Abstract 3284: Influence of Blood Clot Age, Density and Thrombin Content on In Vitro Sonothrombolysis Rate and Efficacy
Sonothrombolysis (STBL) uses ultrasound to lyse thrombi that cause stroke, myocardial infarct and other vascular occlusion diseases; and efficacy is improved markedly by combining tPA and/or microbubbles (MBs) with ultrasound. The efficacy of in vitro STBL varies considerably depending upon how blood clots are formed. However, the relationship between the physical and chemical nature of blood clots and STBL efficacy has not been quantified. This study measured the effects that clot age, density and thrombin content had on STBL efficacy and the ability of tPA and MBs to complement STBL. Blood clots with different characteristics were produced by mixing pooled rabbit plasma with rabbit blood cells, adding varied amounts of thrombin and calcium, followed by incubation at 37oC in glass tubes for 3 h to 72 h. Blood clots were then cured at 5oC for 24 h to 72 h. The volume and mass density of the clots were measured, 9-11 mg pieces were cut from the main clot, weighed and then insonated with 1 MHz, pulsed (20% duty factor) ultrasound in a Mylar chamber through which fresh rabbit serum containing tPA and or MBs was flowed continuously past the clot. STBL efficacy was measured as % of clot mass lysed in 15 min. For clots incubated 3 h to 24 h at 37oC with 5 mM Ca+2 and 5 U of thrombin, maximal STBL with ultrasound plus tPA was observed with 0.1 mg/ml tPA for all ultrasonic intensities tested. Using MBs (1.0-2.5 x108/ml) produced a comparable level of STBL. More effective STBL was produced by combining tPA with MBs, even with reduced tPA concentrations (0.006-0.01 mg/ml). However, increasing clot thrombin, up to 60 U, proportionally reduced STBL efficacy with tPA or MBs, and combining tPA with MBs no longer produced an additive STBL effect. This was also true for clots incubated with 25 mM Ca+2 that did not contract and thus had a fourfold lower mass density. Increasing thrombin levels produced clots whose STBL efficacy was markedly lower with either tPA or MBs, and for which combining tPA with MBs produced no additive STBL effect. Hence, the level of thrombin used during clot formation (and putatively the degree of fibrin networks) was the major factor in determined STBL efficacy and whether or not tPA combined with MBs were additive in potentiating STBL. Surprisingly, the mass density of the clot alone had no effect on STBL efficacy.
- © 2012 by American Heart Association, Inc.