The sthemO 301 is a new hemostasis analyzer developed by Diagnostica Stago, combining simultaneously three different methodologies (i.e. clotting, chromogenic and immunological assays) on a single platform. The objective of this evaluation was to assess the analytical performances of the sthemO 301 and to compare it with the STA R Max on a selection of 20 hemostasis parameters (PT, aPTT with two reagents, Clauss fibrinogen, thrombin time, D-dimer, anti-Xa, factors II, V, VII, X, VIII, IX and XI, antithrombin, protein C and protein S). Within-run and inter-laboratory precisions have been evaluated using quality controls. Method comparisons were performed using a minimum of 100 plasma samples, collected across multiple sites, and compared to the STA R Max analyzer. Reference intervals were determined or verified based on published data. Within-run and inter-laboratory precisions were respectively below 5.2 and 7.6% whatever the parameter and the sample used. Pearson correlation coefficient was above 0.976 for all assays and slopes ranged from 0.94 to 1.07 for all but thrombin time, which was at 0.86. Bias at levels of interest were less than 6% for most routine coagulation parameters, coagulation factors and inhibitors. We reported the precision, accuracy and reference intervals of 20 parameters on the sthemO 301, half of these being reported for the first time, which include anti-Xa, factors II, V, VII, X, VIII, IX, XI, protein S free antigen and activity. The sthemO 301 analyzer offers satisfactory analytical performance and a good comparability with the STA R Max for all parameters.

In thrombosis and haemostasis, coagulation and platelet activation pathways culminate to form solid fibrin clots, which can become vaso-occlusive or prevent excessive bleeding. We report a novel mechanism describing how developing fibrin clots prolong and modulate the reactivity of thrombin, an enzyme propagating platelet and coagulation activation and forming fibrin from fibrinogen. Using immunological and genetic approaches, we delineate how thrombin bound to the A and Baachains of fibrin E-domains regulates lateral fibrin fibre extension. Our data reveal that fibrin-bound thrombin remains active and is temporarily protected against inactivation by antithrombin-III. Immunological displacement of thrombin from fibrin profoundly lowered its capacity, whereas a peptide mimicking the AA-chain binding-site increased its reactivity. In a cohort of patients with congenital dysfibrinogenemia, carrying FGA, FGB or FGG mutations associated with bleeding or thrombosis phenotypes, we noticed a high thrombin capacity and suppressed thrombinantithrombin-III complex formation, pointing to a prolonged active thrombin lifetime, likely due to abnormal formation of thrombin-containing fibrin. In conclusion, the combination of an impaired clotting and increased thrombogenicity may explain the paradoxical bleeding and thrombotic complications observed in such patients. Development of fibrin-directed agents may offer new therapeutic opportunities to normalize hemostasis or prevent thrombosis.

Background: Recurrent venous thromboembolism (VTE) is a major concern after stopping anticoagulation. Thrombin generation (TG) parameters-including thrombomodulin (TM) sensitivity-have been proposed as biomarkers of recurrence, but findings remain inconsistent. The role of thrombin dynamic (TD) parameters, including prothrombin conversion and thrombin inactivation, is unclear. Objectives: This study evaluated whether TG and TD parameters are associated with VTE recurrence. Methods: In 589 patients from the prospective VISTA study, TG was measured during anticoagulation and 1 month after stopping vitamin K antagonist therapy. TM sensitivity was quantified as the percentage inhibition of endogenous thrombin potential (ETP) and peak thrombin concentration. TD parameters were derived computationally from TG curves. Cox regression was used to evaluate associations with recurrent VTE >2 years, including subgroup analyses excluding hormone-related events. Results: During the 2-year follow-up, 63 patients experienced recurrent VTE. In multivariable Cox regression adjusted for age, sex, index event type, and hormone use, reduced thrombin inactivation by antithrombin was associated with recurrence (per 100-unit decrease-hazards ratio [HR], 1.10; 95% CI, 1.00-1.20). In patients without hormone-related VTE, lower ETP and reduced TM-mediated inhibition of peak thrombin concentration were also significantly associated with recurrence (ETP per 100-unit decrease-HR, 1.11; 95% CI, 1.01-1.21; TM sensitivity per 10% decrease-HR, 1.26; 95% CI, 1.00-1.58). Other TG parameters were not significantly associated with recurrence. Conclusion: Reduced ETP, TM sensitivity, and thrombin inactivation were associated with VTE recurrence in people with unprovoked VTE. These biomarkers may have potential to identify patients at higher risk of recurrence.

