Shear-dependent platelet aggregation size

Abstract

Nonsurgical bleeding is the most frequent complication of left ventricular assist device (LVAD) support. Supraphysiologic shear rates generated in LVAD causes impaired platelet aggregation, which increases the risk of bleeding. The effect of shear rate on the formation size of platelet aggregates has never been reported experimentally, although platelet aggregation size can be considered to be directly relevant to bleeding complications. Therefore, this study investigated the impact of shear rate and exposure time on the formation size of platelet aggregates, which is vital in predicting bleeding in patients with an LVAD. Human platelet-poor plasma (containing von Willebrand factor, vWF) and fluorochrome-labeled platelets were subjected to a range of shear rates (0-10 000 s^-1 ) for 0, 5, 10, and 15 minutes using a custom-built blood-shearing device. Formed sizes of platelet aggregates under a range of shear-controlled environment were visualized and measured using microscopy. The loss of high molecular weight (HMW) vWF multimers was quantified using gel electrophoresis and immunoblotting. An inhibition study was also performed to investigate the reduction in platelet aggregation size and HMW vWF multimers caused by either mechanical shear or enzymatic (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13-ADAMTS13, the von Willebrand factor protease) mechanism under low and high shear conditions (360 and 10 000 s^-1 ). We found that the average size of platelet aggregates formed under physiological shear rates of 360-3000 s^-1 (200-300 μm² ) was significantly larger compared to those sheared at >6000 s^-1 (50-100 μm² ). Furthermore, HMW vWF multimers were reduced with increased shear rates. The inhibition study revealed that the reduction in platelet aggregation size and HWM vWF multimers were mainly associated with ADAMTS13. In conclusion, the threshold of shear rate must not exceed >6000 s^-1 in order to maintain the optimal size of platelet aggregates to "plug off" the injury site and stop bleeding.

Keywords: ADAMTS13; exposure time; platelet aggregate; shear rate; von Willebrand factor.

FIGURE 1

A, A custom‐built blood‐shearing device was designed to be incorporated into a microscope. When the motor rotates, two opposite rotational motions are generated and transmitted to the sample chamber using pulleys and transmitting belts. B, Cross‐sectional view of the sample chamber: an acrylic bob and a glass plate that rotates in the opposite direction to generate shear Couette flow. The formation of platelet aggregates in the sample chamber was visualized in real‐time through the ×40 objective lens of the microscope [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 2

The effect of shear rate on average platelet aggregation size measured through microscopy (A‐D). Average platelet aggregation size formed under different shear rates of 0, 360, 1000, 3000, 6000, 10 000 s⁻¹ at different exposure times. Results are described as mean ± standard deviation (SD), n = 5, *P < .05 [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 3

The effect of exposure time on average platelet aggregation size measured through microscopy (A‐D). Average platelet aggregation size formed under different shear rates at different exposure times of 0, 5, 10, and 15 minutes. Results are described as mean ± standard deviation (SD), n = 5, *P < .05 [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 4

A, von Willebrand factor (vWF) multimer analysis demonstrates high molecular weight (HMW) of vWF multimers degraded as the increasing a shear range of 0‐10 000 s⁻¹ for an exposure time of 15 minutes. HMW of vWF multimers degraded (top dotted box) and low molecular weight (LMW) of vWF multimers accumulated as shear increased (bottom dotted box). B, High molecular weight von Willebrand factor (HMW vWF) density obtained from immunoblotting at shear range 0‐10 000 s⁻¹ quantified using densitometry. HMV vWF density (360‐10 000 s⁻¹) were normalized to the static result (0 s⁻¹) as a baseline and described as mean ± standard deviation (SD), n = 5, *P < .05 [Color figure can be viewed at wileyonlinelibrary.com]

FIGURE 5

A, Average platelet aggregation size formed following shearing at 360 and 10 000 s⁻¹ for an exposure time of 15 minutes with and without ethylenediaminetetraacetic acid (EDTA) (ADAMTS13 inhibitor) to determine the contribution between ADAMTS13 and the induced shear force on the reduced platelet aggregation size. Results are described as mean ± standard deviation (SD), n = 5, *P < .05. B, von Willebrand factor (vWF) multimer analysis demonstrates degradation of high molecular weight von Willebrand factor (HMW vWF) multimers following shearing at 360 and 10 000 s⁻¹ for an exposure time of 15 minutes with and without EDTA (ADAMTS13 inhibitor) to determine the contribution between ADAMTS13 and the induced shear force on the degradation of HMW vWF multimers. Results are described as mean ± standard deviation (SD), n = 5, *P < .05 [Color figure can be viewed at wileyonlinelibrary.com]