Quantitative protein mass spectrometry for multiplex measurement of coagulation and fibrinolytic proteins towards clinical application: What, why and how?

Plasma proteins involved in coagulation and fibrinolysis are essential to hemostasis. Consequently, their circulating levels and functionality are critical in bleeding and thrombosis development. Well-established laboratory tests to assess these are available; however, said tests do not allow high multiplicity, require large volumes of plasma and are often costly. A novel technology to quantify plasma proteins is quantitative protein mass spectrometry (QPMS). Aided by stable isotope-labeled internal standards a large number of proteins can be quantified in one single analytical run requiring <30 µL of plasma. This provides an opportunity to improve insight in the etiology and prognosis of bleeding and thrombotic disorders, in which the balance between different proteins plays a crucial role. This manuscript aims to give an overview of the QPMS potential applications in thrombosis and hemostasis research (quantifying the 38 proteins assigned to coagulation and fibrinolysis by the KEGG database), but also to explore the potential and hurdles if designed for clinical practice. Advantages and limitations of QPMS are described and strategies for improved analysis are proposed, using as an example the test requirements for antithrombin. Application of this technology in the future could represent a step towards individualized patient care.

Genetic polymorphisms and major bleeding risk during vitamin K antagonists treatment: The BLEEDS case-cohort.

BACKGROUND: Major bleeding occurs annually in 1%-3% of patients on vitamin K antagonists (VKAs), despite close monitoring. Genetic variants in proteins involved in VKA response may affect this risk. AIM: To determine the association of genetic variants (cytochrome P450 enzymes 2C9 [CYP2C9] and 4F2 [CYP4F2], gamma-glutamyl carboxylase [GGCX]) with major bleeding in VKA users, separately and combined, including vitamin K epoxide reductase complex subunit-1 (VKORC1). METHODS: A case-cohort study was established within the BLEEDS cohort, which includes 16,570 patients who initiated VKAs between 2012 and 2014. We selected all 326 major bleeding cases that occurred during 17,613 years of follow-up and a random subcohort of 978 patients. We determined variants in CYP2C9, CYP4F2, GGCX, VKORC1 and evaluated the interaction between variant genotypes. Hazard ratios for major bleeding with 95% confidence intervals (95% CI) were estimated by weighted Cox regression. RESULTS: Genotype was determined in 256 cases and 783 subcohort members. Phenprocoumon was the most prescribed VKA for both cases and the subcohort (78% and 75%, respectively). Patients with major bleeding were slightly older than subcohort patients. CYP4F2-TT carriership was associated with a 1.6-fold (95% CI 0.9-2.8) increased risk of major bleeding compared with CC-alleles, albeit not statistically significant. For the CYP2C9 and GGCX variants instead, the major bleeding risk was around unity. Carrying at least two variant genotypes in CYP2C9 (poor metabolizer), CYP4F2-TT, and VKORC1-AA was associated with a 4.0-fold (95%CI 1.4-11.4) increased risk, while carriers of both CYP4F2-TT and VKORC1-AA had a particularly increased major bleeding risk (hazard ratio 6.7, 95% CI 1.5-29.8) compared with carriers of CC alleles in CYP4F2 and GG in VKORC1. However, the number of major bleeding cases in carriers of multiple variants was few (8 and 5 patients, respectively). CONCLUSIONS: CYP4F2 polymorphism was associated with major bleeding, especially in combination with VKORC1 genetic variants. These variants could be considered to further personalize anticoagulant treatment.

Coexisting atrial fibrillation and cancer: time trends and associations with mortality in a nationwide Dutch study.

