Professor The College of New Jersey, United States
Introduction:: Procoagulant microparticles are prevalent in many disease states such as hypertension, heart disease, diabetes and cancer. Patients with these conditions are also at greater risk for thrombosis.1 Monocytic derived microparticles carry tissue factor (TF) and are highly procoagulant. Previous studies in animals demonstrated that leukocyte-derived microparticles deposit in growing thrombi and bind to activated platelets.2 However, the transport mechanism and the effect of local flow conditions have not been studied. This process can be observed in real time under various flow conditions using a parallel plate flow chamber that is compatible with confocal microscopy. Thus, the objective of this study was to characterize TFMP deposition in platelet thrombi as a function of flow conditions in order to elucidate the transport mechanisms leading to incorporation of TFMPs in growing thrombi.
Materials and Methods:: Whole blood, anti-coagulated with 10 uM PPACK, was collected from donors (n = 4) via venipuncture (IRB-2021-0415). Platelets were labeled with mepacrine (10 uM) and TFMPS were labeled with eFluor670™. TFMPs, derived from a monocytic cell line (THP-1, ATCC.org) with a standardized level of TF activity were added to blood (25:1000) immediately prior to perfusion over a Type I collagen coated coverslip. Prepared blood was perfused through a vacuum sealed parallel plate flow chamber at various wall shear rates using a withdrawing syringe pump (100 s-1, 200 s-1, 300 s-1, 400 s-1) (Figure 1). Three-dimensional image stacks were captured at 3, 6, and 9 minutes using a Leica confocal microscope and LASX software.
The images were analyzed to obtain the total volume of TFMPs throughout the stack. The TFMP (red channel) images in each 3D stack were converted to binary images. Particles were identified and particle areas were calculated using ImageJ software (Figure 2). TFMP volumes were determined by multiplying TFMP area by slice thickness of the stack (0.3 µm). The effects of shear rate and time on total microparticle volume was evaluated using a two-way repeated measures ANOVA with a post-hoc Bonferroni test. The effect of z-position in the stack and shear rate on microparticle volume was evaluated via a two-way repeated measures ANOVA with a post-hoc Bonferroni test. All statistical tests were performed with α=0.05.
Results, Conclusions, and Discussions:: The results indicate that TFMP deposition increases with increasing wall shear rate and over the first several minutes of platelet deposition (Figure 3). A statistically significant difference in microparticle deposition was found between the 100 s1 and 300 s-1 shear rates (Figure 3). In addition, the study clearly indicated that the lower portion of the image stack (0-25% of total height) had significantly more TFMPs by volume than the rest of the stack (Figure 4). This suggests that MPs deposit more readily in the early stages of platelet deposition. Due to their size, on the order of 200 nm, their transport to the vessel wall is not enhanced by the presence of red blood cells, as occurs with platelets. Thus, at lower wall shear rates, the flux of TFMPs to the vessel wall is likely low. It was expected that the MPs would more likely deposit by impacting the platelet thrombus once it protruded into the flow field. This was the primary mechanism of deposition in a previously published model of microparticles in disturbed flow regimes.3 These findings therefore bring up further questions regarding platelet-microparticle interactions.
Acknowledgements (Optional): : Thank you to our phlebotomist, Carly Baker, and our donors for making these experiments possible.
References (Optional): :
K., Dubois, C., Schäfer, K. Extracellular Vesicles and Thrombosis: Update on the Clinical and Experimental Evidence. International Journal of Molecular Sciences. 22: 9317, 2021.
Falati, S., et al. Accumulation of tissue factor into developing thrombi in vivo is dependent upon microparticle P-selectin glycoprotein ligand 1 and platelet P-selectin. The Journal of experimental medicine, 197(11): 1585-1598, 2003
Hall, C.L., Calt, M. Computational modeling of Thrombotic microparticle deposition in nonparallel flow regimes. Journal of Biomechanical Engineering, 136: 10 pages, 2014.
