Functional Coagulation Assays Research Articles

Characterization of zebrafish coagulation cofactors Fviii and Fv mutants and modeling hemophilia A and factor V deficiency.

The aim of this study is to characterize zebrafish coagulation cofactors fviii and fv mutant fish and assess if they phenocopy classical hemophilia A and factor V deficiency in humans. The embryos from fviii and fv zebrafish heterozygote mutants generated by ENU mutagenesis were purchased from the ZIRC repository. They were reared to adulthood and genotyped. The heterozygote male and female were crossed to get homozygote, heterozygote, and wild-type fish. Functional kinetic coagulation assays and bleeding assays were performed on normal and mutant adult fish, and venous laser injury assays were performed on the larvae. The DNA from fviii and fv mutants were sequenced to confirm if they have a premature stop codon in exon 19, and in exon 2, respectively, and in both mutants, the amino acid glutamine is replaced with a stop codon. Homozygous and heterozygous 5 days post fertilization (dpf) larvae for fviii and fv deficient mutants exhibited prolonged time to occlusion after venous laser injury compared to wild-type controls. The homozygous and heterozygous fviii adult mutants showed modest bleeding and delayed fibrin formation in the kinetic partial thromboplastin time (kPTT) assay with their plasma. fv homozygous larvae had poor survival beyond 12 dpf. However, heterozygous fv mutants exhibited heavy bleeding and prolonged fibrin formation in the kPTT and kPT assay compared with wild-type siblings. Our characterization showed fviii and fv mutants from ZIRC phenocopied to a considerable extent classical hemophilia A and factor V deficiency in humans, respectively. These models should be useful in studying and developing novel drugs that reverse the phenotype and in generating suppressor mutations to identify novel factors that compensate for these deficiencies.

The 2023 ACR/EULAR Antiphospholipid Syndrome Classification Criteria.

To develop new antiphospholipid syndrome (APS) classification criteria with high specificity for use in observational studies and trials, jointly supported by the American College of Rheumatology (ACR) and EULAR. This international multidisciplinary initiative included 4 phases: 1) Phase I, criteria generation by surveys and literature review; 2) Phase II, criteria reduction by modified Delphi and nominal group technique exercises; 3) Phase III, criteria definition, further reduction with the guidance of real-world patient scenarios, and weighting via consensus-based multicriteria decision analysis, and threshold identification; and 4) Phase IV, validation using independent adjudicators' consensus as the gold standard. The 2023 ACR/EULAR APS classification criteria include an entry criterion of at least one positive antiphospholipid antibody (aPL) test within 3 years of identification of an aPL-associated clinical criterion, followed by additive weighted criteria (score range 1-7 points each) clustered into 6 clinical domains (macrovascular venous thromboembolism, macrovascular arterial thrombosis, microvascular, obstetric, cardiac valve, and hematologic) and 2 laboratory domains (lupus anticoagulant functional coagulation assays, and solid-phase enzyme-linked immunosorbent assays for IgG/IgM anticardiolipin and/or IgG/IgM anti-β2 -glycoprotein I antibodies). Patients accumulating at least 3 points each from the clinical and laboratory domains are classified as having APS. In the validation cohort, the new APS criteria versus the 2006 revised Sapporo classification criteria had a specificity of 99% versus 86%, and a sensitivity of 84% versus 99%. These new ACR/EULAR APS classification criteria were developed using rigorous methodology with multidisciplinary international input. Hierarchically clustered, weighted, and risk-stratified criteria reflect the current thinking about APS, providing high specificity and a strong foundation for future APS research.

Association of Thromboelastography with Progression of Hemorrhagic Injury in Children with Traumatic Brain Injury.

