Paradoxical Effect of Nonphysiological Shear Stress on Platelets and von Willebrand Factor

Abstract

Blood can become hypercoagulable by shear-induced platelet activation and generation of microparticles. It has been reported that nonphysiological shear stress (NPSS) could induce shedding of platelet receptor glycoprotein (GP) Ibα, which may result in an opposite effect to hemostasis. The aim of this study was to investigate the influence of the NPSS on platelets and von Willebrand factor (vWF). Human blood was exposed to two levels of NPSS (25 Pa, 125 Pa) with an exposure time of 0.5 s, generated by using a novel blood-shearing device. Platelet activation (P-selectin expression, GPIIb/IIIa activation and generation of microparticles) and shedding of three platelet receptors (GPIbα, GPVI, GPIIb/IIIa) in sheared blood were quantified using flow cytometry. Aggregation capacity of sheared blood induced by ristocetin and collagen was evaluated using an aggregometer. Shear-induced vWF damage was characterized with Western blotting. Consistent with the published data, the NPSS caused significantly more platelets to become activated with increasing NPSS level. Meanwhile, the NPSS induced the shedding of platelet receptors. The loss of the platelet receptors increased with increasing NPSS level. The aggregation capacity of sheared blood induced by ristocetin and collagen decreased. There was a loss of high molecular weight multimers (HMWMs) of vWF in sheared blood. These results suggest that the NPSS induced a paradoxical effect. More activated platelets increase the risk of thrombosis, while the reduction in platelet receptors and the loss of HMWM-vWF increased the propensity of bleeding. The finding might provide a new perspective to understand thrombosis and acquired bleeding disorder in patients supported with blood contacting medical devices.

Keywords: Bleeding; Blood contacting medical devices; Nonphysiological high shear stress; Receptor shedding; Thrombosis.

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

The authors declared that there are no conflicts of interests.

Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

Figures

Figure 1

The blood shearing system; it consisted of a syring pump used to control the flow rate, 140ml syringe, blood shearing device (Hemolyzer-L) including 150μm narrow gap between the inner rotor and the outer housing, waste container, and tubing.

Figure 2

Comparison of P-selectin expression and GP IIb/IIIa activation among baseline blood sample and the sheared samples (0.5s/25pa and 0.5s/125Pa) using flow cytometry. A: Typical flow cytometry dot plots. The platelets population were identified by scatter gating (gating on forward scatter [FSC] and side scatter [SSC]) of flow cytometer. The percent of platelets with P-selectin expression is indicated by the antibody of PE conjugated anti-CD62P and the percent of platelets with GP IIb/IIIa activation is indicated by the antibody of FITC labeled PAC-1. B: Average percentage of activated platelets indicated by the P-selectin surface expression (n=5). C: Average percentage of activated platelets indicated by the GP IIb/IIIa activation (n=5).

Figure 3

A: The flow cytometry dot plots of comparing of PMPs among base sample and sheared samples (0.5s/25pa and 0.5s/125Pa). The PMPs were determined by the FSC characteristics of all CD41 positive events. B: The comparison of average percentage of PMPs (n=5).

Figure 4

A: The typical histograms of the mean fluorescence of the platelet receptor GPIbα expression (FITC conjugated CD42b) in the baseline blood and two sheared blood samples (0.5s/25Pa and 0.5s/125Pa). B: The quantification comparison of the platelet receptor GPIbα expression (mean fluorescence) (n=5).

Figure 5

A: The typical histograms of the mean fluorescence of the platelet receptor GPVI expression (eflour660) in the baseline blood and two sheared blood samples (0.5s/25Pa and 0.5s/125Pa). B: The quantification comparison of the platelet receptor GPVI expression (mean fluorescence) (n=5).

Figure 6

A: The typical histograms of the mean fluorescence of the platelet receptor GP IIb/IIIa (PE conjugated anti-CD41/61) in the baseline blood and two sheared blood samples (0.5s/25Pa and 0.5s/125Pa). B: The quantification comparison of the platelet receptor GP IIb/IIIa (mean fluorescence) (n=5).

Figure 7

Comparison of platelet aggregation ability among baseline blood sample and the sheared samples (0.5s/25pa and 0.5s/125Pa) using the aggregometer. A: The changing of impedance curve (aggregation curve) of the platelets aggregation induced by collagen. The platelet aggregation is indicated by area under the impedance curve. The time course of the platelets aggregation curve changing was recorded for 6 minutes. B: The average comparing of platelet aggregation induced by collagen (n=5). C: The changing of impedance curve (aggregation curve) of the platelets aggregation induced by ristocetin. D: The average comparing of platelet aggregation induced by ristocetin (n=5).

Figure 8

A: The representative images of plasma vWF multimers in the baseline blood and two sheared blood samples (0.5s/25Pa and 0.5s/125Pa). B: The quantitative comparison of plasma HMWM-vWF images optical density ratio (n=5).