Increased circulating platelet-derived extracellular vesicles in severe COVID-19 disease.

Coagulation disturbances are major contributors to COVID-19 pathogenicity, but limited data exist on the involvement of extracellular vesicles (EVs) and residual cells (RCs). Fifty hospitalized COVID-19 patients stratified by their D-dimer levels into high (>1.5 mg/L, n = 15) or low (≤1.5 mg/l, n = 35) and 10 healthy controls were assessed for medium-sized EVs (mEVs; 200-1000 nm) and large EVs/RCs (1000-4000 nm) by high sensitivity flow cytometry. EVs were analyzed for CD61, CD235a, CD45, and CD31, commonly used to detect platelets, red blood cells, leukocytes or endothelial cells, respectively, whilst phosphatidyl serine EVs/RCs were detected by lactadherin-binding implicating procoagulant catalytic surface. Small EV detection (sEVs; 50-200 nm) and CD41a (platelet integrin) colocalization with general EV markers CD9, CD63, and CD81 were performed by single particle interferometric reflectance imaging sensor. Patients with increased D-dimer exhibited the highest number of RCs and sEVs irrespective of cell origin (p < .05). Platelet activation, reflected by increased CD61+ and lactadherin+ mEV and RC levels, associated with coagulation disturbances. Patients with low D-dimer could be discriminated from controls by tetraspanin signatures of the CD41a+ sEVs, suggesting the changes in the circulating platelet sEV subpopulations may offer added prognostic value during COVID progression.

What is the context? Coronavirus disease 19 (COVID-19) frequently leads to blood clotting disturbances, including thromboses.Particles smaller than cells, extracellular vesicles (EVs), and residual cells (RCs) affect blood clotting, but data on their role and diagnostic utility in COVID-19 are sparse.What is new? In this study, we assessed 50 hospitalized COVID-19 patients and 10 healthy controls for their different EV subpopulations and residual cells (50­4000 nm).Blood clotting marker D-dimer, which is elevated in severe COVID-19 infection, was used to characterize disease severity and stratify the patient subgroups. Fifteen patients (30%) with high D-dimer (>1.5 mg/L) were compared to controls, and 35 patients with lower D-dimer (≤1.5 mg/mL).The most topical state-of-the-art methods for detection of EV subpopulations, that is, high sensitivity flow cytometry (hsFCM) and single particle interferometric reflectance imaging sensor (SP-IRIS), were used with markers indicative of platelet, red blood cell, leukocyte or endothelial cells. The subpopulations differentiated by platelet and tetraspanin signatures by hsFCM and SP-IRIS, respectively.The main findings are Patients with high D-dimer systematically exhibited the highest number of platelet EVs in all subpopulations (p < .05).Small EVs subpopulations (differentiated by the tetraspanin signatures) could discriminate patients with low D-dimer (p < .001) from healthy controls.Differences between the two D-dimer groups were seen in the platelet-derived (large and medium EVs and RCs), RBC-derived mEVs and l EVs and RCs, and lactadherin-positive large EVs and RCs (p < .05).What is the impact? Platelet activation, reflected by increased EVs was associated with blood clotting disturbances. Small EVs signatures revealed changes in the EV subpopulations in association with blood clotting during COVID-19. Such signatures may enable identification of severely ill patients before the increase in coagulation is evident by coagulation parameters, for example, by high D-dimer.

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.

Whole Blood Thromboelastometry by ROTEM and Thrombin Generation by Genesia According to the Genotype and Clinical Phenotype in Congenital Fibrinogen Disorders.

