Properties of Plasma Clots in Adult Patients Following Fontan Procedure: Relation to Clot Permeability and Lysis Time—Multicenter Study

Objectives: thromboembolic complications are a major cause of morbidity and mortality following Fontan (FO) surgery. It is also well established that altered FO circulation results in systemic complications, including liver and endothelium damage. We sought to evaluate whether dysfunctions of these sources of hemostatic factors may result in changes of fibrin clot properties. Methods: a permeation coefficient (Ks) and clot lysis time (CLT) were assessed in 66 FO patients, aged 23.0 years [IQR 19.3–27.0], and 59 controls, aged 24.0 years [IQR 19.0–29.0]. Ks was determined using a pressure-driven system. CLT value was measured according to assay described by Pieters et al. Endothelium and liver-derived hemostatic factors along with liver function parameters were evaluated. The median time between FO operation and investigation was 20.5 years [IQR 16.3–22.0]. Results: FO patients had lower Ks (p = 0.005) and prolonged CLT (p < 0.001) compared to that of controls. Ks correlated with CLT (r = −0.28), FVIII (r = −0.30), FIX (r = −0.38), fibrinogen (r = −0.41), ALT (r = −0.25), AST (r = −0.26), GGTP (r = −0.27) and vWF antigen (r = −0.30), (all p < 0.05). CLT correlated with the time between FO operation and investigation (r = 0.29) and FIX (r = 0.25), (all p < 0.05). After adjustment for potential cofounders, TAFI antigen and GGTP were independent predictors of reduced Ks (OR 1.041 per 1% increase, 95% CI 1.009–1.081, p = 0.011 and OR 1.025 per 1 U/L increase, 95% CI 1.005–1.053, p = 0.033, respectively). Protein C and LDL cholesterol predicted prolonged CLT (OR 1.078 per 1% increase, 95% CI 1.027–1.153, p = 0.001 and OR 6.360 per 1 μmol/L increase, 95% CI 1.492–39.894, p = 0.011, respectively). Whereas elevated tPA was associated with lower risk of prolonged CLT (OR 0.550 per 1 ng/mL, 95% CI 0.314–0.854, p = 0.004). GGTP correlated positively with time between FO surgery and investigation (r = 0.25, p = 0.045) and patients with abnormal elevated GGTP activity (n = 28, 42.4%) had decreased Ks, compared to that of the others (5.9 × 10−9 cm2 vs. 6.8 × 10−9 cm2, p = 0.042). Conclusion: our study shows that cellular liver damage and endothelial injury were associated with prothrombotic clot phenotype reflected by Ks and CLT.

Absence of Hyperactivation of Fibrinolysis Explains the Lack of Hemostatic Efficacy of Prophylactic Tranexamic Acid (TXA) in Hypoproliferative Thrombocytopenia

