Assessment of Novel Anti-thrombotic Fusion Proteins for Inhibition of Stenosis in a Porcine Model of Arteriovenous Graft

<div><p>Background</p><p>Hemodialysis arteriovenous synthetic grafts (AVG) provide high volumetric blood flow rates shortly after surgical placement. However, stenosis often develops at the vein-graft anastomosis contributing to thrombosis and early graft failure. Two novel fusion proteins, ANV-6L15 and TAP-ANV, inhibit the tissue factor/factor VIIa coagulation complex and the factor Xa/factor Va complex, respectively. Each inhibitor domain is fused to an annexin V domain that targets the inhibitor activity to sites of vascular injury to locally inhibit thrombosis. This study’s objective was to determine if these antithrombotic proteins are safe and effective in inhibiting AVG stenosis.</p><p>Methods</p><p>A bolus of either TAP-ANV or ANV-6L15 fusion protein was administered intravenously immediately prior to surgical placement of a synthetic graft between the external jugular vein and common carotid artery in a porcine model. At surgery, the vein and artery were irrigated with the anti-thrombotic fusion protein. Control animals received intravenous heparin. At 4 weeks, MRI was performed to evaluate graft patency, the pigs were then euthanized and grafts and attached vessels were explanted for histomorphometric assessment of neointimal hyperplasia at the vein-graft anastomosis. Blood was collected at surgery, immediately after surgery and at euthanasia for serum metabolic panels and coagulation chemistries.</p><p>Results</p><p>No acute thrombosis occurred in the control group or in either experimental group. No abnormal serum chemistries, activated clotting times or PT, PTT values were observed after treatment in experimental or control animals. However, at the vein-graft anastomosis, there was no difference between the control and experimental groups in cross-sectional lumen areas, as measured on MRI, and no difference in hyperplasia areas as determined by histomorphometry. These results suggest that local irrigation of TAP-ANV or ANV-6L15 intra-operatively was as effective in inhibiting acute graft thrombosis as intravenous administration of heparin, but failed to inhibit hyperplasia development and stenosis in AVG.</p></div

Hyperplasia-to-graft (H/G) ratio analysis example.

<p>The dashed line in the cartoon of the vein and graft on the left indicates the approximate region at the vein-graft anastomosis where the histological section, shown on the right, was collected. A line was drawn across the mouth of the graft on a digitized image of the histological section then the areas of the graft and the hyperplasia growing into the lumen of the graft were measured using NIH Image J and are shown on the histology image. The table shows the sum of the areas of the hyperplasia and the graft and then the H/G ratio results for that histological section.</p

Effect of anti-thrombotic fusion protein treatment on open lumen area determined by MR imaging.

<p>Control = heparin-treated. A value of 100 would indicate the graft lumen was completely open in the vein-graft anastomosis cross-section; a value of 0 would indicate the lumen of the graft was completely occluded. There was no significant difference in the open lumen area amongst treatment groups. The bars represent means ± SD.</p

Hyperplasia-to-graft (H/G) ratio analysis example.

<p>The dashed line in the cartoon of the vein and graft on the left indicates the approximate region at the vein-graft anastomosis where the histological section, shown on the right, was collected. A line was drawn across the mouth of the graft on a digitized image of the histological section then the areas of the graft and the hyperplasia growing into the lumen of the graft were measured using NIH Image J and are shown on the histology image. The table shows the sum of the areas of the hyperplasia and the graft and then the H/G ratio results for that histological section.</p

MRI analysis example.

<p>A multiplanar reconstruction of the 2D black-blood scan was performed on a pig with a graft placed between the common carotid artery and external jugular vein in the neck. The sagittal plane (a) and transverse plane (b) of the vein-graft anastomosis region were used to locate the cross-section with the smallest open lumen area at the vein-graft anastomosis (c). The graft wall and vessel lumens appear hypointense (black) in black-blood imaging. The spiral support around the graft is also hypointense and visible along the graft wall in (b). The face of the plane indicated by the dashed line in (a) and (b) is shown in (c). The lumen and graft cross-sectional areas were measured as shown in (d) using the region-of-interest pencil tool in Osirix.</p

Activated clotting times (ACT) of normal porcine whole blood after the addition of TAP-ANV or with no drug.

<p>*p<0.005 when compared with no drug. The bars represent means ± SD.</p

Effect of anti-thrombotic fusion protein treatment on hyperplasia-to-graft (H/G) ratios determined by histomorphometry.

<p>Control = heparin-treated. A value of 1.0 would indicate the graft was fully occluded with hyperplasia. A value of 0.0 would indicate no hyperplasia. There was no significant difference in the H/G ratio amongst the treatments groups. The bars represent means ± SD.</p

Serum chemistries.

<p>ALT = alanine transaminase; WBC = white blood cell count.</p><p>*Pre = plasma collected immediately before surgery and prior to drug administration; Post = plasma collected immediately after surgery. Values are listed as means ± SD;</p><p>†Comparison between plasma values collected before drug administration and immediately after surgery using paired t-test. Results were considered significant at p ≤ 0.05.</p><p>Serum chemistries.</p

Plasma coagulation test results.

<p>PT = prothrombin time; aPTT = activated partial thromboplastin time.</p><p>*Pre = plasma collected immediately before surgery and prior to drug administration;</p><p>Post = plasma collected immediately after surgery. Values are listed as means ± SD;</p><p>†Comparison between plasma values collected before drug administration and immediately after surgery using paired t-test. Results were considered significant at p ≤ 0.05.</p><p>Plasma coagulation test results.</p

Point-of-care activated clotting times (ACT) in whole blood from heparin-treated (control), ANV-6L15-treated and TAP-ANV-treated animals.

<p>Blood was collected before (pre) and immediately after (post) AVG creation surgery. There was no significant difference between paired pre-operative and post-operative values in any group. The bars represent means ± SD.</p