Progression of hemorrhagic injury (PHI) in children with traumatic brain injury portends poor outcomes. The association between thromboelastography (TEG), functional coagulation assays, and PHI is not well characterized in children. This was a retrospective cohort study of children presenting with PHI at a pediatric level I academic trauma center from 2015 to 2020. Inclusion criteria were as follows: age less than 18years, intracranial hemorrhage on admission head computed tomography scan, and admission rapid TEG assay and conventional coagulation tests. PHI was defined by the following radiographic criteria: any expansion of or new intracranial hemorrhage on subsequent head computed tomography scan. Rapid TEG values included Activated Clotting Time (ACT), alpha angle, maximum amplitude, and lysis at 30min. Wilcoxon rank-sum test was used to assess baseline differences between groups with PHI and without PHI, including laboratory assays. Univariate analysis was performed to examine the association between variables of interest and PHI. Patients were dichotomized on the basis of this cut point to generate a "low ACT" group and a "high ACT" group. These variables were included in a multivariable logistic regression model to determine independent association with traumatic brain injury progression. In total, 219 patients met criteria for analysis. In this cohort, the median (interquartile range [IQR]) age = 6 (2-12) years, median (IQR) Injury Severity Score = 21 (11-27), 68% were boys, and 69% sustained blunt injury. The rate of PHI was 25% (54). Median (IQR) time to PHI was 1 (0-4) days. Children with PHI had a higher Injury Severity Score (p < 0.001), lower Glasgow Coma Scale (p < 0.001), greater incidence of shock (p = 0.04), and lower admission hemoglobin (p = 0.02) compared with those without PHI. Children with PHI had a higher International Normalized Ratio (INR) and longer TEG-ACT; other TEG values (alpha angle, maximum amplitude, and lysis at 30min) were not associated with PHI. In the logistic regression model accounting for other covariates associated with PHI, elevated ACT remained an independent predictor of progression (odds ratio = 2.25, 95% confidence interval 1.09-4.66; p = 0.03; area under the receiver operating characteristic curve = 0.76). After adjusting for confounders, INR fell out of the model and was not an independent predictor of progression (odds ratio = 1.32, 95% confidence interval 0.60-2.93; p = 0.49). Although INR was elevated in children with PHI and has been associated with poor clinical outcomes, only admission TEG-ACT was independently associated with PHI. Further study is warranted to determine whether TEG-ACT reflects an actionable therapeutic target.

Laboratory Diagnosis of Antiphospholipid Syndrome: Insights and Hindrances.

Diagnosis of antiphospholipid syndrome (APS) requires the presence of a clinical criterion (thrombosis and/or pregnancy morbidity), combined with persistently circulating antiphospholipid antibodies (aPL). Currently, laboratory criteria aPL consist of lupus anticoagulant (LAC), anticardiolipin antibodies (aCL) IgG/IgM, and anti-β2 glycoprotein I antibodies (aβ2GPI) IgG/IgM. Diagnosis and risk stratification of APS are complex and efforts to standardize and optimize laboratory tests have been ongoing since the initial description of the syndrome. LAC detection is based on functional coagulation assays, while aCL and aβ2GPI are measured with immunological solid-phase assays. LAC assays are especially prone to interference by anticoagulation therapy, but strategies to circumvent this interference are promising. Alternative techniques such as thrombin generation for LAC detection and to estimate LAC pathogenicity have been suggested, but are not applicable yet in routine setting. For aCL and aβ2GPI, a lot of different assays and detection techniques such as enzyme-linked immunosorbent and chemiluminescent assays are available. Furthermore, a lack of universal calibrators or standards results in high variability between the different solid-phase assays. Other non-criteria aPL such as anti-domain I β2 glycoprotein I and antiphosphatidylserine/prothrombin antibodies have been suggested for risk stratification purposes in APS, while their added value to diagnostic criteria seems limited. In this review, we will describe laboratory assays for diagnostic and risk evaluation in APS, integrating applicable guidelines and classification criteria. Current insights and hindrances are addressed with respect to both laboratory and clinical implications.

