Circulating microRNAs targeting coagulation and fibrinolysis in patients with severe COVID-19.

BACKGROUND: Coronavirus-19 disease (COVID-19) frequently causes coagulation disturbances. Data remains limited on the effects of microRNAs (miRNAs) on coagulation during COVID-19 infection. We aimed to analyze the comprehensive miRNA profile as well as coagulation markers and blood count in hospitalized COVID-19 patients. METHODS: Citrated plasma samples from 40 patients (24 men and 16 women) hospitalized for COVID-19 were analyzed. Basic coagulation tests, von Willebrand factor (VWF), ADAMTS13, blood count, C-reactive protein, and 27 miRNAs known to associate with thrombosis or platelet activation were analyzed. MiRNAs were analyzed using quantitative reverse transcription polymerase chain reaction (RT qPCR), with 10 healthy controls serving as a comparator. RESULTS: Among the patients, 15/36 (41%) had platelet count of over 360 × 109/L and 10/36 (28%) had low hemoglobin of < 100 g/L, while 26/37 (72%) had high VWF of over 200 IU/dL. Patients had higher levels of the miRNAs miR-27b-3p, miR-320a-3p, miR-320b-3p, and miR-424-5p, whereas levels of miR-103a-3p and miR-145-5p were lower than those in healthy controls. In total, 11 miRNAs were associated with platelet count. Let-7b-3p was associated with low hemoglobin levels of < 100 g/L. miR-24-3p, miR-27b-3p, miR-126-3p, miR-145-5p and miR-338-5p associated with high VWF. CONCLUSION: COVID-19 patients differentially express miRNAs with target genes involved in fibrinolysis inhibition, coagulation activity, and increased inflammatory response. These findings support the notion that COVID-19 widely affects hemostasis, including platelets, coagulation and fibrinolysis.

Novel Ex Vivo DOAC Removal Methods Reduce Interference in Lupus Anticoagulant Testing.

Direct oral anticoagulants (DOAC) interfere in laboratory coagulation testing. The aim here was to study how commercial DOAC removal methods, DOAC Filter® and DOAC-Stop™, perform to eliminate DOAC concentrations and false positive results in lupus anticoagulant (LAC) testing. We acquired 50 patient samples with high concentrations of DOACs: apixaban (n = 18, range 68-572 ng/mL), dabigatran (n = 8, range 47-154 ng/mL), edoxaban (n = 8, range 35-580 ng/mL) and rivaroxaban (n = 16, range 69-285 ng/mL). DOACs were removed ex vivo with either DOAC Filter® (n = 28) or DOAC-Stop™ (n = 22). Additionally, commercial control and calibrator samples were studied (n = 13 for DOAC Filter®, n = 14 for DOAC-Stop™). LAC screening was performed before and after DOAC removal. Both DOAC Filter® and DOAC-Stop™ were effective in removing DOAC concentrations in samples: DOAC concentrations decreased to median of 0 ng/mL (range 0-48 ng/mL). Only one sample had more than residual 25 ng/mL of DOAC (apixaban). Before DOAC removal, 96% (48/50) of patient samples and over 90% (12/13 DOAC Filter®, 13/14 DOAC-Stop™) of control/calibrator samples were positive in the LAC screening. In patient samples, LAC screening turned negative in 61% (17/28) after DOAC Filter® and 45% (10/22) after DOAC-Stop™ treatment. All control samples became negative after DOAC removal. In conclusion, DOAC removal ex vivo reduces false positives in LAC screening. DOAC removal halved the need for confirmation or mixing tests- Although a subset of patients would require further testing, DOAC removal reduces unnecessary repeated LAC testing.

Anaemia and enhancement of coagulation are associated with severe COVID-19 infection.