BACKGROUND AND AIMS: Coexisting atrial fibrillation (AF) and cancer challenge the management of both. The aim of the study is to comprehensively provide the epidemiology of coexisting AF and cancer. METHODS: Using Dutch nationwide statistics, individuals with incident AF (n = 320 139) or cancer (n = 472 745) were identified during the period 2015-19. Dutch inhabitants without a history of AF (n = 320 135) or cancer (n = 472 741) were matched as control cohorts by demographic characteristics. Prevalence of cancer/AF at baseline, 1-year risk of cancer/AF diagnosis, and their time trends were determined. The association of cancer/AF diagnosis with all-cause mortality among those with AF/cancer was estimated by using time-dependent Cox regression. RESULTS: The rate of prevalence of cancer in the AF cohort was 12.6% (increasing from 11.9% to 13.2%) compared with 5.6% in the controls; 1-year cancer risk was 2.5% (stable over years) compared with 1.8% in the controls [adjusted hazard ratio (aHR) 1.52, 95% confidence interval (CI) 1.46-1.58], which was similar by cancer type. The rate of prevalence of AF in the cancer cohort was 7.5% (increasing from 6.9% to 8.2%) compared with 4.3% in the controls; 1-year AF risk was 2.8% (stable over years) compared with 1.2% in the controls (aHR 2.78, 95% CI 2.69-2.87), but cancers of the oesophagus, lung, stomach, myeloma, and lymphoma were associated with higher hazards of AF than other cancer types. Both cancer diagnosed after incident AF (aHR 7.77, 95% CI 7.45-8.11) and AF diagnosed after incident cancer (aHR 2.55, 95% CI 2.47-2.63) were associated with all-cause mortality, but the strength of the association varied by cancer type. CONCLUSIONS: Atrial fibrillation and cancer were associated bidirectionally and were increasingly coexisting, but AF risk varied by cancer type. Coexisting AF and cancer were negatively associated with survival.

Tailored anticoagulant treatment after a first venous thromboembolism: protocol of the Leiden Thrombosis Recurrence Risk Prevention (L-TRRiP) study - cohort-based randomised controlled trial.

INTRODUCTION: Patients with a first venous thromboembolism (VTE) are at risk of recurrence. Recurrent VTE (rVTE) can be prevented by extended anticoagulant therapy, but this comes at the cost of an increased risk of bleeding. It is still uncertain whether patients with an intermediate recurrence risk or with a high recurrence and high bleeding risk will benefit from extended anticoagulant treatment, and whether a strategy where anticoagulant duration is tailored on the predicted risks of rVTE and bleeding can improve outcomes. The aim of the Leiden Thrombosis Recurrence Risk Prevention (L-TRRiP) study is to evaluate the outcomes of tailored duration of long-term anticoagulant treatment based on individualised assessment of rVTE and major bleeding risks. METHODS AND ANALYSIS: The L-TRRiP study is a multicentre, open-label, cohort-based, randomised controlled trial, including patients with a first VTE. We classify the risk of rVTE and major bleeding using the L-TRRiP and VTE-BLEED scores, respectively. After 3 months of anticoagulant therapy, patients with a low rVTE risk will discontinue anticoagulant treatment, patients with a high rVTE and low bleeding risk will continue anticoagulant treatment, whereas all other patients will be randomised to continue or discontinue anticoagulant treatment. All patients will be followed up for at least 2 years. Inclusion will continue until the randomised group consists of 608 patients; we estimate to include 1600 patients in total. The primary outcome is the combined incidence of rVTE and major bleeding in the randomised group after 2 years of follow-up. Secondary outcomes include the incidence of rVTE and major bleeding, functional outcomes, quality of life and cost-effectiveness in all patients. ETHICS AND DISSEMINATION: The protocol was approved by the Medical Research Ethics Committee Leiden-Den Haag-Delft. Results are expected in 2028 and will be disseminated through peer-reviewed journals and during (inter)national conferences. TRIAL REGISTRATION NUMBER: NCT06087952.

Coagulation profiles during and after anabolic androgenic steroid use: data from the HAARLEM study.

