Peptide-Based Fibrin-Targeting Fluorescent Probes for Murine Stasis Venous Thrombus Resolution

Abstract

Background- The lack of a stasis venous thrombosis model (VT) in female mice has limited investigation of sex differences of anti-VT therapies, such as acetylsalicylic acid (ASA), which appears efficacious in preventing recurrent VT. Here we (i) develop a new extended stasis VT model; and (ii) examine the effects of ASA on stasis VT resolution in both female and male mice using serial intravital microscopy (IVM). Method- To extend stasis thrombus persistence beyond a single ligation (SL) model, the femoral vein was ligated, followed by a second ligation of saphenous vein near popliteal vein (Fig. A). Following FITC-dextran injection (10 mg/kg), FITC light irradiation (475/35nm, 20x) for three minutes across three illumination fields generated stasis VT. Subsequently, Dylight-649-GPIbβ agent was injected (100ug/kg, ex 630nm) to observe platelets. At T30’, fibrin targeting agent-FTP11-CyAm7 (150nmol/kg, ex 750 nm) was injected. ASA pre-treatment was done for 7 days (3mg/kg, n=5-6). Serial epifluorescence IVM images were acquired up to 24 hours (90i, NIS Elements software, Nikon). Thrombus area was quantified (NIH ImageJ). Results- DL thrombus was significantly larger and visualized through 24 hours (Fig B-G) and 48 hours (data not shown) versus SL thrombus (Fig. E). Corresponding sections of thrombus at T60’, T6h and T24h showed formation of a persistent thrombus core consisting platelets and fibrin (Fig. H-J). In the DL model, females developed reproducible stasis VT burden and resolution profiles as in males (Fig. K). ASA pre-treatment resulted in significant reduction in thrombus induction (T0’), and accelerated resolution in both sexes (Fig. L-P). ASA inhibited platelet recruitment to thrombi within T30’, consistent with its known anti-platelet effect via irreversible inhibition of Thromboxane A2. Furthermore, ASA inhibited fibrinogenesis, suggestive of an impaired platelet-fibrin interface. Conclusion- Here we establish a new extended stasis VT model applicable to both female and male mice that develops thrombus for up to 48 hours. Moreover, this model is amenable to study vein wall fibrosis-key aspect of post-thrombotic syndrome. Furthermore, ASA inhibits thrombus formation and accelerates thrombus resolution in a sex-independent manner, supporting its use in prevention of recurrent VT. AHA 953662; NIH 1R21AR081535-01; NIH R01GM126062 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.