Background: Direct oral anticoagulants (DOACs) interfere with coagulation assays, complicating the diagnosis of thrombophilia and lupus anticoagulants. Single-domain antibodies (sdAbs) may be used to neutralize anticoagulant effects in vitro, thereby restoring physiological coagulation for accurate diagnostic testing. Objectives: To develop and validate sdAbs specific to DOACs that inhibit factor Xa, specifically edoxaban, apixaban, and rivaroxaban. Methods: Llama-derived sdAbs specific to DOACs were selected via phage display. To enhance efficacy, bivalent sdAbs were engineered. Both single and bivalent forms were tested in healthy control plasma using various coagulation assays (thrombin generation, prothrombin time [PT], and activated partial thromboplastin time [APTT]), dilute Russell's viper venom time, and international normalized ratio. The ability of these specific sdAbs to reverse the effects of edoxaban, apixaban, and rivaroxaban was also evaluated in plasma samples from patients currently receiving these medications. Results: Antiedoxaban, antiapixaban, and antirivaroxaban sdAbs achieved reversal of edoxaban, apixaban, and rivaroxaban, respectively, as demonstrated by thrombin generation, PT, APTT, dilute Russell's viper venom time, and international normalized ratio assays for both the single and bivalent variants. Specifically, each anti-DOAC sdAb effectively reversed the effects of its respective DOAC on lag time and peak thrombin levels, without overshooting a prothrombotic response. Furthermore, in plasma from edoxaban-, apixaban-, or rivaroxaban-treated patients, the corresponding sdAbs restored both PT and APTT. Conclusions: We selected and validated sdAbs that specifically reverse the inhibitory effects of edoxaban, apixaban, and rivaroxaban, without procoagulant activity. These low-cost reversal agents work directly on plasma or whole blood without incubation or preanalytical steps. They enable rapid, reliable baseline coagulation testing in patients treated with these DOACs.

PURPOSE:Pancreatic ductal adenocarcinoma (PDAC) is associated with a high risk of venous thromboembolism (VTE), which is burdensome and associated with decreased survival. Although neoadjuvant treatment is increasingly used in patients with PDAC, data on VTE in this setting remain scarce. This study evaluated VTE incidence during (neo)adjuvant therapy for resectable and borderline resectable PDAC and its relation to survival. METHODS:This study included patients from the investigator-initiated, multicenter, randomized controlled phase III PREOPANC-2 trial. Patients were randomly assigned to neoadjuvant 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin followed by surgery (FFX arm) or neoadjuvant gemcitabine-based chemoradiotherapy (CRT), followed by surgery and adjuvant gemcitabine (CRT arm). VTE was defined as both incidental and symptomatic lower- or upper-extremity deep vein thrombosis, pulmonary embolism (PE), splanchnic vein thrombosis, and catheter-related thrombosis. VTE occurrence was retrospectively evaluated from random assignment to 12 months after random assignment. The association with overall survival (OS) was analyzed using Cox regression analysis. RESULTS:VTE was diagnosed in 28 of 325 patients (9%): nine (3%) preoperatively and 19 (8%) postoperatively. Most VTEs were symptomatic (54%). Although a higher proportion of patients developed postoperative VTE in the CRT arm (FFX 3% v CRT 12%, P = .02), the 12-month cumulative incidence did not differ between arms (6% v 11%, P = .06). Two patients died from PE-related causes in the CRT arm. VTE was independently associated with reduced OS (adjusted time-varying hazard ratio, 2.13, P = .002). CONCLUSION:VTE occurred in 9% of patients with (borderline) resectable PDAC undergoing (neo)adjuvant treatment in the year after random assignment and was associated with decreased OS. These results underscore the need for standardized reporting of thromboembolic events in clinical trials and future studies assessing the potential benefits of thromboprophylaxis during neoadjuvant therapy.