Background: We previously reported the results of the A-TREAT study (American Trial Using Tranexamic Acid in Thrombocytopenia: NCT02578901). This randomized double-blind placebo-controlled trial demonstrated that TXA administration is not superior to placebo in preventing WHO grade 2 or higher bleeding in severely thrombocytopenic patients requiring supportive platelet transfusion following myeloablative therapy for hematologic disorders (Gernsheimer T., ASH 2020 Plenary Session). Here, we present results of the A-TREAT ancillary study - Fibrinolysis Evaluation in A-TREAT (FEAT). Blood samples were collected from a subset (n=115) of A-TREAT participants just prior to initiation of study drug (when the platelet count was &lt;30,000/µl) and at a later time point when TXA was at a steady state trough level (5 ±2 days following study drug initiation). Using global assays of fibrinolysis in plasma, our a priori hypotheses were that: 1] a baseline 'hyperfibrinolytic' profile would be associated with a higher proportion of grade 2+ bleeding; and 2] trough TXA levels would be associated with a 'hypofibrinolytic' profile and a lower proportion of grade 2+ bleeding. Methods: Fibrinolysis in platelet-free citrated plasma was assessed by 3 global assays: euglobulin clot lysis time (ECLT), plasmin generation (PG), and tPA resistance clot lysis time (tPA-CLT) using previously described methods (Ilich A. RPTH 2020, Miszta A. JTH 2021). Trough plasma TXA concentration was measured using a validated tandem mass spectrometry assay. Individual fibrinolytic analytes (PAI-1, tPA, plasminogen, alpha2-antiplasmin and plasmin-antiplasmin complexes) were quantified by ELISA. Results: Baseline samples did not demonstrate a hyperfibrinolytic profile by ECLT. To the contrary, ECLT values were significantly increased compared to healthy controls (figure 1). Furthermore, none of the measured fibrinolytic parameters (ECLT, tPA-CLT, total PAI-1, tPA, plasminogen, alpha2-antiplasmin or plasmin-antiplasmin complexes) at baseline were associated with a greater risk of grade 2+ bleeding during follow up, regardless of treatment arm. On the follow-up samples, neither pharmacokinetic (trough TXA concentration) nor pharmacodynamic parameters (PG or tPA-CLT) were associated with bleeding severity. A high inter-patient variability of TXA trough concentrations was noted in the treatment arm (min-max: 0.7-10 ug/ml), and drug levels correlated strongly with global fibrinolysis assessment by PG (Spearman r, -0.78, 95% CI -0.88 - -0.62) and tPA-CLT (r, 0.74, 0.56 - 0.85) (figure 2). Conclusions: 1] No evidence of fibrinolytic hyperactivation was observed in these thrombocytopenic patients; 2] trough TXA concentrations varied significantly between patients receiving the same dosing schedule; and 3] tPA-CLT and PG parameters correlated well with TXA plasma concentrations and thus may be used to estimate the extent of fibrinolytic inhibition in patients treated with TXA. Discussion: The absence of hyperactivation of endogenous fibrinolysis in this study is in contrast to our recent findings in trauma. Specifically, we reported that almost half of trauma patients demonstrated evidence of fibrinolytic hyperactivation (by ECLT) on admission (Ilich A. Thromb Res, 2021). Since TXA has been shown to reduce mortality due to bleeding in trauma (CRASH II, Lancet, 2010) and given that baseline hyperfibrinolysis is common in trauma, we hypothesize that the absence of fibrinolytic hyperactivation observed in the A-TREAT study patients likely explains the clinical lack of efficacy of TXA. Figure 1 Figure 1. Disclosures Gernsheimer: Amgen: Honoraria; Novartis: Honoraria; Principia: Research Funding; Rigel: Research Funding; Cellphire: Consultancy; Dova: Consultancy; Sanofi: Consultancy. Triulzi: Fresenius Kabi: Membership on an entity's Board of Directors or advisory committees; Realta: Membership on an entity's Board of Directors or advisory committees. Wolberg: CSL Behring: Consultancy; Bristol Myers Squibb: Research Funding; Takeda: Research Funding. Key: Takeda: Research Funding; BioMarin: Honoraria, Other: Participation as a clinical trial investigator; Sanofi: Consultancy; Grifols: Research Funding; Uniqure: Consultancy, Other: Participation as a clinical trial investigator.

Does Fibrin Structure Contribute to the Increased Risk of Thrombosis in COVID-19 ICU Patients?