Evaluation of Covid-19-Associated Hypercoagulability with Functional Coagulation Assays and Extracellular Vesicles

Most patients affected by the novel coronavirus SARS-CoV-2 — responsible for the Coronavirus disease, COVID-19 — remain asymptomatic or develop mild symptoms. Only a small percentage of cases develop a severe disease that may lead to a fatal outcome. Since the early reports published in the literature by Wuhan colleagues, we have learned that patients hospitalized for acute COVID-19 infection have different clinical and laboratory pictures of coagulopathy. In particular, a marked increase in blood clotting capacity has been reported in most hospitalized patients – COVID-19-associated coagulopathy, CAC – which in turn increases the risk of developing thrombotic complications. The main pathophysiological mechanisms underlying this hypercoagulable state are inflammation, endothelial damage, hypofibrinolysis and hypoxemia. Traditional coagulation tests fail to fully characterize the nature and severity of COVID-19-associated hypercoagulability (CAH). Hence the need for functional coagulation assays (i.e. tromboelastometry/graphy, thrombin generation, platelet function test) and circulating extracellular vesicles to better understand these peculiar conditions. Moreover, it would be very helpful to use these tests to identify patients at increased risk of developing thrombotic complications or with a worse prognosis as well as to ascertain the effectiveness of the anticoagulant treatment. The aim of our narrative review was to describe the main pathophysiologic mechanisms of CAH and to summarize the current knowledge on functional coagulation assays and extracellular vesicles tests in CAH.

Evaluation of the in vitro stability of direct oral anticoagulants in blood samples under different storage conditions

In this study, we evaluated the in vitro stability of direct oral anticoagulants (DOACs) in blood samples of 57 patients under different storage conditions using functional coagulation assays. We determined the analyte concentrations (1) immediately after blood collection (baseline); (2) after storage of citrated whole blood (agitated) at room temperature and citrated plasma at room temperature and at 4 °C for 4, 8, and 24 h, respectively; and (3) after storage of citrated plasma at −20 °C for 30, 60, and 90 days. According to the concept of acceptable change limits (ACL), analytes were considered stable if the mean relative analyte recovery at a given time was >78%. The mean baseline values (range) of dabigatran, rivaroxaban, apixaban, and edoxaban were 115 ng/mL (62–217), 129 ng/mL (31–215), 156 ng/mL (49–362), and 101 ng/mL (33–283), respectively. After applying the analyte stability limit, all four DOACs were stable for 24 h at room temperature and at 4 °C. The mean recovery after 24 h was 102–111% for dabigatran, 88–97% for rivaroxaban, 95–98% for apixaban, and 90–96% for edoxaban. When plasma samples were stored at −20 °C, the mean percentage deviation after 90 days for all four DOACs was ≤10%, even after three freeze-thaw cycles. Thus, for the correct determination of DOAC plasma concentrations, blood samples do not have to be analyzed immediately and can be stored at room temperature for up to 24 h before analysis. In clinical practice, blood sample transport and storage for DOAC measurements appear to be unproblematic.

Phosphatidylserine-exposing blood cells, microparticles and neutrophil extracellular traps increase procoagulant activity in patients with pancreatic cancer

Patients with pancreatic cancer (PC) are at increased risk of venous thrombosis, but the precise mechanisms of hypercoagulable state in PC remain unclear. We aimed to identify how phosphatidylserine positive (PS+) blood cells (BCs), PS+ microparticles (MPs) and neutrophil extracellular traps (NETs) regulate procoagulant activity (PCA) in PC, and to assess the relationship between PCA and PC staging. A total of 83 PC patients with different stages of disease were compared to 30 healthy controls, with confocal microscopy and flow cytometry used to assess MP and cellular PS exposure. MP and cell PCA was determined using both fibrin production assays and procoagulant enzyme complex analyses, and coagulation time was further measured. Patients with stage I PC and healthy controls exhibited significantly lower frequencies of PS+ MPs and BCs relative to those with more advanced disease, which may partly due to the increased levels of inflammation cytokines in advanced disease. Functional coagulation assays indicated that PS+ MPs and BCs derived from patients with stage II/III/IV PC directly contribute to elevated FXa, thrombin, and fibrin formation, and to more rapid coagulation relative to healthy control samples. In inhibition assays, lactadherin, which antagonizes PS, led to a roughly 80% inhibition of PCA. We further used isolated NETs to stimulate endothelial cells, revealing that this led to morphological changes including retraction from cell-cell junctions and a more pro-coagulative phenotype, with DNase I and activated protein C treatment reversing these changes. In patients with stage III PC, curative resection surgery significantly reduced PCA, whereas non-curative surgery did not have a marked impact based on studies of pre- and post-operative samples. These results highlight the pathogenic activity of PS+ cells, MPs, and NETs in promoting a prothrombotic environment within individuals suffering from advanced PC. Targeting PS and NETs in these patients may thus be a viable means of preventing pathological thrombosis.

