Title:
Enhancement of t-PA Induced Fibrinolysis with Laser Energy: In-Vitro Observations.

Abstract:
The solid-state, pulsed-wave, holmium:YAG laser operates within strong water absorption peaks at the mid-infrared optical wavelength. This laser has been shown to be capable of inducing a mechanical, photoacoustic dissolution of fibrin, a major constituent of thrombi. It is not known whether this laser's energy combined with pharmacologic therapy can enhance the rate of fibrinolysis. The aims of this study were (1) to test the hypothesis that mid-infrared laser emission can enhance tissue-type plasminogen-activator (t-PA) mediated fibrinolysis and (2) to test the combined effect of these two methods of fibrinolysis on fibrin clots varying in age.Three in vitro experimental protocols were used. (1) Fibrin clots were treated with 116 000 IU t-PA for 1, 6 and 12 h, respectively, and then exposed to mid-infrared laser energy (solid-state, pulsed-wave, holmium:YAG, 2.1 μm wavelength 250 ms pulse length, 5 Hz repetition rate, 500 mJ/pulse (33 J/cm(2))). (2) Fibrin gels layered with t-PA were exposed to either 25, 50, 75 or 100 J laser energy. t-PA was then allowed to interact with the lased gels for an additional 4 h. (3) The effects of varying clot age (1, 4 or 8 h) on laser (75 J) augmentation of t-PA induced fibrinolysis were tested. Each experimental protocol had control gels and following each experimental manoeuvre, 20 μl of the plasmin inhibitor ε-amino-n caproic acid was added and fibrin degradation products (FDPs), an indicator of fibrinolysis, were measured by latex agglutination.In fibrin clots exposed to t-PA for 6 h, the addition of laser energy significantly increased FDPs released (t-PA alone 40±0 μg/ml, laser plus t-PA 160±0 μg/ml, p<0.001). For gels exposed to t-PA for 12 h, addition of laser energy resulted in complete dissolution of the clot (FDPs with t-PA alone 160±0 μg/ml vs. laser plus t-PA>300 μg/ml, p=0.001). The rise in FDPs was significantly greater with 75 J of laser energy compared to 25 J (160±0 μg/ml vs. 80±0 μg/ml, p=0.0001), however, energy levels greater than 75 J did not further increase the amount of FDPs indicating a plateau phenomenon in dose-response relationship. t-PA had a decreased fibrinolytic effect on 4 and 8 h-old clots (FDPs of 60±20 μg/ml and 30±10 μg/ml, respectively). Laser energy reversed this trend and enhanced fibrinolysis in both 4 and 8 h-old clots. In 4 h-old clots, laser plus t-PA resulted in FDP release of 160±0 μg/ml compared to 60±20 μg/ml for t-PA alone (p=0.007). In 8 h-old clots, FDP release with laser plus t-PA was 160±0 μg/ml compared to 30±10 μg/ml with t-PA alone (p=0.0004).It was concluded that in vitro application of mid-infrared laser energy significantly enhances fibrinolysis in fibrin clots initially treated by t-PA. The in vitro interaction between mid-infrared laser and t-PA is energy dependent, however, at energy levels exceeding 75 J there is a plateau phenomenon in dose-response relationship. This wavelength photoacoustic energy also augments the decreased response of ageing clots to t-PA.