Demonstration in vitro of re-occlusion, and its prevention by fusion constructs

An in vitro re-occlusion model was developed on the basis of available knowledge [42], and tested with the fusion constructs to check the prevention, if any, of clot-bound thrombin-mediated re-occlusion occurring in the controls (into which no fusion construct was added). In this model, three distinct phases were discerned, namely: a) clot formation phase; b) clot lysis phase and c) re-occlusion phase. In brief, 100 μl clots were prepared in flat-bottom microtiter plates by adding plasminogen-free, purified human fibrinogen (final concentration 4 mg/ ml), 20 mM CaCl_2, HBS buffer (0.01 M HEPES, 0.13 M NaCl, pH 7.4) and 3 IUof thrombin (3μl), followed by incubation at 37°C for 2 h, during which absorbance changes at 405 nm were periodically recorded to measure clot formation. The clots were then washed 3 times with 100 ul of HBS buffer to remove excess unbound thrombin. Following this, 1 μM human plasminogen, HBS and either SK, EGF-SK or SK-EGF fusion constructs were added (25 nM each), and final volume made up to 100 μl on the fibrinogen clot mesh (100 μl fibrin clot + 100 μl buffer containing plasminogen, buffer and thrombolytic agent). Clot lysis was monitored by recording change (seen as a decrease) in absorbance at 405 nm. At times corresponding to ~ 40% clot lysis, clots were washed 3 times with 100 μl of HBS buffer in order to remove free plasmin. After that, for the initiation of re-occlusion phase, 100 μl of HBS buffer that contained fibrinogen (final concentration 4 mg/ml) and 0.1μM human α-2 anti-plasmin (to inhibit any traces of plasmin, and to mimic an in vivo situation) were added, and change (seen as an increase) in absorbance at 405 nm was monitored with time. The reformation of clots after the initial lysis by the thrombolytic/s was considered as the re-occlusion phase. All assays were carried out in triplicates. In this assay, after clot formation, in control experiments, HBS with plasminogen was added without any thrombolytic, for a time period for clot lysis taken by other fusion constructs. Clots were then washed three times with 100 μl of HBS buffer. One set of clots was incubated with 100 μl of HBS and α-2 antiplasmin (0.1 μM), and the other set incubated with 100 μl of fibrinogen (4 mg/ml) containing α-2 antiplasmin (0.1μM) in HBS. The change in absorbance at 405 nm was then measured.