High shear induces platelet dysfunction leading to enhanced thrombotic propensity and diminished hemostatic capacity

Abstract

Thrombosis and bleeding are devastating adverse events in patients supported with blood-contacting medical devices (BCMDs). In this study, we delineated that high non-physiological shear stress (NPSS) caused platelet dysfunction that may contribute to both thrombosis and bleeding. Human blood was subjected to NPSS with short exposure time. Levels of platelet surface GPIbα and GPVI receptors as well as activation level of GPIIb/IIIa in NPSS-sheared blood were examined with flow cytometry. Adhesion of sheared platelets on fibrinogen, von Willibrand factor (VWF), and collagen was quantified with fluorescent microscopy. Ristocetin- and collagen-induced platelet aggregation was characterized by aggregometry. NPSS activated platelets in a shear and exposure time-dependent manner. The number of activated platelets increased with increasing levels of NPSS and exposure time, which corresponded well with increased adhesion of sheared platelets on fibrinogen. Concurrently, NPSS caused shedding of GPIbα and GPVI in a manner dependent on shear and exposure time. The loss of intact GPIbα and GPVI increased with increasing levels of NPSS and exposure time. The number of platelets adhered on VWF and collagen decreased with increasing levels of NPSS and exposure time, respectively. The decrease in the number of platelets adhered on VWF and collagen corresponded well with the loss in GPIbα and GPVI on platelet surface. Both ristocetin- and collagen-induced platelet aggregation in sheared blood decreased with increasing levels of NPSS and exposure time. The study clearly demonstrated that high NPSS causes simultaneous platelet activation and receptor shedding, resulting in a paradoxical effect on platelet function via two distinct mechanisms. The results from the study suggested that the NPSS could induce the concurrent propensity for both thrombosis and bleeding in patients.

Keywords: Bleeding; blood-contacting medical devices; non-physiological shear stress; platelet dysfunction; thrombosis.

Figure 1.

Blood shearing system. It includes a syringe pump, a syringe, a blood-shearing device (axial flow-through Couette-type device), a waste blood reservoir and connecting tubes. The syringe pump can be used to control the flow rate to obtain the desired exposure time.

Figure 2.

Platelet adhesion system. It includes a syringe pump which can be used to control the shear rate during the platelet adhesion, a syringe, three glass tubes (0.2mm x 2mm x 25mm, VitroCom, Mountain Lakes, NJ) coated with VWF (100μg/ml, EMD Millipore, Billerica, MA), collagen (1mg/ml, Chrono-log, Havertown, PA) and fibrinogen (1mg/ml, EMD Millipore, Billerica, MAf) respectively and a waste blood reservoir.

Figure 3.

Comparison of platelet activation and platelet adhesion on fibrinogenin the baseline sample and sheared samples. (A)The percentage of activated platelets indicated by the GP IIb/IIIa activation in the baseline and sheared blood samples (n=6, *P<0.05);(B)Representative fluorescence images of platelet adhesion on fibrinogen from the baseline and four sheared samples (magnification, X400). All scale bars = 20μm; (C) The comparison of the area coverage (ten images taken from the each glass tube) of adherent platelets on fibrinogen for the baseline and four sheared samples (n=6, *P<0.05).

Figure 4.

Comparison of GPIbα and GPVIsurface expressionin the baseline sample and sheared samples. (A) Typical histograms of channel fluorescence of the platelet GPIbα surface expression; (B) Typical histograms of channel fluorescence of the platelet GPVI surface expression; (C) The quantification of the platelet GPIbα surface expression in the baseline sample and sheared samples.The ratio of mean fluorescence intensity (MFI) using sheared samples to compare with baseline sample was taken to perform quantitative comparison (n=6, *P<0.05); (D) The quantification of the platelet GPVI surface expression in the baseline sample and sheared samples (n=6, *P<0.05).

Figure 5.

Comparison of platelet adhesion on VWF and collagenin the baseline sample and sheared samples. (A) Representative fluorescence images of adherent platelets on VWF in the baseline and four sheared samples (magnification, X400). All scale bars = 20μm; (B) The comparison of the area coverage (ten images of each glass tube) of adherent platelets on VWF for the baseline and four sheared samples (n=6, *P<0.05); (C) Representative fluorescence images of adherent platelets on collagen in the baseline and four sheared samples (magnification, X400). All scale bars = 20μm; (D) The comparison of the area coverage (ten images of each glass tube) of adherent platelets on collagen for the baseline and four sheared samples (n=6, *P<0.05).

Figure 6.

Comparison of platelet aggregation induced by ristocetin and collagen in the baseline sample and sheared samples. In this aggregation test, 300 μlof whole blood was mixed with 300 μl of saline solution with or without 3mM CaCl₂intest cells and 50 μl of ristocetin (final concentration of 0.77mg/ml) or 25 μl of collagen (final concentration of 3.2ug/ml) was used to induce platelet aggregation. (A) The impedance curves of ristocetin-induced platelet aggregation of the baseline and four sheared samples; (B) The quantitative comparison of ristocetin-induced platelet aggregation of the baseline and sheared blood samples (n=6, *P<0.05); (C) The impedance curves of collagen-induced platelet aggregation of the baseline and four sheared blood samples; (D) The quantitative comparison of collagen-induced platelet aggregation of the baseline and sheared samples (n=6, *P<0.05).