Downstream platelet adhesion and activation under highly elevated upstream shear forces

Abstract

Elevated shear force caused by an anastomotic stenosis is a common complication at the blood vessel-vascular implant interface. Although elevated shear forces were found to cause platelet aggregation around a stenotic region, transient platelet exposure to elevated shear forces and subsequent downstream events occurring under lower shear force were not extensively studied. We hypothesize that effects of elevated shear forces on pre-activation of platelets for downstream adhesion and activation are relevant in understanding the increased thrombotic risk associated with blood-contacting devices. We designed a microfluidic flow system to mimic the hemodynamic environment of vasculature with an upstream anastomotic stenosis with five wall shear strain rates ranging from 1620 s^-1 to 11560 s^-1. Under shear flow conditions, transient exposure of whole blood to elevated shear forces resulted in higher downstream platelet adhesion onto three different immobilized platelet agonists: fibrinogen, collagen, or von Willebrand factor. Platelet expression of four activation markers (P-selectin, GPIIb/IIIa, lysosomal glycoprotein, and phosphatidylserine) significantly increased after transient exposure to higher upstream wall shear strain rates of 2975-11560 s^-1. A significant lysis was observed when platelets were primed by upstream wall shear strain rate of 11560 s^-1. These experimental results could be helpful to understand how altered hemodynamics around an anastomotic stenosis promotes thrombus formation downstream. STATEMENT OF SIGNIFICANCE: Studying the downstream response of platelets following transient exposure to an upstream agonist is important because of significant clinical implications to the implantation of vascular devices. Due to intimal fibrous hyperplasia, vascular biomaterials such as synthetic small-diameter vascular grafts sometimes become stenotic (narrow), leading to transient platelet exposure to elevated shear forces. In this study, a microfluidic flow system was developed to mimic a stenosed vascular graft and to investigate how highly elevated, transient upstream shear forces, typically found in severe stenosis, results in the pre-activation of platelets for downstream adhesion and activation. The findings of the present study have implications for optimizing the design of blood-contacting biomaterials in order to minimize thrombotic risk associated with transiently elevated shear forces. The findings also provide additional insights into the mechanisms of thrombus formation at the post-stenotic regions of vascular implants.

Keywords: Anastomotic stenosis; Flow cytometry; Microfluidics; Platelet adhesion and activation; Platelet lysis.

Figure 1.

Schematic diagram of the flow cell used in the flow-based platelet adhesion assay. Upstream stenotic regions were created by changing the width of the flow channels. Protein agonists were covalently immobilized downstream on the chemically reactive glass substrates by microcontact printing.

Figure 2.

Wall shear strain rate (γ_w) distribution in the microfluidic flow channels. (A) The first 20 mm of each experimental flow channel was simulated using COMSOL Multiphysics to evaluate wall shear strain rates in the anastomotic stenosis. The colors indicate local wall shear strain rates at the channel bottom wall. Centerline wall shear strain rate (γ_c) represents wall shear strain rate across the centerline (i.e., dotted white line in A) of the channel bottom wall. (B) Centerline wall shear strain rates indicate a steep gradient, particularly for 60% and 80% stenotic models. Centerline wall shear strain rates within the stenotic regions were 1620, 2145, 2975, 4860 and 11560 s⁻¹ for 0%, 20%, 40%, 60% and 80% stenotic models, respectively. Downstream centerline wall shear strain rate (1620 s⁻¹) was kept constant in all experiments.

Figure 3.

Platelet adhesion to three downstream capture proteins as a function of centerline wall shear strain rates in the upstream stenosis compared with control sample containing no stenotic region (γ_c = 1620 s⁻¹). Statistical significance was obtained using a paired t-test (n = 40, *p < 0.05, **p < 0.005 and ***p < 0.0005).

Figure 4.

Representative phase contrast images (A-D) show morphological changes of adhered platelets onto fibrinogen coated capture region after passing through the chamber containing no stenotic region (centerline wall shear strain rate of 1620 s⁻¹) and through the chambers containing upstream stenotic region (centerline wall shear strain rates of 2975 – 11560 s⁻¹). Representative images E and F show morphology of platelets adhered onto collagen and vWF coated capture regions, respectively, after passing through the upstream stenotic region with centerline wall shear strain rate of 11560 s⁻¹.

Figure 5.

Flow cytometry results of platelet activation. Expression levels of (A) P-selectin and (B) activated GPIIb/IIIa (PAC−1 receptor) in perfused blood samples were compared with unstimulated (negative control) and thrombin stimulated (positive control) blood samples collected prior to perfusion. Analysis of 100,000 events for each sample was conducted, and events of platelets expressing each marker were recorded. Statistical significance was obtained using a paired t-test (n = 4, *p < 0.05, **p < 0.005 and ***p < 0.0005 relative to “no stimulation” control).

Figure 6.

Flow cytometry results of platelet activation. Expression levels of (A) annexin V receptor (phosphatidylserine) and (B) CD63 (lysosomal glycoprotein) in perfused blood samples were compared with unstimulated (negative control) and thrombin stimulated (positive control) samples collected prior to perfusion. Analysis of 100,000 events for each sample was conducted, and events of platelets expressing each marker were recorded. Statistical significance was obtained using a paired t-test (n = 4, *p < 0.05 and ***p < 0.0005 relative to “no stimulation” control).

Figure 7.

Effect of wall shear strain rates on the release of LDH from platelets in platelet rich plasma after 5 min perfusion through the albumin coated flow channels. Statistical significance was obtained using a paired t-test (n = 4, *p < 0.05 and ***p < 0.0005 relative to “no stimulation” control).