PB0644 - Effect of Novel Thrombolytic Aptamer DTRI-031 on Microfluidic Thrombolysis and VWF Function Dose-Response

Background: The interaction between von Willebrand Factor (VWF) A1 domain and platelet GPIbα is essential for arterial thrombosis. Aptamer DTRI-031 selectively inhibits VWF A1 and has potent thrombolytic properties. The ideal clinical assay to detect DTRI-031 functional efficacy and the corresponding therapeutic range are unknown. We measured dose responsiveness of DTRI-031 in a custom microfluidic model of thrombolysis and currently available clinical assays.

Aims: The aim of this study is to determine the capacity of clinical assays to reflect microfluidic thrombolysis relative to measurable inhibition induced by DTRI-031.

Methods: Healthy human whole blood (WB, N=10) was dosed with vehicle, 423nM, 846nM, 1692nM, or 3384nM DTRI-031 to approximate an in vivo dose range of 0.25-2mg/kg. VWF function was assayed via VWF:Ag, VWF:Ac (Siemens GP1bα-interaction specific assay, unavailable in US), T-TAS, VWF:RCo, ristocetin impedance aggregometry (RIA). WB was also stained with fluorescent CD41 antibody (platelet label) and perfused through a stenotic microfluidic chamber to induce partial thrombotic occlusion (25mmHg pressure increase), followed by DTRI-031 perfusion. Maximum reduction in thrombus surface was calculated using auto-thresholding (Python v3.10) of CD41-positive surface area yielding microfluidic lytic efficacy (MLE). One-way ANOVA and multiple comparisons were used to assess dose response. Pearson correlation coefficients were calculated between assays.

Results: VWF assays were inhibited by DTRI-031 (Figure 1) while VWF:Ag was, as expected, constant. MLE trended higher at the midrange dose. Correlation with dose was highest in VWF:Ac, VWF:RCo, and RIA, while MLE correlated best with T-TAS and VWF:Ag (Figure 2).

Conclusion(s): Inhibition of GPIbα by DTRI-031 is dose-dependent and measurable on currently available clinical assays. In a thrombolysis microfluidic model, 3384nM DTRI-031 was less efficacious than 1692nM DTRI-031, perhaps indicating off-target effects at the highest dose. MLE correlated best with T-TAS, a function-under-flow assay. Further investigation of DTRI-031 mechanisms will provide insight into discrepancies between static assays and flow-based assays.