Science Platforms

Translational / Basic Science Research

Knowledge gaps exist on causal mechanisms for venous thromboembolism (VTE) and, in particular, chronic complications of VTE. The translational/basic science platform is primarily designed to provide CanVECTOR’s basic scientists with translational research opportunities by providing samples that permit exploration of “bench to bedside and back” questions. By piggy-backing translational research questions and biomarker sub-studies onto our current and future clinical studies, the Network will provide a unique, efficient and cost-effective opportunity to test novel hypotheses on the pathogenesis of VTE and may thereby identify new targets for preventing VTE or its complications.

The Team

Platform Co-Leads

Jeffrey Weitz (Hamilton, ON)
Edward Conway (Vancouver, BC)

Working Group

Peter Gross (Hamilton, ON)
Patricia Liaw (Hamilton, ON)

The Projects

1. Explore the roles of the contact and complement systems in the pathogenesis of venous thromboembolism and complications

Study Aims: To test the hypothesis that lowering factor XI levels reduces thrombin generation and complement activation.

Population: Pre- and post-operative plasma samples from knee arthroplasty patients who were randomized to the factor XI ASO or enoxaparin.

Study Timeline: 2016-2017 (study being conducted at McMaster, Weitz lab)

2. Examine the role of inflammation in the post-thrombotic syndrome

Study Aims: To examine the hypothesis that chronic inflammation contributes to the post-thrombotic syndrome (PTS) and determine whether there is more complement activation and histidine-rich glycoprotein (HRG) levels are lower in patients with versus without PTS.

Population: Samples from patients with a first, symptomatic, proximal DVT who participated in the SOX trial.

Study Timeline: 2016-2017 (study being conducted at UBC Centre for Blood Research, Conway lab)

3. Studies of cell-free DNA, thrombin generation, and fibrinolysis in patients with unprovoked VTE

Principal Investigator: Patricia Liaw, PhD

Study Summary

Venous thromboembolism (VTE) is the third leading cause of cardiovascular mortality after myocardial infarction and stroke.  In recent years, neutrophils have been shown to be indispensable for the initiation and propagation of VTE.  Neutrophils contribute to VTE through the delivery of neutrophil extracellular traps (NETs), consisting of cell-free DNA (cfDNA), histones, and neutrophil granular enzymes.  We have previously shown that cfDNA contributes to the prothrombotic potential of NETs by triggering the intrinsic pathway of blood coagulation and by inhibiting plasmin-mediated fibrin degradation.  This study explores the hypothesis that cfDNA is not only a predictor of recurrent VTE in patients with unprovoked VTE, but also contributes to the pathogenesis of VTE by promoting blood coagulation and by inhibiting fibrinolysis.  This is a translational study in collaboration with Dr. Clive Kearon which will utilize plasma samples from the DODS study (D-dimer Optimal Duration Study). 

4. FXIII and EACA increase thrombus stability and decrease pulmonary embolism in an in vivo mouse model of Venous Thromboembolism, but EACA increases thrombus size.

Authors: Shana A. Shaya, Jeff I. Weitz, Peter L. Gross

Affiliation: Thrombosis and Atherosclerosis Research Institute, Department of Medicine, McMaster University.

Background: Whether a patient presents with deep vein thrombosis (DVT) or pulmonary embolism (PE) varies based on clinical factors. The biological mechanisms that determine DVT stability in the progression of DVT to PE are not known.

Anticoagulation is contraindicated in patients presenting with DVT and severe bleeding. Inferior vena cava filters are used instead but associated with high morbidity. Are there alternative treatments that can stabilize clots, minimize embolization and PE burden, without causing further bleeding? We have previously described a mouse model of venous thromboembolism (VTE) that can assess the thrombus size, quantify embolic events that break off the thrombus, and correlate this to the resulting PE burden, and shown the requirement of thrombin-mediated factor XIII (FXIII) activation in this model. 

Aim: Determine the effects of epsilon aminocaproic acid (EACA) and FXIII on thrombus stability and PE burden.

Methods: Platelets were fluorescently labelled using CD41 Fab fragments conjugated to an Alexa Fluor 488. The femoral vein of C57BL/6 female mice was subjected to ferric chloride injury to initiate thrombus formation. Treatment with saline, dalteparin (0.2 IU/g, dabigatran (33 mg/g), EACA (1 mg/kg), or FXIII (1mg/kg) was administered 12 minutes after thrombus formation. Intravital videomicroscopy recorded the thrombus sizes (TSs) and embolic events (EEs) leaving the thrombus for 2 hours. Lungs were harvested, sectioned and stained for the presence of PE.