Pro- and anti-coagulant factors are significantly altered in liver cirrhosis resulting in an increased bleeding and thrombosis risk. The thrombin generation (TG) test often seems unaltered in cirrhosis, indicating that a new hemostatic balance is reached. TG is determined by prothrombin conversion and thrombin inactivation. These processes can be quantified using the computational thrombin dynamics (TD) add-on analysis for the TG test. We studied TD parameters in cirrhosis to explore its predictive potential for bleeding and thrombosis. In the current study, 32 CP-A, 22 CP-B and 41 CP-C cirrhosis patients and 20 healthy controls were enrolled. Cirrhosis was predominantly alcohol-related (52%), while viral and metabolic etiologies accounted for 34% and 14% of cases, respectively. Patients receiving anticoagulant therapy were excluded. TG was measured using PPP Reagent Low in the presence and absence of thrombomodulin. TG peak height and velocity index were higher in patients than controls (+20%,p = 0.039 and +70%,p = 0.002), and time-to-peak was shorter (−18%,p < 0.0001). TD parameters were obtained computationally based on TG and coagulation factor data. The amount of prothrombin converted (PCtot) was significantly reduced in patients (−43%,p < 0.0001), and the magnitude of the reduction was associated with disease severity. The maximum rate of prothrombin conversion (PCmax) showed a trend towards higher levels in CP-A patients. On the anticoagulant slide, the thrombin decay capacity (TDC) was significantly lower in patients (−44%,p < 0.0001), indicating a newly developed balance between pro- and anticoagulant processes that result in a slightly procoagulant TG phenotype. Four patients experienced bleeding and seven experienced thrombosis during follow-up (median = 499 days; CI: 478–524 days). Low maximum prothrombin conversion rates (PCmax) were associated with an increased hazard of bleeding. Patients in the lowest 20% of PCmax had a HR of 12.9 (95% CI 1.34–124.31, p = 0.027) for bleeding. Low thrombin-α2Macroglobulin (T-α2M) complex formation was significantly associated with thrombosis during follow-up. Patients in the lowest quintile of T-α2M had a HR of 6.6 (95% CI 1.47–29.9, p = 0.014) for thrombosis. We conclude that reduced prothrombin conversion and thrombin inactivation result in rebalanced TG in cirrhosis patients. Our results demonstrate the potential clinical usefulness of TD parameters for the stratification of bleeding and thrombotic risk in cirrhosis patients.

Patients with hemorrhage after treatment with direct oral anticoagulants (DOACs) are saved from hematoma expansion by administration of Andexanet alfa, a modified factor Xa analog. A recent clinical trial points to incidental prothrombotic effects after Andexanet alfa treatment. We investigated mechanisms explaining the thrombogenic side effect. Calibrated automated thrombin generation was employed to assess the effect of Andexanet alfa on the coagulant potential of DOAC-treated plasma and whole blood. The study examined anticoagulant inhibition pathways involving tissue factor pathway inhibitor (TFPI), antithrombin, and endothelial cell components. Treatment of control plasmas with rivaroxaban, apixaban, or edoxaban resulted in an impaired thrombin generation, exemplified by a prolonged lag time and reduced thrombin peak height. Andexanet alfa over-converted this anticoagulant activity into a procoagulant effect. Plasma treatment with Andexanet alfa similarly increased thrombin generation in the absence of DOACs, even at 8 nM far below the therapeutic dose. The enhancement was abrogated in the absence or by blocking of TFPI. The Andexanet alfa effect was enhanced in the presence of heparin, thereby reversing the antithrombin protection. In the presence of endothelial cells, Andexanet alfa stimulated coagulation via TFPI and heparin inhibition. Andexanet alfa overconverts the anticoagulant effect of rivaroxaban, apixaban, and edoxaban to achieve a hypercoagulable plasma state. The action mechanism involves TFPI inhibition and neutralization of antithrombin-heparin complexes. This may account for the incidence of thrombosis observed in patients treated with Andexanet alfa.

