aPTT and PT. The GWAS for aPTT was conducted in 9,240 individuals of European ancestry from the Atherosclerosis Risk in Communities (ARIC) study, and the GWAS for PT was conducted in 2,583 participants from the Genetic Study of Three Population Microisolates in South Tyrol (MICROS) and the Lothian Birth Cohorts (LBC) of 1921 and 1936. Replication was assessed in 1,041 to 3,467 individuals. For aPTT, previously reported associations with KNG1, HRG, F11, F12, and ABO were confirmed. A second independent association in ABO was identified and replicated (rs8176704, p = 4.26 × 10(-24)). Pooling the ARIC and replication data yielded two additional loci in F5 (rs6028, p = 3.22 × 10(-9)) and AGBL1 (rs2469184, p = 3.61 × 10(-8)). For PT, significant associations were identified and confirmed in F7 (rs561241, p = 3.71 × 10(-56)) and PROCR/EDEM2 (rs2295888, p = 5.25 × 10(-13)). Assessment of existing gene expression and coronary artery disease (CAD) databases identified associations of five of the GWAS loci with altered gene expression and two with CAD. In summary, eight genetic loci that account for ∼29% of the variance in aPTT and two loci that account for ∼14% of the variance in PT were detected and supported by functional data.

Recombinant plasminogen activator (reteplase) is a third generation thrombolytic agent which has been used on coronary artery thrombosis and acute myocardial infarction. Clot lysis assay is usually considered as a unique method to evaluate biological activity of reteplase. In this study biological activity of reteplase was determined by APTT (activated partial thromboplastin time) lysis method. Validity of this method was evaluated in comparison with reference method, clot lysis time assay. Results of APTT lysis test showed good reproducibility (relative standard deviation (RSD) 3-5% for within day analysis and 4-7% for between day analysis), and accuracy (101.3-102.7%). APTT lysis responses were linear in range of 0.001-0.1 mg/mL reteplase. Therefore, APTT lysis method is applicable for biological activity determination of reteplase. Although more comprehensive studies are required to approve this test as a reference method, APTT lysis method seems to be valuable to receive more attention due to advantages of technical simplicity, sensitivity, applicability, and cost efficiency.

Factor V together with activated factor X forms the prothrombinase complex, which transforms prothrombin into thrombin. The Mus musculus species is characterized by very high levels of this factor and short clotting times, which hinders accurate measurements. For that reason, a detailed characterization of such parameters is indispensable. A method was designed as part of this study to provide an accurate determination and standardization of factor V levels, prothrombin time and activated partial thromboplastin time in Mus musculus. Those parameters were evaluated in a sample of 66 healthy animals using a semi-automated coagulometer and human diagnostic reagents in an attempt to determine the most appropriate time of day for the extractions. A mouse-based protocol was designed, capable of making corrections to the samples at dilutions of 1:100 for factor V and at of 1:3 for prothrombin time. The goal was to smoothen the calibration curves, which often present with steep slopes and narrow measurement ranges between one calibration point and another. It was found that the most stable period for blood sample extraction was that comprised between the first 6 h of light. No clinical differences were observed between the sexes and reference intervals were established for factor V (95.80% ± 18.14; 25.21 s ± 1.34), prothrombin time (104.31% ± 14.52; 16.85 s ± 1.32) and activated partial thromboplastin time (32.86 s ± 3.01). The results obtained are applicable to human or veterinary biomedical research, to transfusional medicine or to pathological models for diseases such as factor V deficiency.

von Willebrand Disease (vWD) is the most prevalent bleeding disorder. Women are more likely to manifest abnormal bleeding symptoms due to physiologic events and menorrhagia is the most common presenting symptom.

A modern diagnostic laboratory offers wide spectrum of coagulation assays utilized in the diagnosis and management of patients with haemostatic disorders, preoperative screening and anticoagulation therapy monitoring. The recent survey conducted among Croatian medical biochemistry and transfusion laboratories showed the existence of different practice policies in particular phases of laboratory process during coagulation testing and highlighted areas that need improvement. Lack of assay standardization together with non-harmonized test results between different measurement methods, can potentially lead to incorrect decisions in patient's treatment. Consequently, patient safety could be compromised. Therefore, recommended procedures related to preanalytical, analytical and postanalytical phases of prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer testing are provided in this review, aiming to help laboratories to generate accurate and reliable test results.

Thromboelastography (TEG) provides a global evaluation of haemostasis. This diagnostic test is widely used in mammals but has not previously been performed in reptiles, mainly due to the limited availability of taxon-specific reagents. The objective of this pilot study was to establish a protocol to perform TEG in sea turtles. Pooled citrated plasma, stored at -80°C, from four green turtles (Chelonia mydas) was assayed on a TEG 5000. Several initiators were evaluated: kaolin (n=2), RapidTEG (n=2), fresh (n=2) and frozen (n=6) thromboplastin extracted from pooled brain tissue from several chelonian species, human recombinant tissue factor at 1:100 (n=1), Reptilase (n=2), and rabbit thromboplastin (n=1). Both fresh and frozen chelonian thromboplastin were superior in producing quantifiable TEG reaction time compared with all other reagents. These findings are consistent with the lack of an intrinsic pathway in turtles and confirmed a lack of coagulation in the turtle samples in response to mammalian thromboplastin. A TEG protocol was subsequently established for harvested species-specific frozen thromboplastin. The frozen thromboplastin reagent remained stable after one year of storage at -80°C. The developed protocol will be useful as a basis for future studies that aim to understand the pathophysiology of haemostatic disorders in various stranding conditions of sea turtles.