Coagulation disturbances are common in severe COVID-19 infection. We examined laboratory markers in COVID-19 patients during the first wave of the pandemic in Finland. We analysed a wide panel of coagulation tests (IL ACL TOP 750/500®) from anonymously collected samples of 78 hospitalized COVID-19 patients in intensive care units (ICUs; n = 34) or medical wards (n = 44) at Helsinki University Hospital in April-May 2020. These coagulation data were supplemented with the laboratory information system results, including complete blood count and C reactive protein (CRP). Coagulation and inflammatory markers were elevated in most: FVIII in 52%, fibrinogen 77%, D-dimer 74%, CRP 94%, platelet count 37%. Anaemia was common, especially in men (73% vs. 44% in women), and overall weakly correlated with FVIII (women R2 = 0.48, men R2 = 0.24). ICU patients had higher fibrinogen and D-dimer levels (p < .01). Men admitted to the ICU also had higher platelet count, leukocytes and FVIII and lower haemoglobin than the non-ICU patients. None of the patients met the disseminated intravascular coagulation (DIC) criteria, but 31% had a D-dimer level of at least 1.5 mg/L. Presence of both anaemia and high D-dimer together with FVIII is independently associated with ICU admission. Antithrombin was reduced in 47% of the patients but did not distinguish severity. Overall, CRP was associated with coagulation activation. Elevated FVIII, fibrinogen and D-dimer reflected a strong inflammatory response and were characteristic of hospitalized COVID-19 patients. The patients were often anaemic, as is typical in severe inflammation, while anaemia was also associated with coagulation activity.

Anticoagulated patients exhibit intact endogenous thrombin potential using ST Genesia unlike the Calibrated Automated Thrombogram.

BACKGROUND: The thrombin generation (TG) assay is a feasible but labor-intensive method for detecting global coagulation. It enables comprehensive assessment of anticoagulation, while drug-specific assays assess only exposure. Traditionally, the Calibrated Automated Thrombogram (CAT) has been used, however the ST Genesia (Diagnostica Stago) allows automated evaluation. OBJECTIVE: We aimed to observe coagulation using the ST Genesia and compare the data with those of CAT in anticoagulated patients. PATIENTS AND METHODS: In total, 43 frozen-thawed samples were studied using DrugScreen to assess direct oral anticoagulants (DOACs), warfarin, and low-molecular-weight heparin. Twenty samples (nine rivaroxaban, five apixaban, three warfarin, and three heparin) were also compared using CAT (5 pM tissue factor). RESULTS: TG reduction in DrugScreen depended on the specific drug and modestly correlated with DOAC levels (lag time R2 = 0.36; peak R2 = 0.50). The best correlation was observed with peak thrombin and rivaroxaban-specified anti-activated factor X (anti-Xa) activity (R2 = 0.60). When comparing ST Genesia with CAT, only the results for apixaban concorded (R2 = 0.97). Unlike CAT, ST Genesia yielded a normal endogenous thrombin potential (ETP) in 77% (24/31) activated factor X inhibitor cases, and it failed to give readouts at international normalized ratio (INR) ≥4.5 and at anti-Xa ≥1.0 IU/mL. CONCLUSION: The ST Genesia data did not correlate with CAT, but it was independently associated with INR, anti-Xa, and DOAC concentrations. The lag time and peak responses were similar; the major differences were that ST Genesia showed no ETP effect of DOACs and failed to give readout at high INR or anti-Xa activity.

Warfarin dose requirement in patients having severe thrombosis or thrombophilia.

AIMS: Warfarin dose requirement varies significantly. We compared the clinically established doses based on international normalized ratio (INR) among patients with severe thrombosis and/or thrombophilia with estimates from genetic dosing algorithms. METHODS: Fifty patients with severe thrombosis and/or thrombophilia requiring permanent anticoagulation, referred to the Helsinki University Hospital Coagulation Center, were screened for thrombophilias and genotyped for CYP2C9*2 (c.430C>T, rs1799853), CYP2C9*3 (c.1075A>C, rs1057910) and VKORC1 c.-1639G>A (rs9923231) variants. The warfarin maintenance doses (target INR 2.0-3.0 in 94%, 2.5-3.5 in 6%) were estimated by the Gage and the International Warfarin Pharmacogenetics Consortium (IWPC) algorithms. The individual warfarin maintenance dose was tailored, supplementing estimates with comprehensive clinical evaluation and INR data. RESULTS: Mean patient age was 47 years (range 20-76), and BMI 27 (SD 6), 68% being women. Forty-six (92%) had previous venous or arterial thrombosis, and 26 (52%) had a thrombophilia, with 22% having concurrent aspirin. A total of 40% carried the CYP2C9*2 or *3 allele and 54% carried the VKORC1-1639A allele. The daily mean maintenance dose of warfarin estimated by the Gage algorithm was 5.4 mg (95% CI 4.9-5.9 mg), and by the IWPC algorithm was 5.2 mg (95% CI 4.7-5.7 mg). The daily warfarin maintenance dose after clinical visits and follow-up was higher than the estimates, mean 6.9 mg (95% CI 5.6-8.2 mg, P < 0.006), with highest dose in patients having multiple thrombophilic factors (P < 0.03). CONCLUSIONS: In severe thrombosis and/or thrombophilia, variation in thrombin generation and pharmacodynamics influences warfarin response. Pharmacogenetic dosing algorithms seem to underestimate dose requirement.