Background: Anabolic androgenic steroids (AAS) are thought to increase venous thromboembolism (VTE) risk. Objectives: We investigated whether AAS influence coagulation parameters associated with VTE by assessing their changes during and after AAS use. Methods: The HAARLEM study enrolled 100 male amateur athletes voluntarily starting an AAS cycle between 2015 and 2018. We measured procoagulant and anticoagulant protein levels, D-dimer levels, endogenous thrombin potential (ETP), and clot lysis time (CLT) at baseline and during 2 years of follow-up. Changes in coagulation during AAS cycle, 3 months after its discontinuation, and 1 year after its inclusion compared with baseline were estimated using linear mixed models. The associations between AAS dose and duration of use with these outcomes were studied through adjusted multivariable linear regression. Results: Participants used AAS for a median of 13 weeks (IQR: 10-23) with a median weekly dose of 901 mg (IQR: 634-1345 mg). Mean levels of multiple coagulation factors (F) increased during use compared with baseline, whereas FVIII and von Willebrand factor levels remained unchanged. Protein S and D-dimer showed the biggest increase (22% [95% CI: 15-29] and 1.3-fold [95% CI: 1.2-1.5], respectively). CLT was 8 minutes longer (95% CI: 5-10) and ETP was 165 nM∗min (95% CI: -205 to -124) lower during the AAS cycle. A high weekly AAS dose and short cycle duration were associated with changes in protein S and ETP during use. All parameters returned to baseline values 3 months after discontinuation and remained similar after. Conclusion: During AAS use, procoagulant and anticoagulant protein levels increased in a reversible manner. The overall balance did not suggest a clear procoagulant state.

Efficacy and safety of intravenous imatinib in COVID-19 ARDS: a randomized, double-blind, placebo-controlled clinical trial.

PURPOSE: A hallmark of acute respiratory distress syndrome (ARDS) is hypoxaemic respiratory failure due to pulmonary vascular hyperpermeability. The tyrosine kinase inhibitor imatinib reversed pulmonary capillary leak in preclinical studies and improved clinical outcomes in hospitalized COVID-19 patients. We investigated the effect of intravenous (IV) imatinib on pulmonary edema in COVID-19 ARDS. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled trial. Invasively ventilated patients with moderate-to-severe COVID-19 ARDS were randomized to 200 mg IV imatinib or placebo twice daily for a maximum of seven days. The primary outcome was the change in extravascular lung water index (∆EVLWi) between days 1 and 4. Secondary outcomes included safety, duration of invasive ventilation, ventilator-free days (VFD) and 28-day mortality. Posthoc analyses were performed in previously identified biological subphenotypes. RESULTS: 66 patients were randomized to imatinib (n = 33) or placebo (n = 33). There was no difference in ∆EVLWi between the groups (0.19 ml/kg, 95% CI - 3.16 to 2.77, p = 0.89). Imatinib treatment did not affect duration of invasive ventilation (p = 0.29), VFD (p = 0.29) or 28-day mortality (p = 0.79). IV imatinib was well-tolerated and appeared safe. In a subgroup of patients characterized by high IL-6, TNFR1 and SP-D levels (n = 20), imatinib significantly decreased EVLWi per treatment day (- 1.17 ml/kg, 95% CI - 1.87 to - 0.44). CONCLUSIONS: IV imatinib did not reduce pulmonary edema or improve clinical outcomes in invasively ventilated COVID-19 patients. While this trial does not support the use of imatinib in the general COVID-19 ARDS population, imatinib reduced pulmonary edema in a subgroup of patients, underscoring the potential value of predictive enrichment in ARDS trials. Trial registration NCT04794088 , registered 11 March 2021. European Clinical Trials Database (EudraCT number: 2020-005447-23).

How to predict recurrent venous thromboembolism and bleeding? A review of recent advances and their implications.

After the first venous thromboembolism (VTE), anticoagulant treatment duration should be based on the balance between the risk of recurrence and bleeding. However, this decision is challenging on the individual level. Prediction models that accurately estimate these risks may help selecting patients that would benefit from either short or indefinite anticoagulant treatment. Currently, 17 models to predict VTE recurrence and 15 models to predict bleeding in VTE patients have been proposed. In addition, 7 models to predict bleeding in anticoagulated patients, mostly for atrial fibrillation, have been evaluated for use in VTE patients. Sex, age, type, and location of the index event and D­dimer levels were the most often included predictors of recurrent VTE, whereas age, history of (major) bleeding, active malignancy, antiplatelet therapy, anemia, and renal insufficiency were most often used for the prediction of bleeding. In this review, we provide a summary of these models and their performance. Notably, these models are rarely used in clinical practice and none of them is incorporated in current guidelines due to insufficient accuracy or insufficient validation. Moreover, evidence supporting the value of implementing these models is still lacking. Before these models can be used in routine care, further refinement may be required, and their added value and feasibility should be proven in both management and implementation studies.