Introduction: SARS-CoV-2 is responsible for a global pandemic, with almost 200 million confirmed cases. SARS-CoV-2 infection can lead to various disease states, from only mild symptoms in the majority of cases to severe disease, which is associated with an increased incidence of venous thromboembolism (VTE). We hypothesized that an altered fibrin network structure contributes to VTE in COVID-19 patients by affecting thrombus stability and fibrinolysis sensitivity. By studying the fibrin network of COVID-19 patients, we aimed to unravel the mechanisms that contribute to the increased risk of VTE in COVID-19 patients. Methods: Between April 2020 and December 2020, we collected plasma samples from patients with COVID-19 admitted to the intensive care unit (ICU) of the Erasmus Medical Center. We included patients with confirmed VTE diagnosed on CT-angiography, and COVID-19 patients without confirmed VTE during ICU admission. Samples were collected on admission to the ICU and after confirmed VTE or at similar time points in ICU patients without confirmed VTE. In addition, we collected plasma from COVID-19 patients at admission to general wards without confirmed VTE and from healthy controls. Clots were formed by mixing citrated plasma with thrombin (final concentration 1 U/ml) and calcium (17 mM). We imaged the clots using stimulated emission depletion (STED) microscopy, a super-resolution technique in which a depletion laser is used to selectively switch off fluorophores surrounding the focal point, thereby increasing the resolution. In these images, fibrin fiber diameters were measured using the Local Thickness plugin of ImageJ. Fiber density was quantified as percentage of area in Z-stacks of confocal microscopy images. Finally, a clot lysis assay based on turbidity was used to determine sensitivity to fibrinolysis (clot lysis time) and clot density (difference between maximum and baseline absorbance). Differences in fibrin network properties between groups were tested using One-Way ANOVA with Bonferroni post-hoc tests and linear regression with and without adjustment for fibrinogen levels. Results: We included 21 COVID-19 ICU patients with confirmed VTE, 20 COVID-19 ICU patients without confirmed VTE, 10 COVID-19 ward patients and 7 healthy controls. Mean age was comparable between the groups, while BMI was higher in COVID-19 patients than in healthy controls (Table 1). Levels of fibrinogen, D-dimer and anti-Xa were significantly higher in COVID-19 ICU patients than in COVID-19 ward patients and healthy controls. FVIII levels were significantly higher in COVID-19 ICU patients than in healthy controls, while FXIII levels were significantly lower. On admission to the ICU, clot density was significantly higher in COVID-19 ICU patients with and without confirmed VTE than in healthy controls (Figure 1 and Table 2). However, after adjustment for fibrinogen levels, this difference disappears. Clot lysis time was significantly longer in clots from COVID-19 ICU patients than in clots from healthy controls, regardless of fibrinogen levels (Table 2). COVID-19 ICU patients with confirmed VTE also showed a significant longer clot lysis time than COVID-19 ward patients. Interestingly, in the clot lysis assay, fibrinolysis did not occur in 25% of COVID-19 ICU patients with VTE versus 9.5% of COVID-19 ICU patients without VTE (Figure 2). This fibrinolysis shutdown was never observed in clots from healthy controls and COVID-19 ward patients. Fibrin fiber diameters were comparable between the groups. In the clots from plasma samples collected at admission to the ICU, there were no differences between COVID-19 ICU patients with and without VTE (Figure 2). However, when comparing clots prepared from plasma collected at the second time point (after VTE or at a similar time point for patients without VTE), we observed significant longer clot lysis times in patients with confirmed VTE (97.4 [88.5-158.8] min) than in patients without confirmed VTE (80.0 [76.0-97.8] min) (p=0.03). Finally, there were no significant changes between clots from plasma before and after VTE or between the two time points in patients without VTE, except for a decreased clot lysis time over time for COVID-19 ICU patients without confirmed VTE. Conclusion: Our results suggest that SARS-CoV-2 infection increases clot density and decreases clot susceptibility to fibrinolysis, and that these changes relate to the severity of the disease. Figure 1 Figure 1. Disclosures Kruip: Daiichi Sankyo: Research Funding; Bayer: Honoraria, Research Funding.

Exact distribution of threshold-crossing times for protein concentrations: Implication for biological timekeeping

Stochastic transcription and translation dynamics of protein accumulation up to some concentration threshold sets the timing of many cellular physiological processes. Here we obtain the exact distribution of first threshold-crossing times of protein concentration, in either Laplace or time domain, and its associated cumulants: mean, variance and skewness. The distribution is asymmetric and its skewness non-monotonically varies with the threshold. We study lysis times of E-coli cells for holin gene mutants of bacteriophage-λ and find a good match with theory. Mutants requiring higher holin thresholds show more skewed lysis time distributions as predicted.

Increased Expression of Extracellular Vesicles Is Associated With the Procoagulant State in Patients With Established Rheumatoid Arthritis

This study sought to identify different subpopulations of extracellular vesicles (EVs) in plasma from female patients with established rheumatoid arthritis (RA) in relation to the activation of coagulation and fibrin formation in these patients. Forty women were included in the study, 20 patients and 20 age-matched healthy controls. The mean disease duration in patients was 13.0 (5.0–25.0) years, with medium to high disease activity despite ongoing treatment with low-dose prednisolone and methotrexate. There were no differences between the investigated groups regarding the presence of traditional cardiovascular risk factors. The concentration of phosphatidylserine-positive (PS+) EVs; platelet (CD42a+), leucocyte (CD45+), monocyte (CD14+), and endothelial (CD144+)-derived EVs; and EVs-expressing tissue factor (CD142+), P-selectin (CD62P+), and E-selectin (CD62E+) were determined by flow cytometry analysis. Overall hemostasis potential (OHP) was assessed to follow the hemostatic disturbances, including the parameters for overall coagulation potential (OCP) and overall fibrinolytic potential (OFP). Fibrin clot turbidity was measured together with clot lysis time, and scanning electron microscopy was performed. Increased concentrations of PS+, CD42a+, CD142+, CD45+, CD14+, and CD62P+ EVs were found in plasma from patients with RA compared to healthy controls, and the concentrations of PS+, CD42a+, CD14+, and CD62P+ EVs were positively correlated with the inflammatory parameters in RA patients. Positive correlations were also found between the levels of PS+ and CD42a+ EVs and OCP as well as between the levels of PS+, CD42a+, and CD62P+EVs and OHP. The levels of PS+, CD42a+, CD14+, CD62P+, and CD62E+ EVs were negatively correlated with OFP. Elevated levels of circulating EVs of different cell origins were found in patients with established RA, in relation to the inflammatory burden and coagulation activation in the disease.