The presence of endometriosis determines an inflammatory response locally. The objective of this validation study and systematic review was to assess systemic levels of coagulation and inflammatory parameters in women with or without the disease. We conducted a retrospective analysis of a database prospectively collected from January 2017 to February 2020 including n = 572 women who underwent laparoscopic surgery for endometriosis (cases, n = 324) or other benign gynecologic diseases (controls, n = 248). Inflammatory markers and coagulation parameters were determined. An advanced systematic search of the literature on the same parameters was conducted up to April 2020. A significantly higher neutrophil count was found in endometriosis patients. Patients with endometriomas and stage III-IV disease had a significantly lower absolute lymphocyte count and shortened activated partial thromboplastin time (aPTT) values. In the final regression model, aPTT retained significant predictive value for stage III-IV endometriosis (odds ratio (OR) = 0.002, 95% confidence interval (CI) = 0.00-0.445; p = 0.024). Results from the n = 14 included studies in the systematic review are characterized by a high variability, but some consistency has been found for alterations in thrombin time, platelet-to-lymphocyte ratio, and neutrophil count associated with endometriosis. Modest systemic changes of some inflammatory and coagulation parameters are associated with endometriosis. Indeed, all the modifications detected are still within the normal reference intervals, explaining the high heterogeneity among studies.

The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC50 value of ~8 μg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis-Menten kinetics, LSAS decreased the VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa-antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure-activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.

Currently, the greatest challenge in hemophilia treatment is managing hemophilia patients with inhibitors. The two main bypassing agents that are used to treat hemophilia patients with inhibitors are activated prothrombin complex concentrates (APCC) and recombinant factor VIIa (rFVIIa). Hemophilia patients with inhibitors can develop bleeding episodes, that are refractory to monotherapy with either APCC or rFVIIa and thus are often difficult to manage.

To assess the correlation between standard laboratory indicators at admission and severe maternal complications due to placental abruption (PA) with intrauterine fetal death (IUFD) after 24 weeks.

Heparins extracted from different animal sources have been conventionally considered effective anticoagulant and antithrombotic agents despite of their pharmacological dissimilarities. We performed herein a systematic analysis on the physicochemical properties, disaccharide composition, in vitro anticoagulant potency and in vivo antithrombotic and bleeding effects of several batches of pharmaceutical grade heparins obtained from porcine intestine, bovine intestine and bovine lung. Each of these three heparin types unambiguously presented differences in their chemical structures, physicochemical properties and/or haemostatic effects. We also prepared derivatives of these heparins with similar molecular weight differing exclusively in their disaccharide composition. The derivatives from porcine intestinal and bovine lung heparins were structurally more similar with each other and hence presented close anticoagulant activities whereas the derivative from bovine intestinal heparin had a higher proportion of 6-desulfated α-glucosamine units and about half anticoagulant activity. Our findings reasonably indicate that pharmaceutical preparations of heparin from different animal sources constitute distinct drugs, thus requiring specific regulatory rules and therapeutic evaluations.

Transfusion of defective platelets could contribute to the inefficiency of platelet transfusion in preventing or stopping bleeding.

Introduction: Measurement of thrombolytic activity is crucial for research and development of novel thrombolytics. It is a key factor in the assessment of the effectiveness of conventionally used thrombolytic therapies in the clinic. Previous methods used for the assessment of thrombolytic activity are often associated with some drawbacks such as being costly, time-consuming, complex with low accuracy. Here, we introduce a simple, economic, relatively accurate and fast method of spectrophotometric analysis of thrombolytic activity (SATA) assay, standardized by tissue plasminogen activator (tPA), which can quantitatively measure in vitro thrombolytic activity. Methods: Blood clots were formed, uniformly, by mixing citrated whole blood with partial thromboplastin time (PTT) reagent, together with calcium chloride. Then, designated concentrations of tPA were added to the samples, and the released red blood cells from each clot were quantified using spectrophotometry (λmax=405nm) as an indicator of thrombolytic activity. The accuracy of the method was tested by assessment of dose-responsibility against R2 value obtained by linear equation and measurement of the limit of detection (LOD) and limit of quantification (LOQ). The SATA assay was validated in comparison with some currently used techniques. Results: A linear relationship was obtained between different concentrations of tPA versus the spectrophotometric absorbance of the related dilutions of lysed clots, at λmax=405nm. Calculated R2 values were greater than 0.9; with LOD of 0.90 µg/mL of tPA (436.50IU) and LOQ of 2.99 µg/mL of tPA (1450.15IU). Conclusion: Conclusively, the SATA assay is a very simple quantitative method with repeatable and reproducible results for estimating the potency of an unknown thrombolytic agent, and calculating the activity as delicate as 1 µg/mL of tPA (485 IU/mL of thrombolytic dose).