Adherence to protocols for the use of reversal agents in patients treated with direct oral anticoagulants.

OBJECTIVES: This study aimed to evaluate the adherence to protocols for the use of reversal agents in direct oral anticoagulant (DOAC) users in Dutch hospitals. METHODS: A retrospective cohort study was conducted in seven hospitals in the Netherlands. Treatment protocols for bleeding and (urgent) procedures in patients on DOAC were collected from each hospital. All patient data on the use of reversal agents were retrospectively collected from September 2021 to April 2022 and compared to the protocols. The degree of per-protocol adherence (compliance score) was categorized into four levels as follows: poor (<45%), moderate (45-79%), high (80-89%), and full (> 90%) adherence rates. RESULTS: A total of 290 patients were included in our study. In patients with bleeding under DOAC, the protocol adherence for prothrombin complex concentrate (PCC) was "moderate" (61%). In the remaining cases (39%), non-adherence was mainly caused by underdosing (68%), overdosing (12%), and a lack of indication (14%). Furthermore, idarucizumab was administered for bleeding with "full" adherence (96%). For andexanet alfa, adherence to the hospital bleeding protocol was "moderate" (67%), with a lack of indication being the only reason for non-adherence. In case of reversal for an urgent procedure, the protocol adherence for PCC was "low" (45%), with underdosing, a lack of indication, and missing lab data being the main reasons for non-adherence. Missing lab data on dabigatran plasma concentration before reversal was the main reason for "low" adherence (26%) in idarucizumab. The adherence for andexanet alfa was also "low" (0%). CONCLUSION: In case of reversal for bleeding under DOAC, overall adherence to the protocol was "moderate"; however, in patients needing an urgent procedure, it was "low." The major reasons for non-adherence were underdosing, off-label use, and a lack of specific lab testing. The results of this study can assist in improving the implementation of hospital protocols.

Influence of rosuvastatin on apolipoproteins and coagulation factor levels: Results from the STAtin Reduce Thrombophilia trial.

Background: The STAtins Reduce Thrombophilia trial showed that, in patients with prior venous thrombosis, rosuvastatin decreased various coagulation factor levels. Objectives: Here, we investigated the hypothesis that statins decrease coagulation factor levels through shared mechanisms of synthesis or regulatory pathways with apolipoproteins. Methods: We measured the levels of apolipoprotein (Apo)A-I, A-II, A-IV, (a), B-100, B-total, C-I, C-II, C-III, and E in patients (n = 126) randomized to 28 days of rosuvastatin use. We assessed the association between apolipoproteins and coagulation factors at baseline using linear regression. The mean difference in apolipoprotein levels between baseline and after 28 days of rosuvastatin use was determined through linear regression, adjusting for age, sex, and body mass index. Coagulation factors were added to this model to determine if the lowering of apolipoproteins by rosuvastatin was linked with coagulation factor levels. Results: At baseline, levels of all apolipoproteins, except Apo(a), were positively associated with FVII, FIX, and FXI. Apolipoproteins levels, except for ApoA-I, A-IV, and Apo(a), were decreased after 28 days of rosuvastatin. ApoB-100 showed the largest mean decrease of -0.43 g/L (95% CI = -0.46 to -0.40). The decrease in ApoC-I and C-III levels was associated with a decrease in FVII, whereas the decrease in apoA-II, B-100, and B-total was associated with a decrease in FXI. The decrease in apolipoproteins was neither associated with FVIII or vWF decrease nor with endogenous thrombin potential changes. Conclusions: Rosuvastatin decreases the level of several apolipoproteins, but this decrease was associated only with a decrease in FVII and XI and not with FVIII/vWF.

Selective Serotonin Reuptake Inhibitor Use and Risk of Major Bleeding during Treatment with Vitamin K Antagonists: Results of A Cohort Study.