The effect of unfractionated heparin, enoxaparin, and danaparoid on lupus anticoagulant testing: Can activated carbon eliminate false‐positive results?

BackgroundHeparins and heparinoids interfere with functional clotting assays used for lupus anticoagulant (LAC) detection. However, current guidelines for LAC testing do not provide clear guidance on this matter. ObjectivesWe aimed to assess to effect of unfractionated heparin (UFH), enoxaparin, and danaparoid on LAC assays over broad anti‐Xa activity ranges and to evaluate whether activated carbon (AC) is able to neutralize these effects. MethodsUFH (0.1‐3.0 IU/mL), enoxaparin (0.2‐2.9 IU/mL), and danaparoid (0.6‐2.2 IU/mL) were spiked to normal pooled plasma. AC was added at multiple activity levels. Anti‐Xa assays and LAC tests were performed on all samples using Stago analyzers and reagents. ResultsAbnormal activated partial thromboplastin time (APTT) screening and mixing tests were obtained at the lowest levels for all compounds. Abnormal APTT confirmation tests were seen from 2.5 and 1.9 anti‐Xa IU/mL for enoxaparin and danaparoid, respectively. Abnormal dilute Russell’s viper venom test (dRVVT) screening tests were obtained from 1.6, 1.4, and 1.1 anti‐Xa IU/mL for UFH, enoxaparin, and danaparoid, respectively. Mixing tests were abnormal from 2.5 and 1.3 anti‐Xa IU/mL for enoxaparin and danaparoid, respectively. Abnormal dRVVT confirmation results were seen for danaparoid only from 1.9 anti‐Xa IU/mL. AC was unable to neutralize anti‐Xa activity in plasma and overcome the effect of the tested anticoagulants on LAC assays but may cause prolongation of APTT clotting times. ConclusionsUFH, enoxaparin, and danaparoid clearly affected LA tests; however, false‐positive LAC conclusions were obtained at supratherapeutic enoxaparin and danaparoid levels only. AC may prolong APTT screen clotting times, requiring 3‐step testing to avoid potential misdiagnosis of LAC.

PROC Promoter Single Nucleotide Polymorphisms Associated With Low Protein C Activity But Not Increased Risk of Thromboembolism in Pediatric Population.

Protein C (PC) deficiency, caused by mutations of the PROC gene, is a common inherited risk factor of thromboembolism (TE) among Thai people. This study aimed to investigate the association of 3 single nucleotide polymorphisms (SNPs; −1654 C/T, −1641 A/G, −1461A/T) at the PROC promoter region with PC activity and the risk of developing TE. A total of 216 patient s with TE, diagnosed at aged 0 to 20 years, and 102 healthy adults were enrolled. The SNPs were identified by Sanger sequencing. Protein C activity was measured using an automated functional clotting assay. Linear and logistic regression analyses were used to determine the association of SNPs with PC activity and the risk of TE. Patients and controls with homozygous TAA (119.6% ± 26.1%) and CGT haplotypes (102.7% ± 22.6%) had significantly lower PC activity than those with a homozygous CAA haplotype (140.4% ± 44.9%); P = .027 and .016, respectively. However, none of these haplotypes increased the risk of TE. This study suggested that the 3 PROC promoter SNPs were shown to be associated with lower PC activity but did not increase the risk of TE.

Tranexamic Acid for Acute Hemorrhage: A Narrative Review of Landmark Studies and a Critical Reappraisal of Its Use Over the Last Decade.

