Transport Limitations of Nitric Oxide Inhibition of Platelet Aggregation under Flow

Abstract

Nitric oxide (NO) inhibits platelet aggregation at and near the site of a vascular injury by upregulation of cyclic guanosine monophosphate, which reduces the dimerization of the integrin α(IIb)β₃. The magnitude of NO flux from the vessel wall and the NO concentration that is necessary to inhibit platelet aggregation under physiological flow conditions is unknown. In this study, a NO releasing polymer, diazeniumdiolated dibutylhexanediamine, was integrated into a microfluidic flow assay to determine the relationship between NO wall flux and collagen mediated platelet adhesion, activation and aggregation. A NO flux equal to or greater than 2.5 × 10⁻¹⁰ mol cm⁻² min⁻¹ was found to abrogate aggregation, but not initial platelet adhesion, on collagen at 200 and 500 s⁻¹ as effectively as the α(IIb)β₃ antagonist abciximab. The dynamic range of NO fluxes found to induce measurable inhibition of platelet aggregation spanned from 0.33 × 10⁻¹⁰ to 2.5 × 10⁻¹⁰ mol cm⁻² min⁻¹ at 200-500 s⁻¹. These fluxes correspond to near-wall NO concentrations of 3-90 nM based on a computational model of NO transport. The model predicts that NO concentration in the platelet rich layer near the wall is kinetically limited, while NO penetration into the lumen is mass transfer limited.

Utility of microfluidic devices to study the platelet–endothelium interface

“…For instance, these platforms allow for the control of the spatial organization of surface coatings of adhesive or ECM proteins, cells and the controlled release of agonists and inhibitors. [7,69–72] As an example, in one study human umbilical ECs were focally injured by an electrode in order to study the interaction of platelets with activated and adjacent healthy ECs. [73] The results showed that while activated ECs lost their tight junctions and released their Weibel-Palade bodies to promote platelet recruitment and adhesion, ECs upstream and downstream of the site of focal injury maintained their quiescence state and barrier function under physiological flow conditions.…”

“…[6] The roles of the pro/anticoagulants produced by the endothelium have been studied in reductionist approaches through, for example, the addition of a NO donor, soluble TM and other reagents in microfluidic platforms in order to study their individual roles in maintaining vessel patency and platelets in a quiescent state. [7–9]…”

Emerging microengineered tools for functional analysis and phenotyping of blood cells

“…Microchannels made of NO releasing polymers were fabricated to independently tune NO concentration and shear rate [70]. These assays revealed that platelet adhesion and aggregation would diminish when NO flux exceeded a certain threshold and would completely disappear if NO flux became too high.…”

Systems Biology of Platelet–Vessel Wall Interactions

“…Interestingly, vWF release from the endothelial Weibel–Palade bodies is dependent on essential autophagy genes Atg5 or Atg7, and pharmacological inhibitors of autophagic flux lead to increased bleeding time [38]. On the other hand, the endothelium also releases substances that are inhibitory to platelet activation, by secreting nitric oxide (NO) and the downstream modulation of cGMP levels [39–43], or by secreting prostacyclin(PGI2) [44]. …”