Background: One-third of patients undergoing transcatheter aortic valve implantation (TAVI) have a concomitant indication for oral anticoagulation (OAC). Previous studies suggested that periprocedural continuation of OAC could reduce transient hyper-coagulation after TAVI. Objective: To evaluate the effects of continuation versus interruption of OAC on periprocedural hemostasis function. Methods: Patients were randomized 1:1 to OAC continuation vs interruption. Plasma samples were taken at 6 time points, and hemostasis function was assessed by measuring thrombin and plasmin generation, as well as biomarkers of coagulation, fibrinolysis, platelet activation, and endothelial function. Results: A total of 167 patients were included: 82 were assigned to OAC continuation and 85 to OAC interruption. Pre-TAVI, a significantly lower endogenous thrombin potential (ETP) was observed in the continuation group compared with the interruption group. In both groups, ETP was reduced immediately post-TAVI, and its restoration was significantly lower in the continuation group. In contrast, levels of prothrombin fragment 1 + 2 were similar between the groups. A significantly lower endogenous plasmin potential was observed in the continuation group from pre-TAVI to 8 hours post-TAVI. In both groups, fibrin degradation products increased immediately post-TAVI, while this increase was significantly lower in the continuation group. Markers of endothelial and platelet activation were unaffected by the randomized strategy. Conclusion: Continuation of OAC during TAVI modestly reduced the acute activation of coagulation and fibrinolysis compared with the interruption of OAC. This did not translate into any apparent differences in thromboembolic risk in the main trial. However, continuation of OAC resulted in a marked suppression of ETP and an increased risk of bleeding.

BACKGROUND:Coronary artery disease plaque rupture leads to occlusive thrombosis, causing acute myocardial infarction requiring immediate life-saving treatment. The blood clot is supported by a mesh of fibrin fibers that are generated through the polymerization of fibrin. We recently showed that fibrin also produces a film on the surface of blood clots. OBJECTIVES:This study aimed to examine whether fibrin film occurs intravascularly on thrombi from patients with myocardial infarction. METHODS:In this observational study, we recruited 42 patients with acute ST-elevation myocardial infarction and obtained intracoronary thrombi using catheter-guided aspiration thrombectomy. We used a protocol that avoids air contact and immediately fixes the sample upon thrombectomy. Thrombi were analyzed by tiled scanning electron micrographs to analyze percentage coverage by fibrin film. RESULTS:We found that all thrombi showed surface areas covered with fibrin film. Some film coverage was discontinuous. Total film coverage was on average 24.1% ± 17.0% (range, 4.6%-77.2%). Percentage of film coverage correlated positively with the time from onset of symptoms to thrombectomy (ischemia duration) and negatively with in vitro clot formation time. Discontinuous film did not correlate with plasmin generation. CONCLUSION:These findings show that fibrin film is an integral structural feature that covers around one-quarter of the surface of all coronary thrombi and that older thrombi contain more film. This new structure on thrombi may have important implications for clot contraction, resistance to thrombolysis, and mechanical clot extraction.

Background:The thrombin dynamics method has been used as an add-on analysis method for the thrombin generation test over the past years to study the balance between pro- and anticoagulant pathways. Thrombin generation test results are known to be age-dependent and gender-dependent. Objectives:In this study, we investigated the association of thrombin dynamics parameters with age and gender. Methods:Thrombin generation was measured in 17,530 individuals of the Moli-sani cohort using the Calibrated Automated Thrombinography method. Thrombin dynamics analysis was performed to quantify parameters of prothrombin conversion and thrombin inactivation. Differences in thrombin dynamics parameters in men and women and in different age groups were investigated. Additionally, reference ranges for each age and gender category were established. Results:Thrombin dynamics parameters change throughout life, and these changes differ between men and women. The total amount of prothrombin conversion decreased with increasing age (-14.4% difference between 35-44 and 85+ years of age; R = -0.234; P < .001), although the maximum rate of prothrombin conversion increased with age in women (+18.2%; R = 0.079; P < .001). The ability to inactivate thrombin decreases with age as well (-13.7%; R = -0.320; P < .001), indicating a shift towards a more procoagulant phenotype with advancing age. This is reinforced by the diminished inhibitory actions of thrombomodulin on the procoagulant pathway. Conclusion:The balance between prothrombin conversion and thrombin inactivation becomes more procoagulant with increasing age, but only in women. Therefore, age and gender appropriate reference values for thrombin dynamics parameters would be of interest for the clinical implementation of thrombin generation and thrombin dynamics analysis.