P–706 Impaired fibrinolysis during estrogen substitution in relation to frozen-thawed embryo transfer

Study question Is the clot lysis time prolonged in women undergoing estrogen substitution in artificial cycle during frozen-thawed embryo transfer (AC-FET)? Summary answer Women receiving AC-FET have a prolonged clot lysis time that could result in increased venous thromboembolic risk during estrogen substitution. What is known already High doses of estrogen are used for women treated with AC-FET; this in contrast to women treated with natural cycle frozen-thawed embryo transfer (NC-FET). Based on previous research on hormone replacement therapy in other settings, estrogen substitution is probably associated with an increased risk of thromboembolism. Moreover, it has formerly been shown that pregnant women followed assisted reproductive technology (ART) treatment as compared to natural fertilization, has an increased risk of thrombosis. However, changes in fibrinolysis has never been examined in women undergoing estrogen substitution during treatment with AC-FET. Study design, size, duration Prospective cohort study of women receiving AC-FET with oestrogen/progesterone substitution or NC-FET. Blood samples were obtained four times: 1) prior to hormone substitution (baseline), 2) confirmation of biochemical pregnancy, 3) gestational week 8 and 4) gestational week 13 (5 weeks after cessation of hormone substitution). Inclusion criteria: women aged &gt; 18 years assigned for FET. Exclusion criteria: egg donor recipients, known bleeding disorders, indication for thromboprophylaxis and treatment with anti-platelet medication or non-steroid-anti-inflammatory drugs. Participants/materials, setting, methods We included women at the Department of Obstetrics and Gynaecology, Horsens Fertility Clinic, Denmark, from August 2019 – November 2020. In total, 34 participants were included: 19 women treated with AC-FET and 15 women receiving NC-FET. We examined fibrinolysis measured by a dynamic fibrin clot lysis assay that can assess the capacity for fibrin formation and fibrinolysis. This detailed information of the fibrinolytic activity are used as a surrogat marker of thromboembolic risk. Main results and the role of chance Our results showed a significantly longer lysis time (908 ± 234 vs 1157 ± 218) (p &lt; 0.001) within the AC-FET group after hormone substitution compared to baseline. Moreover, we found a higher area under the curve (AUC) (919 ± 305 vs 1167 ± 391) (p = 0.006) within the AC-FET group. However, we observed no changes in mean lag phase or maximum absorbency after estrogen substitution within the AC-FET group. Since we observed a significantly higher AUC within the AC-FET group after estrogen substitution, this is probably due to the prolonged lysis time, indicating hypofibrinolysis. No significant changes was found comparing the NC-FET group with the AC-FET group. Limitations, reasons for caution Our data are based on a small study population. Additionally, we cannot exclude that the coagulation parameters could be affected by estrogen prior to study enrollment as we had no wash out period. Wider implications of the findings: Our findings indicate prothrombotic changes in the AC-FET group. It is relevant to individually consider the indication for AC-FET and restrict the use of unnecessary hormone exposure. These data should be followed by a populations-based study to clarify how this potentially increased venous thromboembolic risk will manifest itself clinically. Trial registration number NCT04359576

The influence of sample quantity and lysis parameters on the success of ancient DNA extraction from skeletal remains

DNA extraction is of utmost importance in archaeobiology, as it determines the success of further DNA analyses. This study concentrates on the success of ancient DNA extraction using silica spin columns and PCR-based analysis from archaeological skeletal material and investigates the influence of sample quantity, lysis time and lysis temperature during sample preparation. The results show that lysis times ranging from 2 to 48 h are suitable, and that lysis should be carried out at a constant temperature of 56°C. Concerning sample quantity, 10 mg for mitochondrial DNA and 50 mg for chromosomal DNA are sufficient for high quality analyses. Thus invaluable sample material can be saved, and time of sample preparation can be reduced considerably.

