Shear Stress Induces Spatial and Temporal Alterations of Platelet Membrane Fluidity- Implications for Mechanoceutical Design

 In All Presenters, Miller-Gutierrez, Samuel

 Presenting Author: Samuel Miller-Gutierrez

Institution: University of Arizona

Additional Authors: Alice Sweedo, Yana Roka-Moiia, J. Sheriff, Danny Bluestein, Marvin J. Slepian

Background/Purpose: Mechanical circulatory support devices are the mainstay of therapy for patients with advanced and end-stage heart failure. While hemodynamically effective, these devices impart significant shear to blood resulting in device-related thrombosis and thromboembolic events, despite the use of antiplatelet and anticoagulant therapies. We have previously shown that platelet fluidity is an important determinant of shear-mediated platelet activation (SMPA) and that its manipulation may have therapeutic value in reducing SMPA. Here we investigate to identify how shear alters geographic distribution of fluidity in both the platelet membrane’s hydrophilic surface and hydrophobic core, and alteration occurring upon exposure to shear.

Methods: Gel filtered platelets (20,000plts/uL), obtained from human donors under informed consent, were exposed to shear of 70 dynes/cm2 at incremental time points (0, 0.5, 1.5, 2, 5, and 10 min) for stress accumulation of 0, 2100, 6300, 8400, 21000, and 42000 dynes*s/cm2, using a hemodynamic shearing device. Platelets were then incubated with either TMA-DPH (trimethylammonium diphenylhexatriene) (1uM, 5 min. at 37 C) or DPH (diphenylhexatriene) (500uM, 15 min. at 37 C).

Results: Shear stress exposure led to a rapid decrease in surface anisotropy as measured with hydrophilic TMA-DPH, revealing changes after only 30 seconds of exposure to 70 dynes/cm2 and further anisotropy decrease as shear exposure was increased (5.7% at 42,000 dynes*s/cm2). Hydrophobic DPH also indicated a rapid change with shear exposure, with results indicating that deeper regions of the membrane undergo a stiffening phase at lower levels of exposure (3.5% increase at 8400 dynes*s/cm2 before increasing in fluidity (3.2% decrease in anisotropy at 42,000 dynes*s/cm2). Both probes demonstrated a period of fluctuation below 8400 dynes*s/cm2.

Conclusion: Platelet exposure to shear led to an asynchronous fluidity shifts in distinct membrane domains. Short-term shear exposure led to surface fluidizing more rapidly than the core region. Prolonged shear accumulation resulted in fluidity increase throughout the membrane. Our observations further suggest the platelet’s ability to mitigate shear forces is reduced by prolonged shear exposure. This work contributes to defining platelet structural changes that occur as a result of continued exposure to shear, which may be useful for developing therapeutic strategies to reduce SMPA.

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