Background: Patients with cirrhosis develop multiple hemostatic alterations. Although fibrinolysis is also affected by liver disease, studies have produced conflicting results, highlighting the need for a reliable fibrinolysis assay. Assessing the kinetics of plasmin generation (PG) is a new method to study the fibrinolytic state of cirrhosis patients. Objectives: This study aimed to compare fibrinolysis between patients with cirrhosis and healthy subjects. Methods: This single-center cohort study included cirrhosis patients from the Padova University Hospital. Fibrinolysis and hemostasis were assessed with PG, thrombin generation (TG), and clot lysis time. To quantify malalignment between TG and PG, ratios were calculated. Results: In total, 101 patients with cirrhosis (Child-Pugh A/B/C: 36/24/41) and 20 healthy subjects were included. Compared with healthy subjects, patients showed a significantly lower endogenous plasmin potential and plasmin peak. The PG capacity decreased with liver disease severity. The lag time to PG was prolonged in patients. No differences in endogenous thrombin potential and lag time were found when comparing TG profiles. Patients had a shorter clot lysis time. Increased TG/PG ratios for the endogenous plasmin potential and plasmin peak were found in patients compared with that in controls. TG/PG ratios increased with liver disease severity. Conclusion: Patients with cirrhosis have a complex fibrinolytic profile, with a delayed and decreased capacity to generate plasmin and a more rapid clot lysis. A disbalance was found between coagulation and fibrinolysis, with a normal-to-increased TG capacity and a decreased PG capacity. These results support the theory that cirrhosis patients are in a prothrombotic state.

BACKGROUND:Venous thromboembolism (VTE) can recur shortly after stopping anticoagulation, highlighting the need for reliable biomarkers to identify high-risk patients during treatment. OBJECTIVES:To determine whether platelet and endothelial markers predict VTE recurrence. METHODS:We used data and samples from the randomized controlled VISTA trial (2011-2015), which included patients with unprovoked VTE who were treated with vitamin K antagonists for 6 months. After treatment cessation, patients were followed for 2 years to assess recurrence. This dataset formed the basis for the current prospective analysis, in which plasma levels of von Willebrand factor (VWF), active VWF (aVWF), soluble P-selectin, soluble thrombomodulin, CXCL4, and CXCL7 were measured before and after cessation of anticoagulation. Associations between biomarker levels and recurrence were analyzed using Cox regression. RESULTS:Plasma samples from 629 patients with a first unprovoked VTE who discontinued vitamin K antagonists were analyzed. Recurrence occurred in 75 patients (12%); 11 (15%) within 1 month, 29 (39%) within 3 months, and 46 (61%) after 3 months. Elevated levels of VWF and aVWF were associated with an increased risk of recurrence per 10-unit increase (VWF: hazard ratio, 1.03; 95% CI, 1.01-1.06; aVWF: hazard ratio, 1.02; 95% CI, 1.00-1.05). Associations were stronger for early recurrence (<3 months), while (a)VWF levels were not associated with late recurrence (>3 months). Stratification showed that VWF and aVWF were associated with increased recurrence risk in men, but not in women. CONCLUSION:Elevated levels of VWF and aVWF during anticoagulation were associated with early recurrence in men and might serve as biomarkers for predicting VTE recurrence.