The publication of the Clinical Randomization of an Antifibrinolytic in Significant Hemorrhage-2 (CRASH-2) study and its intense dissemination prompted a renaissance for the use of the antifibrinolytic agent tranexamic acid (TXA) in acute trauma hemorrhage. Subsequent studies led to its widespread use as a therapeutic as well as prophylactic agent across different clinical scenarios involving bleeding, such as trauma, postpartum, and orthopedic surgery. However, results from the existing studies are confounded by methodological and statistical ambiguities and are open to varied interpretations. Substantial knowledge gaps remain on dosing, pharmacokinetics, mechanism of action, and clinical applications for TXA. The risk for potential thromboembolic complications with the use of TXA must be balanced against its clinical benefits. The present article aims to provide a critical reappraisal of TXA use over the last decade and a "thought exercise" in the potential downsides of TXA. A more selective and individualized use of TXA, guided by extended and functional coagulation assays, is advocated in the context of the evolving concept of precision medicine.

A comparative study of viscoelastic hemostatic assays and conventional coagulation tests in trauma patients receiving fibrinogen concentrate

BackgroundBoth thrombelastography (TEG) and rotational thromboelastometry (ROTEM) have been investigated for diagnosis of coagulopathy and guidance of resuscitation in trauma and surgery. Given similarities between the two systems, it is important to determine whether one is superior to the other and how comparable they are to conventional coagulation tests (CCTs). Therefore, we conducted a comparative study of functional fibrinogen and coagulation assays using TEG and ROTEM and CCTs to determine their capability to monitor coagulation profiles, diagnose coagulopathy and predict blood transfusion requirements in trauma patients. MethodsBlood samples were collected from 45 patients at admission and during 48-h hospitalization as part of a randomized control trial on early fibrinogen replacement in trauma. Functional fibrinogen (FF) TEG, ROTEM FIBTEM and EXTEM, and CCTs were performed and compared. ResultsWe found significant differences between the placebo and fibrinogen groups over hospitalization time in FF TEG MA, ROTEM CT, MCF and LI30. FF TEG MA and ROTEM FIBTEM MCF mirrored plasma fibrinogen profiles, reached a maximum difference between the two groups 1–3 h after fibrinogen administration. In comparison, CCTs detected minimal hemostatic changes by fibrinogen treatment. TEG and ROTEM showed various degrees of correlations with CCTs. TEG MA and ROTEM MCF provided better predictions for plasma and RBC transfusions than CCTs, but poor accuracy for cryoprecipitate transfusion. Both TEG and ROTEM well predicted hypofibrinogenemia (fibrinogen concentration < 1 g/L), but poorly detected coagulopathy (INR ≥ 1.2). ConclusionsTEG and ROTEM detected increases in clot strength following early use of fibrinogen. ROTEM also detected changes in coagulation time and clot lysis. Both were better than CCTs for monitoring coagulation profiles and predicting transfusion requirements.

Increased Phosphatidylserine on Blood Cells in Oral Squamous Cell Carcinoma

The specific function of phosphatidylserine (PS) in the context of the development of a hypercoagulable state among individuals with oral squamous cell carcinoma (OSCC) is uncertain. The goal of this study was therefore to assess the exposure of PS on microparticles (MPs) as well as on endothelial and blood cells and to assess procoagulant activity (PCA) as a function of the stage of OSCC progression. We recruited patients with OSCC (n = 63) as well as healthy controls (n = 26) to participate in this study. PS exposure was then assessed via confocal microscopy and flow cytometry, revealing that patients with stage III/IV OSCC exhibited higher frequencies of PS-exposing blood cells, MPs, and serum-cultured endothelial cells (ECs) than did patients with stage I/II OSCC or healthy controls. When we conducted functional coagulation assays, we discovered that PS+ blood cells, MPs, and serum-cultured ECs from patients with stage III/IV OSCC mediated more rapid coagulation and more substantial production of FXa, thrombin, and fibrin as compared with controls. When samples were treated with the PS antagonist lactadherin, this resulted in an 80% disruption of PCA. Strikingly, when pre- and postoperative samples were compared from patients with stage III/IV OSCC undergoing resective surgery, PCA was significantly reduced in the postoperative samples. After stimulating ECs with inflammatory cytokines, we found by confocal microscopy that they expose PS on their cell membranes, thus generating FVa and FXa binding sites and mediating the formation of fibrin. Together our findings provide evidence that PS+ blood cells and MPs are important mediators of the development of a hypercoagulable and prothrombotic state among individuals afflicted by advanced-stage OSCC. As such, a PS blockade may be a viable therapeutic strategy for treating such patients.