BACKGROUND: Selective serotonin reuptake inhibitors (SSRIs) may increase the risk of major bleeding by decreasing platelet function or decreasing vitamin K antagonist (VKA) metabolism via cytochrome P450 (CYP) inhibition. AIMS: To determine whether SSRIs are associated with major bleeding during VKA treatment and investigate the possible mechanisms. METHODS: In this cohort study, information on SSRI use and bleeding complications was obtained from patient records of VKA initiators between 2006 and 2018 from two anticoagulation clinics. Conditional logistic regression and time-dependent Cox regression were used to estimate the effect of SSRIs on a high international normalized ratio (INR ≥ 5) within 2 months after SSRI initiation and on major bleeding during the entire period of SSRI use, respectively. SSRI use was stratified for (non-)CYP2C9 inhibitors. RESULTS: A total of 58,918 patients were included, of whom 1,504 were SSRI users. SSRI initiation versus nonuse was associated with a 2.41-fold (95% confidence interval [CI]: 2.01-2.89) increased risk for a high INR, which was 3.14-fold (95% CI: 1.33-7.43) among CYP2C9-inhibiting SSRI users. The adjusted hazard ratio of major bleeding was 1.22 (95% CI: 0.99-1.50) in all SSRI users and 1.31 (95% CI: 0.62-2.72) in CYP2C9-inhibiting SSRI users compared with nonusers. CONCLUSION: SSRI use is associated with an increased risk of high INR and might be associated with major bleeding. The risk of a high INR was slightly more elevated for CYP2C9-inhibiting SSRI users, suggesting there might be a pharmacokinetic interaction (by CYP2C9 inhibition) next to a pharmacodynamic effect of SSRIs on platelet activation.

Clinical Outcomes in Patients With Dilated Cardiomyopathy and Ventricular Tachycardia.

BACKGROUND: Recurrent ventricular tachycardia (VT) due to dilated cardiomyopathy (DCM) is difficult to treat, and long-term outcome data are limited. OBJECTIVES: The aim of this study was to identify predictors of mortality or heart transplantation (HTx) and VT recurrence. METHODS: Consecutive patients with DCM accepted for radiofrequency catheter ablation (RFCA) of VT at 9 centers were prospectively enrolled and followed. RESULTS: Of 281 consecutive patients (mean age 60 ± 13 years, 85% men, mean left ventricular ejection fraction [LVEF] 36% ± 12%), 35% had VT storm, 20% had incessant VT, and amiodarone was unsuccessful in 68%. During follow-up of 21 months (IQR: 6-30 months), 67 patients (24%) died or underwent HTx, and 138 (49%) had VT recurrence (45 within 30 days, defined as early); the 4-year rate of VT recurrence or mortality or HTx was 70%. Independent predictors of mortality or HTx were early VT recurrence (HR: 2.92; 95% CI: 1.37-6.21; P < 0.01), amiodarone at discharge (HR: 3.23; 95% CI: 1.43-7.33; P < 0.01), renal dysfunction (HR: 1.92; 95% CI: 1.01-3.64; P = 0.046), and LVEF (HR: 1.36; 95% CI: 1.0-1.84; P = 0.052). LVEF ≤32% identified patients at risk for mortality or HTx (area under the curve: 0.75). Mortality or HTx per 100 person-years was 40.4 events after early, compared with 14.2 events after later VT recurrence and 8.5 events with no VT recurrence after RFCA (P < 0.01 for both). Patients with early recurrence and LVEFs ≤32% had a 1-year rate of mortality or HTx of 55%. VT recurrence was predicted by prior implantable cardioverter-defibrillator shocks, basal anteroseptal VT origin, and procedural failure but not LVEF. CONCLUSIONS: Patients with DCM needing RFCA for VT are a high-risk group. Following RFCA, approximately one-half remain free of VT recurrence. Early VT recurrence with LVEF ≤32% identifies those at very high risk for mortality or HTx, and screening for mechanical support or HTx should be considered. Late VT recurrence after RFCA does not predict worse outcome.