The outcome of congenital fibrinogen defects (CFD) is often unpredictable. Standard coagulation assays fail to predict the clinical phenotype. We aimed to assess the pheno- and genotypic associations of thrombin generation (TG) and ROTEM in CFD. We measured fibrinogen (Fg) activity and antigen, prothrombin fragments F1+2, and TG by ST Genesia® with both Bleed- and ThromboScreen in 22 patients. ROTEM was available for 11 patients. All patients were genotyped for fibrinogen mutations. Ten patients were diagnosed with hypofibrinogenemia, nine with dysfibrinogenemia, and three with hypodysfibrinogenemia. Among the 17 mutations, eight were affecting the Fg γ chain, four the Fg Bß chain, and five the Fg Aα chain. No statistical difference according to the clinical phenotypes was observed among FGG and FGA mutations. Median F1+2 and TG levels were normal among the different groups. Fg levels correlated negatively with F1+2 and peak height, and positively with lag time and time to peak. The pheno- and genotypes of the patients did not associate with TG. FIBTEM by ROTEM detected hypofibrinogenemia. Our study suggests an inverse link between low fibrinogen activity levels and enhanced TG, which could modify the structure-function relationship of fibrin to support hemostasis.

Factor XIII deficiency enhances thrombin generation due to impaired fibrin polymerization - An effect corrected by Factor XIII replacement.

INTRODUCTION: Factor XIII (FXIII) cross-links fibrin, completing blood coagulation. Congenital FXIII deficiency is managed with plasma-derived FXIII (pdFXIII) or recombinant FXIII (rFXIII) concentrates. AIM: As the mechanisms protecting patients with low FXIII levels (<5IU/dL) from spontaneous bleeds remain unknown we assessed the interplay between thrombin generation (TG), fibrin formation and clot kinetics before and after FXIII administration in three patients with FXIII deficiency. METHODS: Patients received initially rFXIII (35IU/kg, A-subunit) following with pdFXIII at 1250IU or 2500IU (12-30IU/kg) monthly. TG (CAT), thromboelastometry (ROTEM), prothrombin fragments F1+2, fibrinogen and FXIII activity (FXIII:C) were measured at baseline and one-hour recovery. RESULTS: FXIII was at the target level of 20±6IU/dL at the 4-week trough. rFXIII corrected FXIII to 98±15 and high-dose pdFXIII to a level of 90±6, whereas low-dose/half dose pdFXIII reached 45±4IU/dL. Although fibrinogen (Clauss Method) was normal, coagulation in FIBTEM was impaired, which FXIII administration tended to correct. CAT implied 1.6- to 1.9-fold enhanced TG, which FXIII administration normalized. Inhibition of fibrin polymerization by Gly-Pro-Arg-Pro peptide mimicked FXIII deficiency in CAT by enhancing TG both in control and FXIII recovery plasma. Antithrombin, α2-macroblobulin-thrombin complex and prothrombin were normal, whereas F1+2 were elevated compatible with in vivo TG. DISCUSSION: FXIII deficiency impairs fibrinogen function and fibrin formation simultaneously enhancing TG on the poorly polymerizing fibrin strands, when fibrin's antithrombin I -like function is absent. Our study suggests an inverse link between low FXIII levels and enhanced TG modifying structure-function relationship of fibrin to support hemostasis.

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.

Enhanced thrombin generation and reduced intact protein S in processed solvent detergent plasma.

BACKGROUND: Standardized solvent/detergent (S/D)-treated plasma has been developed as an improved alternative to fresh frozen-plasma (FFP) in the management of severe bleeds. This study aimed at exploring compositional modifications that may influence the general applicability of S/D-treated plasma. MATERIALS AND METHODS: S/D-treated plasma and FFP were compared in procoagulant microparticles and concentration of coagulation factors and inhibitors. Compositional differences were correlated with hemostatic and fibrinolytic characteristics as measured by PT, APTT, thrombin generation and thromboelastography. RESULTS: Procoagulant microparticles were absent in S/D-treated plasma. Procoagulant factors were within the normal range. Antithrombin, TFPI and protein S antigen may be normal or slightly reduced depending on the duration of the S/D-treatment, but S/D-treated plasmas had only 12-14% intact functional protein S. Thrombin generation was subsequently increased, especially at low tissue factor concentration (1 pM). Plasma coagulation times in PT and APTT were normal, but 1.5-fold reduced in thromboelastography at low TF (1 pM). α2-antiplasmin was reduced with a concomitant 3-4 fold shortened clot lysis time measured by thromboelastography in the presence of TF (10 pM) and tissue-type plasminogen activator (0.2µg/ml). Enhanced fibrin degradation could be normalised with tranexamic acid. CONCLUSIONS: S/D-treatment seems to induce a procoagulant phenotype that results from a strongly reduced level of intact single chain protein S. Whether this may correct the apparent hemostatic imbalance as suggested from the increased fibrinolysis remains to be established. Our findings may bear implications in patients with deficiencies of natural anticoagulants. Co-administration of tranexamic acid appears beneficial to control enhanced fibrinolysis.