Results: EACA increases TS significantly and therefore would not be a feasible treatment as it will increase DVT size. FXIII marginally increased TS. Treatment with FXIII decreases total and large EEs in saline, dalteparin or dabigatran-treated mice, similar to EACA-treated mice. The number of emboli per lung slice was reduced after treatment with FXIII and EACA compared to non-treated mice. PE burden was not significantly different between FXIII anticoagulated mice or EACA-treated mice. The large EEs correlate positively with PE burden.

Conclusion: Attenuating fibrinolysis with EACA, but not FXIII, increases TS, but both increase DVT stability and decrease PE burden. This suggests that administration of FXIII is a better treatment option for DVT patients who are bleeding than EACA, since EACA may increase DVT size.

Acknowledgment:  We thank Canvector for partial funding of this study

5. An assay to measure anti-FXa direct oral anticoagulants in VTE patients

Venous thromboembolism (VTE) is a potentially fatal condition caused by blood clot formation in the large deep vein of the leg (deep vein thrombosis, DVT). One debilitating complication is when a piece of the clot breaks off and become lodged in the pulmonary circulation, leading to pulmonary embolism (PE). New class of small molecules referred to as direct oral anticoagulants (DOAC) that target the two central enzyme of clot formation, thrombin and factor Xa (FXa) have recently been developed to replace warfarin as a standard long-term anticoagulant for the prevention and treatment of VTE. Thus, there are two classes of DOACs: 1) thrombin-specific DOAC (e.g. dabigatran), and 2) FXa-specific DOACs (e.g. apixaban, rivaroxaban, edoxaban). These compounds are of great interest because unlike warfarin, DOACs do not require on-going monitoring of anticoagulation due to their predictable pharmacokinetics.

There still remain instances where DOAC measurements are needed such as assessing their potential contribution to bleeding and timing urgent surgery or interventions. Moreover, DOAC plasma levels may be needed in instances such as major trauma, thrombolysis in acute thromboembolism, hemorrhage, drug interactions, accidental or intended overdose, and diminishing/changing renal function. However, there is a lack of methods for quantifying low levels of DOACs with accuracy, especially for the anti-FXa DOACs. While there are currently two considerable methods to quantify levels of anti-FXa DOAC levels in the blood samples of patients, these methods are not without major limitations. HPLC can only be conducted in specialized research facilities requiring high-level of expertise, thus rendering it impractical for routine clinical use. The chromogenic assay lacks the sensitivity to detect DOAC levels less than 20 ng/mL (~45 nM), which is problematic when trying to assess residual DOAC levels upon cessation of anticoagulation in preparation for surgery. We have recently developed a novel assay that is capable of measuring anti-FXa DOAC levels in the plasma or whole blood at levels below 20 ng/mL accurately. Our objective is to measure anti-FXa DOACs in the blood samples of VTE patients. We will do so in collaboration with Drs. James Douketis and Peter Gross, as a part of the PAUSE study (Thromb Haemost. (2017) 117(12):2415).

Aim 1: Quantifying DOAC levels in the blood samples from VTE patients: Plasma is isolated from patients with VTE or are at risk for VTE recurrence, that were assessed for DOAC levels using the chromogenic assay. We will verify these findings using our novel assay. We will compare the quantified DOAC values between the two assays and subsequently correlate these values with clinical outcome (e.g. bleeding events).

Aim 2: Development of alternative FXa substrates: To enhance the sensitivity of this assay in the whole blood, we will modify our current substrate by using a different fluorescent probe that conflicts less with the red blood cells. While the current method to circumvent quenching is to dilute out the whole blood (1:50), which is sufficient to monitor on-going anticoagulation, but are not ideal when measuring low levels of DOACs upon anticoagulation cessation. Therefore, we aim to improve measuring low DOAC levels in the whole blood. Impact: Thus, the use of our assay can offer individualized assessment of anticoagulation in determining whether the patient is suitable for emergency surgical intervention. Furthermore, development of our assay as a standard point-of-care would streamline the monitoring process thus improving efficiency, cost, and care in the treatment of patients with VTE who are taking anti-FXa DOACs.