The INVENT COVID trial: a structured protocol for a randomized controlled trial investigating the efficacy and safety of intravenous imatinib mesylate (Impentri®) in subjects with acute respiratory distress syndrome induced by COVID-19.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has led to a disruptive increase in the number of intensive care unit (ICU) admissions with acute respiratory distress syndrome (ARDS). ARDS is a severe, life-threatening medical condition characterized by widespread inflammation and vascular leak in the lungs. Although there is no proven therapy to reduce pulmonary vascular leak in ARDS, recent studies demonstrated that the tyrosine kinase inhibitor imatinib reinforces the endothelial barrier and prevents vascular leak in inflammatory conditions, while leaving the immune response intact. METHODS: This is a randomized, double-blind, parallel-group, placebo-controlled, multicenter clinical trial of intravenous (IV) imatinib mesylate in 90 mechanically ventilated subjects with COVID-19-induced ARDS. Subjects are 18 years or older, admitted to the ICU for mechanical ventilation, meeting the Berlin criteria for moderate-severe ARDS with a positive polymerase chain reaction test for SARS-CoV2. Participants will be randomized in a 1:1 ratio to either imatinib (as mesylate) 200 mg bis in die (b.i.d.) or placebo IV infusion for 7 days, or until ICU discharge or death. The primary study outcome is the change in Extravascular Lung Water Index (EVLWi) between day 1 and day 4. Secondary outcome parameters include changes in oxygenation and ventilation parameters, duration of invasive mechanical ventilation, number of ventilator-free days during the 28-day study period, length of ICU stay, and mortality during 28 days after randomization. Additional secondary parameters include safety, tolerability, and pharmacokinetics. DISCUSSION: The current study aims to investigate the efficacy and safety of IV imatinib in mechanically ventilated subjects with COVID-19-related ARDS. We hypothesize that imatinib decreases pulmonary edema, as measured by extravascular lung water using a PiCCO catheter. The reduction in pulmonary edema may reverse hypoxemic respiratory failure and hasten recovery. As pulmonary edema is an important contributor to ARDS, we further hypothesize that imatinib reduces disease severity, reflected by a reduction in 28-day mortality, duration of mechanical ventilation, and ICU length of stay. TRIAL STATUS: Protocol version and date: V3.1, 16 April 2021. Recruitment started on 09 March 2021. Estimated recruitment period of approximately 40 weeks. TRIAL REGISTRATION: ClinicalTrials.gov NCT04794088 . Registered on 11 March 2021.

Nonsustained Ventricular Tachycardia Is Independently Associated With Sustained Ventricular Arrhythmias in Nonischemic Dilated Cardiomyopathy.

BACKGROUND: Spontaneous nonsustained ventricular tachycardia (NSVT) on Holter, VT inducibility during electrophysiology study, and late gadolinium enhancement (LGE) on cardiac magnetic resonance (CMR) have been associated with sustained ventricular arrhythmias (SVAs) in nonischemic dilated cardiomyopathy (DCM). This study aimed to analyze whether these parameters carry independent prognostic value for spontaneous SVA in DCM. METHODS: Between 2011 and 2018, patients with the DCM clinical spectrum and documented SVA, suspected SVA, or considered to be at intermediate or high risk for SVA were enrolled in the prospective Leiden Nonischemic Cardiomyopathy Study. Patients underwent a comprehensive evaluation including 24-hour Holter, LGE-CMR, and electrophysiology study. Holters were assessed for the presence of NSVT (≥3 beats; rate, ≥120 bpm; lasting <30 s) and NSVT characteristics (coupling interval, duration, cycle length, morphology, regularity). Patients were followed at 6 to 12 monthly intervals. RESULTS: Of all 115 patients (age, 59±12 years; 77% men; left ventricular ejection fraction, 33±13%; history of SVA, 36%; LGE in 63%; median LGE mass, 13 g; interquartile range, 8-23 g), 62 (54%) had NSVT on Holter, and sustained monomorphic VT was inducible in 34 of 114 patients (30%). NSVT was not associated with LGE on CMR or VT inducibility during electrophysiology study nor were its features (all P>0.05). During 4.0±1.8 years of follow-up, SVA occurred in 39 patients (34%). NSVT (HR, 4.47 [95% CI, 1.87-10.72]; P=0.001) and VT inducibility (HR, 3.08 [95% CI, 1.08-8.81]; P=0.036) were independently associated with SVA during follow-up. A bivariable model including only noninvasively acquired parameters also allowed identification of a high-risk subgroup (ie, those with both NSVT and LGE on CMR). The findings remained similar when only patients without prior SVA were included. CONCLUSIONS: In patients with DCM, NSVT on Holter and VT inducibility during electrophysiology study predict SVA during follow-up independent of LGE on CMR. NSVTs may serve as an initiator, and sustained VT inducibility indicates the presence of the substrate for SVA in DCM. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01940081.