Hemostatic profile under fluid resuscitation during rivaroxaban anticoagulation: an in vitro survey.

BACKGROUND: Uncontrollable bleeding is the leading cause of death in traumatically injured patients. The extent to which direct factor Xa inhibitors interfere with the applied resuscitation measures is presently unknown. STUDY DESIGN AND METHODS: In this study, we investigated the effect of the resuscitation fluids saline, albumin, fresh frozen plasma (FFP) and solvent/detergent (S/D)-treated plasma, fibrinogen concentrate, prothrombin complex concentrate (PCC), and combinations thereof on the hemostatic profile of rivaroxaban-anticoagulated whole blood and plasma. We used rivaroxaban-spiked whole blood and plasma from healthy donors, as well as plasma from patients on rivaroxaban, and mimicked a resuscitation approach in a 50% plasma dilution setting. Thromboelastography, thrombin generation, and fibrin generation clot lysis test were assessed using tissue factor to initiate coagulation and tissue plasminogen activator to induce clot lysis. RESULTS: Rivaroxaban resulted in a hypocoagulant state that remained largely unaltered upon subsequent 50% dilution with S/D-treated plasma or FFP. Using S/D-treated plasma as a diluent, clot stability decreased due to its low α2 -antiplasmin. Dilution with saline and albumin induced a profibrinolytic state and further deteriorated the impaired hemostatic potential of rivaroxaban-anticoagulated blood, even after PCC and fibrinogen support. Combined use of plasma (either FFP or S/D treated) and PCC, however, considerably improved both coagulation and clot stability. CONCLUSION: In the setting of rivaroxaban anticoagulation and major blood loss, transfusing plasma together with PCC may provide the most effective resuscitation approach with the notion that additional antifibrinolytic drug support (e.g., tranexamic acid) is likely required.

Effects of thromboprophylactic doses of apixaban and rivaroxaban on coagulation and thrombin generation in association with total hip replacement.

Factor Xa inhibitors (FXaI) apixaban and rivaroxaban are used for thromboprophylaxis after major elective orthopaedic surgery. Because few patient sample studies exist, we postoperatively assessed patients undergoing unilateral total hip arthroplasty, including 22 treated with apixaban (2.5 mg BID) and 20 treated with rivaroxaban (10 mg OD). We collected blood samples before and 3 h after drug intake at 4 time points, preoperatively, as well as on day 1, week 1 (day 2-8) and day 28 post-operation. APTT and PT were immediately analysed. Calibrated anti-FXa activity, Russel's Viper Venom Time (RVVT) and thrombin generation (TG; Calibrated Automated Thrombogram®) captured the effects of FXaI on coagulation and TG. APTT and PT remained within the reference interval throughout, and did not correlate with FXaI levels (PT R2 = 0.44, APTT R2 = 0.07). Mean apixaban concentration at the peak varied by eightfold (19-153 ng/mL), but rivaroxaban only by 1.5-fold (111-183 ng/mL). Rivaroxaban, but not apixaban prolonged RVVT at peak levels. Both FXaIs had a prolonged lag time of TG (p < 0.001). Rivaroxaban decreased ETP peak at all time points and reached a minimum at day 28 (540 nM/min at rivaroxaban 184 ng/mL, p < 0.001), while rivaroxaban trough levels were low and ETP values normal. However, with apixaban, after an initial decrease, ETP did not differ between peak and trough levels until decreasing on day 28 at peak (990 nM/min at apixaban 112 ng/mL, p = 0.005). In conclusion, due to different dosing and pharmacology rivaroxaban and apixaban distinctly inhibited TG under postoperative conditions.