Laboratory-monitored fondaparinux and coagulation activity in association with total hip replacement.

Fondaparinux, indirect factor Xa (FXa) inhibitor, is recommended for thromboprophylaxis for high-risk patients undergoing major orthopedic surgery. We evaluated the prothrombotic state and anticoagulant intensity of fondaparinux (2.5 mg daily) after total hip replacement (THR). Twenty patients underwent THR - seven bilateral and 13 unilateral. Blood samples were collected preoperatively and at 6 h, 8 h (2 h after fondaparinux), 1 day (12-14 h after fondaparinux), and 4 weeks (12-14 h after fondaparinux) postoperatively. Antithrombin (AT), fibrinogen, factor VIII activity, coagulation times, thrombin-AT (TAT) complex, D-dimer, C-reactive protein, prothrombinase-induced clotting time (PiCT) and anti-Xa activity were measured. The latter two were also tested after plasma spiking with fondaparinux 0-1.25 µg/ml. In spiked prophylactic fondaparinux samples (0-0.25 µg/ml), PiCT and anti-Xa activity correlated (r = 0.84) better than in the patient samples (r = 0.35). On the first day, anti-Xa activity and PiCT dissociated, and PiCT lost sensitivity for fondaparinux. AT decreased but stayed within the normal range, whereas TAT complex and D-dimer peaked at 6 h as signs of thrombin generation. On the first postoperative day, TAT and D-dimer halved. Bilateral THR associated with higher TAT and D-dimer levels up to 4 weeks. Perioperative FVIII levels were not affected, but were elevated in both groups (range 191-211%) after 4 weeks. Anti-Xa activity detected prophylactic fondaparinux with higher sensitivity than PiCT in vitro, but even more so in vivo. Thus, PiCT is not the method of choice to assess fondaparinux at least in association with THR. THR, bilateral more than unilateral, increased thrombin generation and D-dimer 7-11-fold early after surgery. Factor VIII activity and D-dimer remained elevated even after 4 weeks despite the compliant thromboprophylaxis with fondaparinux.

Unaltered blood coagulation and platelet function in healthy subjects exposed to acute hypoxia.

INTRODUCTION: Acute hypoxia has been suspected to cause blood coagulation and platelet activation. Our aim was to study blood coagulation and platelet function during a short hypoxic exposure. METHODS: Healthy nonsmoking men (N = 10) inhaled a normobaric hypoxic gas mixture containing 8% of oxygen (92% nitrogen) for 7 min via a face mask. Venous blood was collected 5 min before and during the 5 to 7 min of hypoxia exposure (i.e., pretest and hypoxia samples, respectively) while monitoring arterial oxygen saturation (SaO2) with pulse oximetry. Blood sampling was completed in 2 min and the face mask was removed. Venous epinephrine and norepinephrine, complete blood counts, and a panel of coagulation markers were analyzed. Platelet aggregation induced by ristocetin, adenosine diphosphate (ADP), arachidonic acid, and thrombin receptor activating peptide was studied with Multiplate and shear force-dependent functions with PFA-100R (collagen/epinephrine and collagen/ADP cartridges), both assays in whole blood. RESULTS: During hypoxia, SaO2 declined from 98 to 58% (ranges 97-99% vs. 42-85%), while heart rate increased from 69/min to 94/min (SD 11 vs. SD 13). Venous epinephrine and norepinephrine levels also increased. This short hypoxia induced minor but uniform increases in red cells, reticulocytes, and leukocytes and decreases in platelet counts. Plateletfunctions and prothrombin time, APTT, thrombin time, D-dimer, fibrinogen levels or von Willebrand factor (VWF), antithrombin, factor V (FV) or FVIII activities did not change. DISCUSSION: Profound acute hypoxia failed to affect blood coagulation or platelet functions in healthy individuals.