Inter- and intra-individual concentrations of direct oral anticoagulants: The KIDOAC study.

BACKGROUND: Direct oral anticoagulants (DOACs) do not require concentration monitoring. However, whether DOAC concentrations are stable and their variation between and within patients is not well studied. METHODS: Patients on vitamin K antagonists (VKA) who switched to rivaroxaban, apixaban, or dabigatran were included between 2018 and 2020. Blood was drawn at DOAC trough and peak concentrations at week 0, 2, and 8. Plasma drug concentrations were determined by anti-factor Xa concentrations (rivaroxaban, apixaban) or diluted thrombin time (dabigatran). Inter- and intra-individual variability was assessed by calculating the coefficient of variation (CV). Linear regression models were employed to evaluate associations between DOAC trough concentrations and previous VKA dosage, creatinine clearance, and body mass index (BMI). RESULTS: One hundred fifty-two patients were included, of whom 96 (63%) were male and with a mean age of 73.9 ± 8.4 years. For the inter-individual variability, the CV ranged between 48% and 81% for trough values and between 25% and 69% for peak values among patients using the recommended DOAC dose. Intra-individual variability was substantially lower, as here the CV ranged between 18% and 33% for trough values and between 15% and 29% for peak values among patients using the recommended DOAC dose. Previous VKA dosage and creatinine clearance were inversely associated with DOAC trough concentrations. No association was found between BMI and DOAC trough concentrations. CONCLUSION: Inter-individual variability of DOAC concentrations was higher than intra-individual variability. Lower previous VKA dosage and creatinine clearance were associated with higher DOAC trough concentrations. These findings support further study into an optimal target range, in which the risks of both bleeding and thrombosis are minimal.

Major bleeding during oral anticoagulant therapy associated with factor V activation by factor Xa.

OBJECTIVE: Plasma thrombin generation (TG) provides important information on coagulation status; however, current TG output parameters do not predict major bleeding of patients on anticoagulants. We recently reported that factor V (FV) activation by factor X (FX)a contributes importantly to the initiation phase of TG. Here we investigated how this pathway varies in the normal population and whether FXa-mediated activation of FV is associated with major bleeding in patients on anticoagulant therapy. APPROACH: We employed TIX-5, a specific inhibitor of FV activation by FXa, to estimate the contribution of FXa-mediated FV activation to tissue factor (TF)-initiated TG. RESULTS: We show that the contribution of this pathway to plasma TG varies considerably in the normal population, as measured by the time needed to form the first traces of thrombin (TG lag time; mean prolongation by TIX-5 40%, range 0%-116%). Comparing patients on vitamin K antagonists (VKA) of the BLEED study (263 patients with and 538 patients without major bleeding), showed a marked prolongation in the median TG lag time in the presence of TIX-5 in cases (12.83 versus 11.00 minutes, P = 0.0030), while the TG lag time without TIX-5 only showed a minor although significant difference (5.83 vs. 5.67 minutes, P = 0.0198). The TIX-5 sensitivity (lag time + TIX-5/lag time + vehicle) in the upper quartile was associated with a 1.62-fold (95% confidence interval 1.04-2.52) increased risk of major bleeding compared to the lowest quartile. CONCLUSION: A greater dependence on FXa-mediated activation of FV of TG is associated with increased risk of major bleeding during VKA therapy.

Stability of vitamin K antagonist anticoagulation after COVID-19 diagnosis.