The hemostatic process relies on platelet and coagulation activation, with additional roles of red blood cells and the vessel wall. By systematic screening of databases for gene-linked information on hemostasis, we collected phenotypic profiles of 3474 orthologous human and mouse genes regarding bleeding, arterial thrombosis, thrombophilia, platelet traits, coagulation, and erythrocytes. Comparisons showed that defects in 252 mouse genes led to increased bleeding combined with platelet dysfunction or thrombocytopenia, in addition to 150 human orthologs that are registered for familial bleeding disorders, based on panel sequencing. Additionally, 139 mouse genes contributed to arterial thrombosis without bleeding phenotype. To further investigate the role of platelets in hemostasis, we integrated multiple genome-wide RNA-sequencing transcriptomes and proteomes from healthy subjects and C57BL/6 mice. This provided reference levels for 54 790 (54 247) transcripts and 6379 (4563) proteins in human (mouse) platelets. Orthologous transcripts in human and mouse platelets correlated with R=0.75, whereas orthologous platelet proteins correlated with R=0.87. Comparison with the phenotypic analysis revealed the following: (i) overall high qualitative similarity of human and mouse platelets regarding composition and function; (ii) presence of transcripts in platelets for most of the 3474 phenotyped genes; (iii) preponderance of syndromic platelet-expressed genes; and (iv) 20-40% overlap with genes from genome-wide association studies. For 42 mouse genes, among which receptors, signaling proteins, and transcription regulators (ASXL1, ERG, GATA2, MEIS1, NFE2, and TAL1), we confirmed novel links with human platelet function or count. This interspecies comparison can serve as a valuable resource for researchers and clinicians studying the genetics of blood-borne hemostasis and thrombosis.

BackgroundRecent developments have made the thrombin generation (TG) test accessible to the clinical laboratory. Therefore, the clinical interpretation of TG parameters has become of increasing interest, and reference values are required. Age and sex have been shown to affect TG parameters, but no consensus has been reached on the subject. We investigated the effect of age and sex on TG parameters to determine the need for age and sex specific reference values for TG.MethodsTG was measured in 22,014 individuals of the Moli-sani study, a cross-sectional cohort of the general adult Italian population. TG was measured using Calibrated Automated Thrombinography in platelet poor plasma (PPP) using PPP reagent Low and PPP reagent, in the presence and absence of thrombomodulin (TM).ResultsIndividuals were grouped into 6 age categories: 35–44 years of age (n = 5,073), 45–54 (n = 6,448), 55–64 (n = 5,516), 65–74 (n = 3,539), 75–84 (n = 1,261), and 85 years of age and older (n = 106). Men and women were distributed evenly in the age categories. Smoking was more common at younger age, whereas cardiovascular diseases, hypertension, hypercholesterolemia, and diabetes were more common at older age (p < 0.001). Lag time and velocity index increased with age, whereas the endogenous thrombin potential (ETP) and time-to-peak decreased. The inhibitory effect of TM was reduced at higher age (p < 0.001). The TG lag time was shorter in women than men at younger age (6%–7% lower in women), and the ETP was lower in women. The activated protein C (APC) pathway was desensitized in women and older individuals.ConclusionThe TG profile becomes more “procoagulant” at older age, especially in women. The sensitivity of the APC pathway to TM is reduced with increasing age in men and women. Therefore, age and sex appropriate reference values for TG parameters would be of interest for the ongoing clinical implementation of the TG assay.

Abstract:Thrombocytopenia at admission predicts mortality in multiple myeloma (MM) and might link to disease progression. Although thrombocytopenia is known to be associated with MM, a possible thrombopathy is clinically less known. We conducted a case-control study comparing platelet responses of MM patients to controls via flow cytometry, integrin αIIbβ3 activation and P-selectin exposure, and a bioluminescent assay, ATP release. No difference was found at baseline, but upon platelet stimulation, MM patients had decreased αIIbβ3 activation, partly impaired P-selectin exposure, and reduced δ-granule (ATP) secretion. Aspirin treatment in patients did not account for these diminished platelet responses. In total, 29% of patients had thrombocytopenia, while 60% had decreased αIIbβ3 activation and 67% had reduced platelet secretion capacity. Importantly, as secretion capacity was corrected for platelet count, granule release per platelet was reduced in patients versus controls. Of 6 patients with thrombocytopenia 4 displayed a thrombopathy, while for 15 patients with normal count, 64% had reduced αIIbβ3 activation and 73% had reduced platelet secretion capacity. Of all patients, 10% had thrombocytopenia combined with reduced αIIbβ3 activation plus low secretion capacity (one patient showed no qualitative or quantitative platelet defect). Our data suggest that beyond the known thrombocytopenia, MM patients also have reduced platelet function, which could reflect impaired platelet vitality. Combined measurement of platelet count and function, especially secretion capacity, gives a more comprehensive view of platelet phenotype than count alone. Large prospective follow-up studies are needed to confirm the importance of the acquired platelet secretion defect on the prognosis of MM patients.