Pediatric trauma-related coagulopathy: Balanced resuscitation, goal-directed therapy and viscoelastic assays

The improved understanding of trauma-induced coagulopathy in adults has led to an evolution in the strategies of damage-control resuscitation. While its impact on the care of pediatric trauma patients is of tremendous interest, the evidence is sparse, and a great deal of research is still needed in this domain. Areas of particular interest include age-related differences in hemostasis and balanced resuscitation, advances in functional coagulation assays and effective adjunctive medications, such as tranexamic acid, for hemorrhage control. This review examines the available pediatric data, reviews applicable adult data, and introduces areas of investigation that will impact pediatric trauma care in the future.

Multianalyte Determination of NOACs Using LC-MS/MS and Comparison with Functional Coagulation Assays.

Detection of new oral anticoagulant (NOAC) levels by screening, special and global tests, and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) is important in clinical situations when the cause of bleeding needs to be determined. We compared a routine coagulation test, special function test for NOACs, global coagulation test, and an LC-MS/MS method that enables simultaneous determination of apixaban, dabigatran and rivaroxaban in human plasma within one analysis to determine the optimal indication of the comparison methods, including their limitations and interferences. This study was conducted on a set of blood samples from 116 patients treated with NOACs. The results of both specific dilute thrombin time (dTT) tests for dabigatran provided the same results as the activated partial thromboplastin time (aPTT) screening test in comparison with LC-MS/MS as a reference. The dTT assay HemosIL® showed better results for low concentrations when compared to LC-MS/MS than dTT HYPHEN® as HemosIL® uses a non-linear calibration curve. Results of the specific anti-Xa assay yielded better results than the prothrombin time test in comparison with LC-MS/MS as a reference, especially for apixaban, but also for rivaroxaban. Our LC MS/MS method is simply feasible, but only in a specialized laboratory. The method is easy-to-use for the simultaneous determination of all dabigatran, apixaban and rivaroxaban by LC-MS/MS within three minutes with a concentration range of 1 to 500 µg/L without dilution. In the normal practice of the coagulation laboratory, it is advisable to use specific tests for NOAC determination as screening and global assays are not sufficiently specific. The dTT test is the optimal choice for dabigatran determination and for xabans to determine anti-Xa activity. The LC-MS/MS method is suitable as an arbitration method for serious conditions.

Inflammation and Its Consequences After Surgical Versus Transcatheter Aortic Valve Replacement.

Symptomatic aortic stenosis can be treated by surgical aortic valve replacement (SAVR) or transcatheter aortic valve replacement (TAVR), the latter of which is regarded as a minimally invasive procedure. Differences between these procedures regarding immune responses or changes in coagulation and neurocognitive function have thus far been evaluated only sparsely. We carried out a prospective, single-center, nonrandomized explorative study with 38 patients. Thirteen patients were subjected to either SAVR or transfemoral (TF-) TAVR, and 12 patients underwent transapical (TA-) TAVR. Plasma cytokines (IL-6, -8, -18, presepsin) and acute-phase proteins (C-reactive protein, procalcitonin), markers of coagulation and platelet function, and neurocognitive function (via various standard tests) were assessed before and at five-time points during a 72-h follow-up after surgery. SAVR and TA-TAVR patients responded similarly to the procedure in terms of C-reactive protein, leukocyte numbers, and IL-6, whereas these responses were substantially lower in TF-TAVR patients. Only SAVR patients showed measurable IL-10 levels. SAVR patients without prior anticoagulation experienced a robust and transient restoration of platelet function after surgery, with no hypercoagulation observable in functional coagulation assays. None of the procedures led to an immediate improvement of hand and leg coordination, but patients after TA-TAVR had decreased neurocognitive function. Patients after SAVR or TA-TAVR exhibit a robust pro-inflammatory response, which is-on the cytokine level-counterbalanced only in SAVR patients. Our results point toward a greater impact of TA-TAVR on neurocognitive function and indicate a potentially detrimental activation of platelets in some patients after SAVR.