BACKGROUND: Coagulopathy has been reported in severely ill patients with coronavirus disease 2019 (COVID-19). It is unclear whether outpatients with COVID-19 who are treated with vitamin K antagonists (VKAs) have unstable anticoagulation. OBJECTIVE: To assess the stability of VKA therapy in patients with COVID-19 through a case-crossover study. METHODS: Between February and July 2020, we included patients who tested positive for COVID-19 from two anticoagulant clinics in the Netherlands. We collected international normalized ratios (INRs) determined between 26 weeks before infection and 12 weeks after. Time in therapeutic range (TTR) and the variance growth rate (VGR) were calculated within patients. RESULTS: Fifty-one patients with COVID-19 (mean age, 84 years) were included, of whom 15 (29%) were men. Mean TTR in the 26 weeks before COVID-19 was 80% (95% confidence interval [CI], 75-85) compared to 59% (95% CI, 51-68) in the 6 weeks after infection. Mean TTR difference was -23% (95% CI, -32 to -14) with a time above therapeutic range of 38% (95% CI, 30-47) in the 6 weeks after infection. The TTR rose again to 79% (95% CI, 69-89) between 6 and 12 weeks after infection. Also, VGR increased, with a mean increase of 4.8 (95% CI, 2.1-7.5) in the 6 weeks after infection. In the 26 weeks before infection, we registered 19 of 641 (3%) of INR ≥5.0 compared with 35 of 247 (14%) in the 6 weeks after (risk ratio, 4.4; 95% CI, 2.7-7.3). CONCLUSIONS: COVID-19 is associated with a strong decrease in TTR and in therapeutic stability in patients taking VKAs. Additional monitoring in these patients is advised to maximize therapeutic stability.

Adherence to direct oral anticoagulant treatment for atrial fibrillation in the Netherlands: A surveillance study.

BACKGROUND: Adherence to direct oral anticoagulants (DOACs) in patients with atrial fibrillation in every day practice may be less than in clinical trials. AIMS: To assess adherence to DOACs in atrial fibrillation patients in every day practice and identify predictors for non-adherence. METHODS: Individual linked dispensing data of atrial fibrillation patients who used DOACs were obtained from the Foundation for Pharmaceutical Statistics covering the Netherlands between 2012 and 2016. One year adherence to DOAC was calculated for initial DOAC as proportion of days covered (PDC) ≥80% and the association between clinical variables and adherence was assessed using logistic regression. In addition, we measured non-persistence, that is, patients who completely stopped their initial DOAC within 1 year follow-up. RESULTS: A total of 4797 apixaban-, 20 454 rivaroxaban- and 18 477 dabigatran users were included. The mean age was 69 years (n = 43 910), which was similar for the DOAC types. The overall proportion of patients with PDC ≥80% was 76%, which was highest for apixaban- (87%), followed by dabigatran- (80%) and rivaroxaban (69%) users. Multivariable analyses revealed that age ≤60 years, no concomitant drug use were predictors for non-adherence. Of atrial fibrillation patients who continued treatment, 97% had a PDC ≥80%, compared with only 56% for those who discontinued their DOAC treatment within 1 year. CONCLUSIONS: Non-adherence to DOACs was associated with age ≤60 years and no concomitant drugs use. Non-adherence was higher in patients who later discontinued DOAC treatment. Results of our study support research into interventions to improve adherence.

High Soluble Thrombomodulin Is Associated with an Increased Risk of Major Bleeding during Treatment with Oral Anticoagulants: A Case-Cohort Study.

BACKGROUND: Major bleeding occurs in 1 to 3% of patients treated with oral anticoagulants per year. Biomarkers may help to identify high-risk patients. A proposed marker for major bleeding while using anticoagulants is soluble thrombomodulin (sTM). METHODS: Plasma was available from 16,570 patients of the BLEEDS cohort that consisted of patients who started treatment with vitamin K antagonists between 2012 and 2014. A case-cohort study was performed including all patients with a major bleed (n = 326) during follow-up and a random sample of individuals selected at baseline (n = 652). Plasma sTM levels were measured and stratified by percentiles. Patients were also categorized by international normalized ratio (INR). Adjusted hazard ratios (for age, sex, hypertension, and diabetes) with 95% confidence intervals (CIs) were estimated by means of Cox regression. RESULTS: Plasma sTM levels were available for 263 patients with a major bleed and 538 control subjects. sTM levels were dose-dependently associated with risk of major bleeding, with a 1.9-fold increased risk (95% CI: 1.1-3.1) for levels above the 85th percentile versus the <25th percentile. A high INR (≥4) in the presence of high (≥70th percentile) sTM levels was associated with a 7.1-fold (95% CI: 4.1-12.3) increased risk of major bleeding, corresponding with a bleeding rate of 14.1 per 100 patient-years. CONCLUSION: High sTM levels at the start of treatment are associated with major bleeding during vitamin K antagonist treatment, particularly in the presence of a high INR.