A new way to treat proximal deep venous thrombosis using E-selectin inhibition

Abstract

Objective: There is an inter-relationship between thrombosis and inflammation. Previously, we have shown the importance of P-selectin in thrombogenesis and thrombus resolution in many preclinical animal models. The role of E-selectin has been explored in rodent models and in a small pilot study of clinical calf vein deep venous thrombosis. The purpose of this study was to determine the role of E-selectin in thrombosis in a primate model of proximal iliac vein thrombosis, a model close to the human condition.

Methods: Iliac vein thrombosis was induced with a well-characterized primate model. Through a transplant incision, the hypogastric vein and iliac vein branches were ligated. Thrombus was induced by balloon occlusion of the proximal and distal iliac vein for 6 hours. The balloons were then deflated, and the primates recovered. Starting on postocclusion day 2, animals were treated with the E-selectin inhibitor GMI-1271, 25 mg/kg subcutaneously, once daily until day 21 (n = 4). Nontreated control animals received no treatment (n = 5). All animals were evaluated by magnetic resonance venography (MRV); evaluation of vessel area by ultrasound, protein analysis, hematology (complete blood count), and coagulation tests (bleeding time, prothrombin time, activated partial thromboplastin time, fibrinogen, and thromboelastography) were performed at baseline, day 2, day 7, day 14, and day 21 with euthanasia. In addition, platelet function and CD44 expression on leukocytes were determined.

Results: E-selectin inhibition by GMI-1271 significantly increased vein recanalization by MRV vs control animals on day 14 (P < .05) and day 21 (P < .0001). GMI-1271 significantly decreased vein wall inflammation by MRV with gadolinium vein wall enhancement vs control also on day 14 (P < .0001) and day 21 (P < .0001). The thromboelastographic measure of clot strength (maximum amplitude) showed significant decreases in animals treated with GMI-1271 vs controls at day 2 (P < .05) and day 7 (P < .05). Animals treated with GMI-1271 had significant vessel area increase by day 21 vs controls (P < .05) by ultrasound. Vein wall intimal thickening (P < .001) and intimal fibrosis (P < .05) scores were significantly decreased in GMI-1271-treated animals vs controls. Importantly, no significant differences in hematology or coagulation test results were noted between all groups, suggesting that E-selectin inhibition carries no bleeding potential. GMI-1271 did not affect platelet function or aggregation or CD44 expression on leukocytes. In addition, no episodes of bleeding were noted in either group.

Conclusions: This study suggests that E-selectin modulates venous thrombus progression and that its inhibition will increase thrombus recanalization and decrease vein wall inflammation, without affecting coagulation. The use of an E-selectin inhibitor such as GMI-1271 could potentially change how we treat deep venous thrombosis.

Keywords: Animal models; Coagulation; E-selectin inhibition; Glycomimetic; Inflammation; Nonhuman primate; Venous thrombosis.

Figure 1:. Baboon model of iliac vein venous thrombosis and experimental design

A. This model used a 6 hour temporary balloon occlusion in juvenile baboons. On surgical day 0, standard contrast venography and ultrasound was performed in order to acquire baseline information regarding the vein to be thrombosed. A thrombus is created in the iliac vein by threading one balloon catheter via the internal jugular vein caudally down the inferior vena cava by fluoroscopy, to just below the iliac bifurcation. Another catheter was moved cranially up the femoral vein near the pelvic crest. This isolated a vein segment of approximately 3.0 cm in length. During the 6 hours of venous occlusion, the area between balloons were monitored hourly for absence of flow and increasing echogenicity representative of thrombus formation, using ultrasonography. After 6 hours, the balloon catheters were removed, incisions closed, an ultrasound performed to confirm the presence of a thrombus within the iliac vein and the animal monitored for post-operative recovery. B. Animals were evaluated on Days 2, 7, 14, and 21; hematology and coagulation tests, venous duplex ultrasound imaging, magnetic resonance venography (MRV), and standard contrast venography. C. On Day 21, the animals were humanely euthanized and vein samples harvested from the thrombosed iliac (experimental samples) and from the non-thrombosed iliac (control samples) for histology.

Figure 2:. Magnetic resonance venography (MVR).

A) Figure 2A demonstrates vein recanalization by magnetic resonance venography, time offlight imaging, and percent vein recanalization analysis. B) Figure 2B demonstrates vein wall inflammation as determined by capillary leakage in the vein wall using gadolinium (Gd) contrast.

Figure 3:

Thromboelastography measure of clot strength (MA).

Figure 4:

Ultrasound Analysis-Open Lumen on day 21.

Figure 5:

Scoring of the right iliac vein wall intimal thickness (IT) and intimal fibrosis (IF) on Day 21 post venous thrombosis.

Figure 6:. Clinical scenarios that can affect iliac valve reflux times in this study.

All animals were evaluated for thrombus burden and valve reflux times via duplex ultrasound imaging. The study area of the right iliac vein consisted of a proximal (Prox.), middle (Mid.) and distal (Dist.) segments for each animal which all together made up an iliac vein segment approximately 25 mm in length. Panel A. Control animal number 5 had a large thrombus burden in the proximal iliac segment that did not incorporate the valve yielding a viable reflux time with a small flow channel present. These animals had decrease lumen patency and increased vein wall inflammation. Panel B. Control animals 3 and 4 had non-occlusive middle iliac segment thrombus that incorporated the iliac valve through day 14 post thrombosis. However, by day 21, these animals had chronic anterior and posterior thrombus present with competent valves via duplex ultrasound. Treated animals 3 and 4 had non-occlusive middle iliac thrombus that incorporated the iliac valve through day 7 and 14 post thrombosis respectively. However, by day 21, these animals had complete resolution of thrombus burden but non-competent valves via duplex ultrasound. Panel C. Control animals 1 and 2; Treated animals 1 and 2 had venous thrombi in the distal iliac segment that did not incorporate the iliac valve. The treated animals however had lower thrombus burden than the controls animals leading to increased lumen patency and decreased vein wall inflammation. Note: Secondary venous valves above and/or below interrogated segment and/or technical error should be considered.