Effect of Clot Stiffness on Recombinant Tissue Plasminogen Activator Lytic Susceptibility in Vitro

Abstract

The lytic recombinant tissue plasminogen activator (rt-PA) is the only drug approved by the Food and Drug Administration for treating ischemic stroke. Less than 40% of patients with large vessel occlusions who are treated with rt-PA have improved blood flow. However, up to 6% of all patients receiving rt-PA develop intracerebral hemorrhage. Predicting the efficacy of rt-PA treatment a priori could help guide therapeutic decision-making, such that rt-PA is administered only to those individuals who would benefit from this treatment. Clot composition and structure affect the lytic efficacy of rt-PA and have an impact on elasticity. However, the relationship between clot elasticity and rt-PA lytic susceptibility has not been adequately investigated. The goal of this study was to quantify the relationship between clot elasticity and rt-PA susceptibility in vitro. Human and porcine highly retracted and mildly retracted clots were fabricated in glass pipettes. The rt-PA lytic susceptibility was evaluated in vitro using the percent clot mass loss. The Young’s moduli of the clots were estimated using ultrasound-based single-track-location shear wave elasticity imaging. The percent mass loss in mildly retracted porcine and human clots (28.9 ± 6.1% and 45.2 ± 7.1%, respectively) were significantly higher (p < 0.05) than those in highly retracted porcine and human clots (10.9 ± 2.1% and 25.5 ± 10.0%, respectively). Furthermore, the Young’s moduli of highly retracted porcine and human clots (5.33 ± 0.92 kPa and 3.21 ± 1.97 kPa, respectively) were significantly higher (p < 0.05) than those of mildly retracted porcine and human clots (2.66 ± 0.55 kPa and 0.79 ± 0.21 kPa, respectively). The results revealed an inverse relationship between the percent clot mass loss and Young’s modulus. These findings motivate continued investigation of ultrasound-based methods to assess clot stiffness in order to predict rt-PA thrombolytic efficacy.

In vitro characterization of sonothrombolysis and echocontrast agents to treat ischemic stroke

“…Retraction produces a tighter fibrin network clot permeability and the amount of soluble plasminogen, thereby increasing resistance to rt-PA 90,91 . Moreover, we characterized the fibrin content, porosity, and red blood cell content of the clots prepared using this protocol and showed that these values lie within the ranges reported for clots retrieved from stroke patients 82 .…”

“…Although, in general, acute venous clots are easier to lyse than arterial clots, the coagulation cascade can be modulated to obtain retracted, rt-PA-resistant clots using venous blood. We have demonstrated the use of borosilicate glass to form clots that are more resistant to rt-PA thrombolysis than those prepared using soda lime or flint glass 82,83 . Under aqueous conditions, borosilicate glass acquires a negative surface charge 84 .…”

In vitroassessment of stiffness-dependent histotripsy bubble cloud activity in gel phantoms and blood clots

“…The threshold acoustic emissions or grayscale for phantoms were assessed via receiver operating characteristic (ROC) curve analysis. Bubble activity was also assessed in clots formed in vitro with varying degrees of retraction, and therefore varying clot stiffnesses (Mercado-Shekhar et al 2018). The degree of clot liquefaction was assessed histologically, and the power of bubble cloud emissions generated by the histotripsy pulse was recorded.…”

Accelerated sonothrombolysis with Definity in a xenographic porcine cerebral thromboembolism model

“…However, the effects of human whole blood clots in the porcine vasculature over longer periods (greater than 8 h) are unclear. Additionally, the highly retracted, fibrin-dominant exterior of the clots may limit the presentation of foreign antigens in the porcine blood, reducing the incidence of immunologic reactions 31 , 47 , 48 . The methylprednisolone and carprofen, given to prevent porcine hypersensitivity to Definity, may also have suppressed the immune response to the human whole blood clots.…”

“…Additionally these clots are older and larger than the 2–6 h old autologous venous clots used by Gao et al or Culp et al in a similar porcine APA model, although important to note that these studies were not investigating the effects of rt-PA mediated thrombolysis, only ultrasound and microbubbles 24 , 28 , 29 . These fresh autologous clots are softer and more prone to lysis even in the absence of rt-PA 48 . Brown et al and Florres et al used human clots in a rabbit model, which were much smaller (1.0 mm sections in both), but assessed infarct volume rather than recanalization 23 , 52 , 53 .…”