Abstract
Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s−1 through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (LagT), the rate of platelet accumulation (VPLT), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to VPLT and SC at all wall shear rates. A longer LagT for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s−1 and 300 s−1. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.
Highlights
The central role of shear stress in thrombus formation is well documented
These channel dimensions were chosen based on previous work that showed that this channel aspect ratio (10:1 width:height) yields a blunted shear stress profile that results in uniform platelet deposition [21]
The purpose of this study was to measure the variability of platelet accumulation in Microfluidic flow assays (MFA) within the normal population and associate that variability with common factors known to effect platelet function
Summary
The central role of shear stress in thrombus formation is well documented. Platelets can adhere to fibrinogen and collagen at venous shear stresses, but von Willebrand factor (VWF) is necessary to promote rolling prior to firm adhesion at arterial shear stresses [1,2]. The binding of VWF to collagen is shear stress dependent where high shear stress exposes the A1 domain, which can substitute for the collagen binding site in the A3 domain [3]. Recent studies have shown that VWF multimer size is regulated by shear stress where a threshold shear stress gradient exposes the A2 domain allowing cleavage by ADAMTS13 [4]. Fibrin formation is inhibited at high shear rates because fibrin monomers and thrombin are washed out before fibrin fibers can form [5]. Despite these numerous shear stress and shear rate dependent mechanisms, there is no accepted clinical method to evaluate thrombus formation under physiological shear stresses
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