Benchtop von Willebrand Factor Testing: Comparison of Commercially Available Ventricular Assist Devices and Evaluation of Variables for a Standardized Test Method.

Gastrointestinal bleeding occurs in 20-30% of patients receiving ventricular assist devices (VADs) due, in part, to acquired von Willebrand syndrome. We examined factors to optimize a benchtop method to quantify changes in von Willebrand Factor (VWF) multimer distribution and function in VADs, then applied them to evaluate commercially available devices. Human plasma was circulated through flow loops with VADs. Several experimental conditions were examined, including temperature, viscosity, and enzyme inhibition. Samples were analyzed for VWF collagen-binding activity (VWF:CB) and VWF antigen level. von Willebrand Factor multimer profiles were quantified using gel electrophoresis, near-infrared in-gel visualization, and densitometric analysis. The VWF:CB/antigen ratio in the HeartMate II, CentriMag, and HVAD exhibited average decreases of 46%, 44%, and 36% from baseline after 360 minutes of operation. High molecular weight (hVWF) multimer loss occurred within 30 minutes, although the Levacor and control loop profiles were unchanged. Varying temperature and viscosity altered hVWF degradation rate, but not the final results. Inhibition of a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13 (ADAMTS13) can potentially distinguish mechanoenzymatic cleavage of VWF from mechanical degradation. We developed a repeatable benchtop method to evaluate VWF compatibility of VADs similar to hemolysis testing that can be adopted for preclinical VAD evaluation.

Effects of microfluidic channel geometry on leukocyte rolling assays.

Microfluidic cell adhesion assays have emerged as a means to increase throughput as well as reduce the amount of costly reagents. However as dimensions of the flow chamber are reduced and approach the diameter of a cell (D(c)), theoretical models have predicted that mechanical stress, force, and torque on a cell will be amplified. We fabricated a series of microfluidic devices that have a constant width:height ratio (10:1) but with varying heights. The smallest microfluidic device (200 µm ×20 µm) requires perfusion rates as low as 40 nL/min to generate wall shear stresses of 0.5 dynes/cm(2). When neutrophils were perfused through P-selectin coated chambers at equivalent wall shear stress, rolling velocities decreased by approximately 70 % as the ratio of cell diameter to chamber height (D(c)/H) increased from 0.08 (H = 100 µm) to 0.40 (H = 20 µm). Three-dimensional numerical simulations of neutrophil rolling in channels of different heights showed a similar trend. Complementary studies with PSGL-1 coated microspheres and paraformaldehyde-fixed neutrophils suggested that changes in rolling velocity were related to cell deformability. Using interference reflection microscopy, we observed increases in neutrophil contact area with increasing chamber height (9-33 %) and increasing wall shear stress (28-56 %). Our results suggest that rolling velocity is dependent not only on wall shear stress but also on the shear stress gradient experienced by the rolling cell. These results point to the D(c)/H ratio as an important design parameter of leukocyte microfluidic assays, and should be applicable to rolling assays that involve other cell types such as platelets or cancer cells.

Signal-dependent slow leukocyte rolling does not require cytoskeletal anchorage of P-selectin glycoprotein ligand-1 (PSGL-1) or integrin αLß2.

In inflamed venules, neutrophils roll on P- or E-selectin, engage P-selectin glycoprotein ligand-1 (PSGL-1), and signal extension of integrin α(L)ß(2) in a low affinity state to slow rolling on intercellular adhesion molecule-1 (ICAM-1). Cytoskeleton-dependent receptor clustering often triggers signaling, and it has been hypothesized that the cytoplasmic domain links PSGL-1 to the cytoskeleton. Chemokines cause rolling neutrophils to fully activate α(L)ß(2), leading to arrest on ICAM-1. Cytoskeletal anchorage of α(L)ß(2) has been linked to chemokine-triggered extension and force-regulated conversion to the high affinity state. We asked whether PSGL-1 must interact with the cytoskeleton to initiate signaling and whether α(L)ß(2) must interact with the cytoskeleton to extend. Fluorescence recovery after photobleaching of transfected cells documented cytoskeletal restraint of PSGL-1. The lateral mobility of PSGL-1 similarly increased by depolymerizing actin filaments with latrunculin B or by mutating the cytoplasmic tail to impair binding to the cytoskeleton. Converting dimeric PSGL-1 to a monomer by replacing its transmembrane domain did not alter its mobility. By transducing retroviruses expressing WT or mutant PSGL-1 into bone marrow-derived macrophages from PSGL-1-deficient mice, we show that PSGL-1 required neither dimerization nor cytoskeletal anchorage to signal ß(2) integrin-dependent slow rolling on P-selectin and ICAM-1. Depolymerizing actin filaments or decreasing actomyosin tension in neutrophils did not impair PSGL-1- or chemokine-mediated integrin extension. Unlike chemokines, PSGL-1 did not signal cytoskeleton-dependent swing out of the ß(2)-hybrid domain associated with the high affinity state. The cytoskeletal independence of PSGL-1-initiated, α(L)ß(2)-mediated slow rolling differs markedly from the cytoskeletal dependence of chemokine-initiated, α(L)ß(2)-mediated arrest.