Effects of upstream shear forces on priming of platelets for downstream adhesion and activation

Abstract

Platelets in flowing blood are sometimes exposed to elevated shear forces caused by anastomotic stenosis at the blood vessel-vascular implant interface. The objective of this study was to determine how effective upstream shear forces are in priming platelets for downstream adhesion and activation. Flow chambers with upstream stenotic regions (shear rates of 400-1000 s^-1) were manufactured by relief molding of polydimethylsiloxane. Downstream from the stenotic regions, microcontact printing was used to covalently immobilize three different proteins (fibrinogen, collagen, or von Willebrand factor) to serve as platelet capture agents. Anticoagulated whole blood was perfused through the flow chambers and platelet adhesion to the downstream capture region was quantified. It was found that transient exposure of platelets to increased shear forces resulted in higher platelet adhesion on all three proteins. The duration of the platelet exposure to elevated shear forces was varied by changing the length of the stenotic regions. The results indicated that, in addition to the magnitude of shear forces, the duration of exposure to these forces was also an important factor in priming platelets. The effect of upstream shear forces on platelet activation was assessed by quantifying P-selectin, integrin α_IIbβ₃, lysosomal glycoprotein, and phosphatidylserine exposure using flow cytometry. The results suggested that increased shear forces were capable of increasing the priming of platelets for downstream activation. This study implicates the anastomotic region(s) of vascular implants as a locus of platelet pre-activation that may lead to thrombus formation downstream.

Statement of significance: A synthetic small-diameter vascular graft can often become stenotic due to intimal fibrous hyperplasia, either generally along the inside of the graft or at the anastomotic regions, leading to an increased shear force on flowing platelets. Our lab is studying how the upstream platelet preactivation (aka "priming") in flowing blood affects their downstream adhesion and activation. This manuscript describes a study in which priming of platelets is achieved by upstream stenotic narrowing in a microfluidic flow chamber. Such experimental design was intended to mimic a vascular implant with stenotic upstream anastomosis and downstream exposed platelet protein agonists. Understanding how the pre-activated platelets respond to imperfect vascular implant surfaces downstream is an important factor in designing better vascular implants.

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

Figure 1

Schematic diagram of a parallel flow assay. The upstream stenotic priming region was created by controlling the width of flow cells. Proteins were immobilized downstream on a reactive glass substrate by μCP to serve as platelet capture regions.

Figure 2

Platelet adhesion to three downstream capture proteins as a function of shear forces in the upstream stenosis compared with control sample containing no stenotic region (shear rate of 400 s⁻¹). Statistical significance for each downstream protein was obtained using paired t-test (n = 30, *p < 0.05, **p < 0.005 and ***p < 0.0005).

Figure 3

Representative fluorescence images showing platelet adhered to the three downstream proteins after passing through the upstream stenotic region or the chamber containing no stenotic region (shear rate of 400 s⁻¹). Scale bar represents 20 μm.

Figure 4

Velocity profiles in four different flow chambers. The first 20 mm of each experimental flow chamber was simulated using COMSOL Multiphysics to visualize the velocity profiles in the anastomotic regions. The profiles show the velocity cross-section at the middle height of the channel (at the depth of 0.05 mm).

Figure 5

Platelet adhesion to three downstream capture proteins at upstream shear rates of (A) 500 s⁻¹ and (B) 1000 s⁻¹ for three different lengths of stenotic regions. Statistical significance was obtained using paired t-test (n = 30, *p < 0.05, **p < 0.005 and ***p < 0.0005).

Figure 6

Flow cytometry analysis of platelet activation. Expression levels of (A) P-selectin and (B) PAC-1 receptor (integrin α_IIbβ₃) 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 paired t-test (n = 3, *p < 0.05 and **p < 0.005 relative to “no stimulation” control).

Figure 7

Flow cytometry analysis of platelet activation. Expression levels of (A) CD63 (lysosomal glycoprotein) and (B) phosphatidylserine (via annexin V binding) 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 paired t-test (n = 3, *p < 0.05 and ***p < 0.0005 relative to “no stimulation” control).