From laboratory to clinical practice: Dabigatran effects on thrombin generation and coagulation in patient samples.

INTRODUCTION: Dabigatran (Dabi) is not routinely monitored. However, in emergency cases quantitative assessment is required and laboratories must provide suitable tests at all hours. Little is known about Dabi effects on thrombin generation. MATERIALS AND METHODS: Patient samples (n=241) were analyzed for functional Dabi concentrations (Dabi-TT) using a combination of the Hemoclot Thrombin Inhibitors assay (HTI®) and, for samples with low levels, undiluted thrombin time (TT). Results were compared to prothrombin time (PT) and activated partial thromboplastin time (APTT). In 49 samples Dabi effects were further investigated with Calibrated Automated Thrombogram (CAT®) for thrombin generation and with Russell's viper venom time (RVVT), prothrombinase-induced clotting time (PiCT®), chromogenic Anti-IIa® and ecarin clotting assay (ECA®). Fibrinogen and D dimer were assessed to reflect the coagulation status of the patient. A subset of these samples (n=21) were also analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). RESULTS: Dabi-TT correlated with RVVT (R(2)=0.49), PiCT® (R(2)=0.73), ECA® (R(2)=0.89), Anti-IIa® (R(2)=0.90) and LC-MS/MS (R(2)=0.81). APTT correlated curvi-linearly with Dabi-TT (R(2)=0.71), but was normal in many cases (18/70) despite Dabi-TT>40ng/mL. There was no association between Dabi-TT and fibrinogen or D dimer levels. Increasing Dabi concentrations prolonged lag time (R(2)=0.54) and, surprisingly, elevated the ETP and Peak of CAT® (p<0.001). CONCLUSIONS: Thrombin-specific tests measure Dabi accurately, whereas coagulation time based assays depend more on other factors. The enhanced thrombin generation in Dabi-treated patients may predict clinically relevant hypercoagulability and warrants further investigation.

Laboratory assessment of novel oral anticoagulants: method suitability and variability between coagulation laboratories.

BACKGROUND: Laboratory tests to assess novel oral anticoagulants (NOACs) are under evaluation. Routine monitoring is unnecessary, but under special circumstances bioactivity assessment becomes crucial. We analyzed the effects of NOACs on coagulation tests and the availability of specific assays at different laboratories. METHODS: Plasma samples spiked with dabigatran (Dabi; 120 and 300 µg/L) or rivaroxaban (Riva; 60, 146, and 305 µg/L) were sent to 115 and 38 European laboratories, respectively. International normalized ratio (INR) and activated partial thromboplastin time (APTT) were analyzed for all samples; thrombin time (TT) was analyzed specifically for Dabi and calibrated anti-activated factor X (anti-Xa) activity for Riva. We compared the results with patient samples. RESULTS: Results of Dabi samples were reported by 73 laboratories (13 INR and 9 APTT reagents) and Riva samples by 22 laboratories (5 INR and 4 APTT reagents). Both NOACs increased INR values; the increase was modest, albeit larger, for Dabi, with higher CV, especially with Quick (vs Owren) methods. Both NOACs dose-dependently prolonged the APTT. Again, the prolongation and CVs were larger for Dabi. The INR and APTT results varied reagent-dependently (P < 0.005), with less prolongation in patient samples. TT results (Dabi) and calibrated anti-Xa results (Riva) were reported by only 11 and 8 laboratories, respectively. CONCLUSIONS: The screening tests INR and APTT are suboptimal in assessing NOACs, having high reagent dependence and low sensitivity and specificity. They may provide information, if laboratories recognize their limitations. The variation will likely increase and the sensitivity differ in clinical samples. Specific assays measure NOACs accurately; however, few laboratories applied them.