Reference ranges for rotational thromboelastometry in male Sprague Dawley rats

BackgroundA functional coagulation assay was used to investigate the extrinsic pathway of coagulation on citrated whole blood samples from healthy adult male Sprague Dawley rats using the mini cup and pin system.MethodsReference values for coagulation parameters from forty-three animals were calculated using data obtained from the ROTEM® delta hemostasis analyzer with the EXTEM test.ResultsThe following ranges, presented as the 2.5–97.5 percentiles, were established: CT [18–77], CFT [20–80], α [73–86], MCF [53–70], and ML [1–22], along with others.ConclusionsThese reference ranges can be used in future studies in rats to identify clinically significant coagulopathies.

Procoagulant extracellular vesicles in amniotic fluid

Embolization of amniotic fluid (AF) into the blood circulation leads to disseminated intravascular coagulation (DIC). Procoagulant phosphatidylserine (PS)-and tissue factor (TF)-exposing extracellular vesicles (EVs) might play an important role in AF embolism-induced DIC. It was the aim of the present study to perform analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry. We applied a prothrombinase assay (that quantifies PS exposure on EVs), an EV-associated TF activity assay, a fibrin generation assay, a thrombin generation assay, a whole blood clotting model, and flow cytometry in AF and control plasma. We found that PS exposure on EVs was 21-fold increased in AF compared with plasma. Also, EV-associated TF activity was highly increased in AF compared with plasma. AF-derived EVs activated the blood coagulation cascade via PS and TF in the fibrin and thrombin generation assays. In a whole blood clottingmodel, AF-derived EVs significantly shortened the clotting time from 734±139seconds in the presence to 232±139seconds in the absence of an anti-TF antibody. The contact activation pathway via factor XII (FXII) was not affected. Applying flow cytometry, a subpopulation of PS+ and TF+ EVs was identified in AF but not in control plasma. In conclusion, we investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine their procoagulant potential. Taken together, our data further delineate the pathomechanisms underlying AF-induced coagulopathy.

Comparison of Ufh and Enoxaparin Originated from Bovine, Ovine and Porcine Mucosa with Functional Coagulation Assays

Background Heparin is a widely used clinical anticoagulant. It is a linearglycosaminoglycan with an average mass between 10 and 20kDa and is primarily composed oftrisulfated disaccharides comprised of 1,4-linkediduronic acid and glucosamine residues containing someglucuronic acid residues.Heparin is biosynthesized in the Golgi of mast cells commonly found in the liver, intestines, and lungs. Pharmaceuticalheparin currently used in the United States is primarily extracted from porcine intestines. Other sources ofheparin including bovine or ovine intestine are being examined as potential substitutes for porcine intestinalheparin. These additional sources are intended to serve to diversify theheparin supply, making this lifesaving drug more available.Aim. The present in vitro study examines bovine, ovine and porcineunfractionated heparin (UFH) and respective low molecular weight heparinenoxaparin and compares these to thecommercialy available heparins.Methods. Independent samples of platelet poor (PPP) and platelet rich plasma (PRP) from 5 healthy volunteers were spiked with increasing concentrations (from 1 to 10 μg/ml) UFH or enoxaparin of bovine, ovine or porcine origin. Commercially available UFH (Calciparin¨) and enoxaparin (Lovenox¨/Clexan¨) were used as comparators. The studied agents were added in plasma at gravimetrically equal concentrations. The specific anti-Xa activity was measured in PPP (using the STA-Liquid anti-Xa assay) and thrombin generation was assessed in PPP and PRP with the Calibrated Automated Thrombogram (CAT¨) from Diagnostica Stago (France). Thrombin generation was triggered with PPP-reagent¨ or PRP-reagent¨ or by the presence of pancreatic cancer cells BXPC3 or breast cancer cells MCF7 as described elsewhere (Rousseau et al Thromb Res. 2015;136:1273-9). The activated partial thromboplastin clotting time (aPTT) was measured with the conventional assays on STA-R¨ instrument.Results. The anti-Xa activity per microgram of the studied preparations of UFH andenoxaparin as well as theconcentrations which induced a 2-fold prolongation of theaPTT are shown in Table 1. The studied compounds had different anti-Xa activity when compared at gravimetrically equivalent basis. When compared at anti-Xa equivalent basis the studied compounds had the similar potency to double theaPTT. The UFH from bovine, ovine or porcine origin as well asCalciparine¨ at the concentration of 0.6 anti-Xa IU/ml as well asenoxaparin of bovine, ovine or porcine origin andLovenox¨/Clexan¨ completely abrogated thrombin generation induced by physiologically relevant tissue factor andprocoagulantphospholipid concentration in PPP or PRP. At the same concentration (0.6 anti-Xa IU/ml) the studied preparation of UFH andenoxaparin inhibited thrombin generation triggered by pancreatic cancer cells or breast cancer cells.Conclusions. UFH orenoxaparin from different species demonstrate similar functional properties on the inhibition of blood coagulation independent of theprocoagulant trigger, when compared on the basis of equivalent anti-Xa activity. The establishment of functional criteria for the evaluation heparins originated from different species is warranted to demonstrate theirbiosimilarity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Highly Procoagulant Extracellular Vesicles in Amniotic Fluid

