Dynamic analysis of platelet deposition to resolve platelet adhesion receptor activity in whole blood at arterial shear rate

Abstract

Platelet activation is traditionally quantified using turbidimetric aggregometry, which reflects integrin αIIbβ3 activity, an important determinant of platelet function during pathophysiological thrombus formation. However, aggregometry does not recreate the shear conditions prevailing during thrombosis in vivo. Here we describe novel whole-frame analysis of real-time video microscopy to quantify platelet adhesion receptor activity under shear in parallel-plate flow chambers. We demonstrate that the rate of change of surface coverage (designated Platelet Population Mobility, PM) quantifies platelet mobility. On collagen, PM falls exponentially to a low level, corresponding to firm platelet adhesion, while on other substrates, PM remains high. Different receptor-specific thrombogenic surfaces reveal that the PM time constant reflects real-time changes in integrins αIIbβ3 and α2β1 activity. This ensemble kinetic analysis has the potential to provide valuable diagnostic information about platelet thrombus formation with both academic and clinical applications.

Keywords: Activation; aggregation; platelets; shear.

Figure 1.

PM quantifies platelet adhesion under flow conditions. (A) Whole blood was perfused at 1000 s⁻¹ over type I collagen fibers (○) or VWF-III (•) and images were acquired at 0.2 Hz for 5 min. Image processing yielded PM measurements throughout the 5-min perfusion, which are plotted vs. time. PM curves were modeled as exponential decays, yielding the parameter of Plateau (B) (see Supplementary material for details). End-point image analysis yielded ZV₅₀ (C).

Figure 2.

PM quantifies integrin activation under flow conditions. Whole blood was pre-treated with carrier (A), 300 µM DM-BAPTA-AM (B) or 5 µM GR144053 (C), before perfusion at 1000 s⁻¹ over coverslips coated with combinations of CRP, VWF-III and one of a panel of integrin α₂β₁-adhesive peptides of varying affinities. PM was calculated as before. For clarity, only GFOGER, (○), GMOGER (•) and GPP₁₀ (•) are shown. Other data sets (GLOGER and GAOGER) are shown in Supplementary Figure S2. End-point measurements were calculated as described in the text, Plateau (D), Decay constant (E) and ZV₅₀ (F). Blood was pre-treated with: Carrier (□), 5 µM GR144053 (▪), 300 µM DM-BAPTA-AM (▪).