Determination of Ocular Irritancy Potential of Ophthalmic products using HET-CAM Method

Objective: The research is about the stability of ophthalmic products as they start to produce irritancy effects during their shelf life. The aim of the present study is to assess the stability of certain ophthalmic products by the HET-CAM (Hen’s Egg Test-Chorioallatonic membrane) Method. Method: Incubated Eggs were utilized for performing the HET-CAM assay according to the protocol prescribed by ICCVAM (The Inter agency Coordinating Committee on the Validation of Alternative Methods). Results: Calculation of various irritancy factors like hemorrhage time, vascular lysis time and coagulation time and to calculate HET-CAM score. Statistical interpretation of the data obtained by one-way ANOVA. Conclusion: Results have shown that the few ophthalmic products are slight and moderate irritants. None of the products are strong irritants. However further studies to be conducted to confirm the potential and safety of the products.

POS0874 CLOT LYSIS TIME PREDICTS RECURRENT DIGITAL ULCERS IN SYSTEMIC SCLEROSIS AFTER ONE YEAR OF FOLLOW-UP: A NESTED CASE-CONTROL STUDY

Background:Digital ulcers (DU) are a common visible manifestation of vasculopathy in systemic sclerosis (SSc), which could be recurrent and associated with disability and mortality (1). Although vasculopathy is connected with impaired coagulation/fibrinolysis system and aberrant expression of adhesion molecules, there are few data about their role in developing recurrent DU.Objectives:To evaluate the possible role of Fibrin generation/Fibrinolysis parameters and adhesion molecules in the prediction of new ischaemic DU oncet during a 1-year follow-up and their impact on the time to new DU onset (TD).Methods:From 58 consecutive patients with SSc who fulfilled the 2013 ACR/EULAR SSc criteria and have never been treated with endothelin receptor antagonist, phosphodiesterase 5 inhibitors or prostanoids, a total of 38 patients with ever had DU, either active at inclusion or in past, were enrolled in a prospective cohort study. Each patient was given a “DU diary”. Demografic, clinical and serologycal data were recorded. The serum concentration of ICAM1 and E selectin were measured by ELISA. Haemostatic potential parameters: overall haemostasis (OHP), overall coagulation (OCP) and overall fibrinolysis (OFP) potential were assessed. Maximum absorbance (Cmax), reflects the fibrin clot density and clot lysis time (Lys50t0, time from initiation of clot formation to the time at which a 50% fall in absorbance from Cmax in the lysis assay), reflects fibrinolytic susceptibility, were calculated (2). Fibrin structure was visualised using scanning electron microscopy (SEM).Results:Over the follow-up period,18 patients (45.5%) developed new DU with the average TD of 7.4±2.9 months. There was no differance in ASA and CCB treatment among two groups (p>0.05). The OFP value was significantly decreased (p<0.01), Lys50t0 prolonged (p<0.05), while OHP was increased (p<0.05) in patients experienced new DU. Lys50t0 showed good validity in identifying patients with new DU oncet (AUC 0.683 95% CI 0.5 - 0.9). By multivariate analysis including clinical data in model the Lys50t0 (HR 1.2, 95% CI 1.1-1.3, p=0.018) and active DU at enrollment (HR 9.6, 95% CI 1.4-66.8, p=0.022) were identified as independent risk factors for the occurrence of new DU. TD was inversly correlated with ICAM1(p<0.001), E selectin (p<0.05), Cmax (p <0.05) and Lys50t0 (p<0.001). Model explaining 81.6% of the TD variability included Lys50t0 (β=- 0.55,p=0.003), E selectin (β=- 0.44,p=0.014) and fibrinogen (β=- 0.49,p=0.017). SEM revealed denser fibrin clots with thinner fibres in group experienced new DU compared to clots formed in plasma of patient without DU.Conclusion:Our results provide evidence that impaired fibrinolysis has critical role in the progression of microvascular disease, identifing clot lysis time as a strong predictor in the oncet of new digital ulcers in Systemic sclerosis. The higher level of E selectin and the longer lysis time are, the shorter is time until the onset of new digital ulcer. These results could be used in selection of patients at high risk of developing recurrent digital ulcer and therefore allow earlier therapeutic intervention.References:[1]Allanore Y, Distler O, Matucci-Cerinic M, Denton CP. Review: Defining a Unified Vascular Phenotype in Systemic Sclerosis. Arthritis Rheumatol. 2018 Feb;70(2):162-170[2]Carter AM, et al. Heritability of clot formation, morphology, and lysis: the EuroCLOT study. Arterioscler Thromb Vasc Biol. 2007 Dec;27(12):2783-9.Figure 1.SEM images of fibrin network in 1 representative sample from a SSc patient with (A) and 1from patient without (B) new DU oncetDisclosure of Interests:None declared