Application of a strain rate gradient microfluidic device to von Willebrand's disease screening

Abstract

Von Willebrand's disease (VWD) is the most common inherited bleeding disorder caused by either quantitative or qualitative defects of von Willebrand factor (VWF). Current tests for VWD require relatively large blood volumes, have low throughput, are time-consuming, and do not incorporate the physiologically relevant effects of haemodynamic forces. We developed a microfluidic device incorporating micro-contractions that harnesses well-defined haemodynamic strain gradients to initiate platelet aggregation in citrated whole blood. The microchannel architecture has been specifically designed to allow for continuous real-time imaging of platelet aggregation dynamics. Subjects aged ≥18 years with previously diagnosed VWD or who presented for evaluation of a bleeding disorder, where the possible diagnosis included VWD, were tested. Samples were obtained for device characterization as well as for pathology-based testing. Platelet aggregation in the microfluidic device is independent of platelet amplification loops but dependent on low-level platelet activation, GPIb/IX/V and integrin αβ engagement. Microfluidic output directly correlates with VWF antigen levels and is able to sensitively detect aggregation defects associated with VWD subtypes. Testing demonstrated a strong correlation with standard clinical laboratory-based tests. Head-to-head comparison with PFA100® demonstrated equivalent, if not improved, sensitivity for screening aggregation defects associated with VWD. This strain rate gradient microfluidic prototype has the potential to be a clinically useful, rapid and high throughput-screening tool for VWD as well as other strain-dependent platelet disorders. In addition, the microfluidic device represents a novel approach to examine the effects of high magnitude/short duration (ms) strain rate gradients on platelet function.

The PI 3‐kinase PI3KC2α regulates mouse platelet membrane structure and function independently of membrane lipid composition

“…Given that platelets from these PI3KC2a-deficient mice have a structural defect that is specific to the OCSa reserve of membrane thought to be utilized for platelet shape change and spreading during activation and aggregation [16] we examined platelet membrane function in both static and blood flow environments. We utilized this microfluidic blood flow assay because of its incorporation of a pronounced stenosis that has been shown to cause a substantial shear gradient (from very high at the point of stenotic narrowing, to very low immediately downstream of the stenosis) [10][11][12]. Here, filopodia production in suspension-activated platelets or under flow were unaffected by loss of PI3KC2a, yet in a microfluidic assay incorporating a high shear gradient-inducing stenosis, PI3KC2a-deficiency led to a marked reduction in platelet deposition.…”

“…Importantly, in vivo studies of platelet behaviour following exposure to such a shear gradient environment (via a vascular stenosis) have shown that initial platelet deposition occurs largely independently of cellular activation by soluble agonists [12]. Indeed, such shear gradients appear to drive initial platelet deposition via the formation of membrane tethers [10][11][12]. As a result, the significant reduction in platelet deposition in this system using blood from PI3KC2a mice may suggest that PI3KC2a is important for the earliest stages of platelet adhesion, where membrane tethers, presumably derived from the OCS, are pulled from platelets without detectable cell activation via an interaction between von Willebrand factor and the platelet adhesion receptor GPIba [17].…”

“…Platelet aggregation was performed using a previously described method modified for use in this study with mouse whole blood [11]. Briefly, a microfluidic device consisting of 200 lm wide channels with inset 40 lm stenosis was fabricated using standard photolithography techniques [10].…”

“…Given the impaired arterial thrombus formation previously observed in PI3KC2a-deficient mice, we examined platelet function in a microfluidic whole blood flow assay known to be dependent on membrane tether formation [10][11][12]. Mouse blood was perfused through a von Willebrand factor-coated microfluidic channel incorporating a shear force gradient-inducing stenosis [10][11][12]. Platelet aggregation occurred downstream of the stenosis and the size of these platelet aggregates was quantified, using the epifluorescent signal of the GFP-positive platelets (Fig.…”

“…These changes occur without major alterations in the lipid composition of these platelets. Surprisingly, the structural changes to the platelet OCS membrane have no impact on the formation of filopodia by platelets activated in suspension, yet markedly impacts on the ability of these platelets to form thrombi in a blood flow environment involving a prominent shear gradient that produces membrane tether formation [10][11][12]. Together, these findings indicate that PI3KC2a modulates the structure of the platelet internal membrane (OCS) independently of changes to membrane composition.…”