Background: The pathomechanisms underlying disseminated intravascular coagulation (DIC) following amniotic fluid (AF) embolism remain to be fully elucidated. Highly procoagulant phosphatidylserine (PS)- and tissue factor (TF) expressing extracellular vesicles (EVs) might play a central role.Objective: To perform extensive analyses of the procoagulant properties of AF with a panel of functional coagulation assays and flow cytometry to investigate the pathogenesis of AF induced DIC.Methods: A prothrombinase assay, an EV-TF dependent factor Xa (FXa) generation assay, a modified thrombin- and fibrin-generation assay, a whole blood clotting model and flow cytometry were applied in AF- and control plasma.Results: Phosphatidylserine expression was 21-fold increased in AF compared to plasma. Factor Xa generation was extremely high when TF-expressing EVs from AF were co-incubated with recombinant FVIIa. In the thrombin- and fibrin generation assay AF-derived EVs strongly activated the blood coagulation cascade via PS and TF. In a whole blood clotting model AF-derived TF-expressing EVs significantly shortened the clotting time from 734 ± 139 seconds in the presence- to 232 ± 139 seconds in the absence of an anti-TF antibody. The contact activation pathway via factor FXII was not affected. Applying flow cytometry, a sub-population of PS- and TF co-expressing EVs was clearly identified in AF.Conclusions: We thoroughly investigated the effect of AF on blood coagulation and found that PS+ and TF+ EVs determine its procoagulant potential. Taken together our data further delineate the pathomechanisms underlying AF-induced coagulopathy, which could improve diagnostic- and treatment modalities. DisclosuresNo relevant conflicts of interest to declare.

Thrombotic risk assessment in antiphospholipid syndrome: the role of new antibody specificities and thrombin generation assay

Antiphospholipid syndrome (APS) is an autoimmune condition characterized by the presence of antiphospholipid antibodies (aPL) in subjects presenting with thrombosis and/or pregnancy loss. The currently used classification criteria were updated in the international consensus held in Sidney in 2005. Vascular events seem to result of local procoagulative alterations upon triggers influence (the so called “second-hit theory”), while placental thrombosis and complement activation seem to lead to pregnancy morbidity. The laboratory tests suggested by the current classification criteria include lupus anticoagulant, a functional coagulation assay, and anticardiolipin and anti-β2-glycoprotein-I antibodies, generally detected by solid phase enzyme-linked immunosorbent assay. The real challenge for treating physicians is understanding what is the actual weight of aPL in provoking clinical manifestations in each case. As thrombosis has a multi-factorial cause, each patient needs a risk-stratified approach. In this review we discuss the role of thrombotic risk assessment in primary and secondary prevention of venous and arterial thromboembolic disease in patients with APS, focusing on new antibody specificities, available risk scoring models and new coagulation assays.