Hemodynamic force triggers rapid NETosis within sterile thrombotic occlusions

Abstract

Essentials Neutrophil extracellular traps (NETs) are generated during thrombosis and sepsis. The effect of hemodynamics on NETosis during sterile thrombosis was studied using microfluidics. Pressure gradients > 70 mmHg per mm-clot across sterile occlusions drive shear-induced NETosis. High interstitial hemodynamic forces trigger rapid NET release.

Summary: Background Neutrophil extracellular traps (NETs) are released when neutrophils encounter infectious pathogens, especially during sepsis. Additionally, NETosis occurs during venous and arterial thrombosis, disseminated intravascular coagulation, and trauma. Objective To determine whether hemodynamic forces trigger NETosis during sterile thrombosis. Methods NETs were imaged with Sytox Green during microfluidic perfusion of activated factor XII-inhibited or thrombin-inhibited human whole blood over fibrillar collagen (with or without tissue factor). Results For perfusions at initial inlet venous or arterial wall shear rates (100 s^-1 or 1000 s^-1 ), platelets rapidly accumulated and occluded microchannels with subsequent neutrophil infiltration under either flow condition; however, NETosis was detected only in the arterial condition. The level of shear-induced NETs (SINs) at 30 min was > 150-fold higher in the arterial condition in the absence of thrombin and > 80-fold greater in the presence of thrombin than the level in the venous condition. With or without thrombin, venous perfusion for 15 min generated no NETs, but an abrupt shift-up to arterial perfusion triggered NETosis within 2 min, NETs eventually reaching levels 15 min later that were 60-fold greater than that in microchannels without perfusion shift-up. SINs contained citrullinated histone H3 and myeloperoxidase, and were DNase-sensitive, but were not blocked by inhibitors of platelet-neutrophil adhesion, high-mobility group protein box 1-receptor for advanced glycation end products interaction, cyclooxygenase, ATP/ADP, or peptidylarginine deiminase 4. For measured pressure gradients exceeding 70 mmHg per millimeter of clot across NET-generating occlusions to drive interstitial flow, the calculated fluid shear stress on neutrophils exceeded the known lytic value of 150 dyne cm^-2 . Conclusions High interstitial hemodynamic forces can drive physically entrapped neutrophils to rapidly release NETs during sterile occlusive thrombosis.

Keywords: extracellular traps; hemodynamics; histone; neutrophil; thrombosis.