Pursuing Ultra-Low VWF Adsorption for Blood-Contacting Devices Under Shear
Authors: Yang Wei , Chen Junmei , Le Jennie , Lopez Jose A. 
Published: October 2019

Abstract
Many human diseases are treated with blood-contacting medical devices. These devices, which include artificial lung and heart machines and extracorporeal membrane oxygenators (ECMO), save or extend many lives every year. However, the devices are often compromised because of blood proteins that accumulate on their surfaces. Such accumulation activates both platelet activation and blood coagulation, and patients supported by blood-contacting devices are at high risk of both bleeding and clotting. Attempts have been made to create nonfouling surfaces, and many have in fact have been produced that resist fouling by plasma when tested under non-flow conditions. However, blood passing through medical devices is often subjected to high shear stresses (especially near surfaces) and flow acceleration, conditions that we have shown promote rapid deposition of the large plasma glycoprotein von Willebrand factor (VWF) onto uncoated plastic surfaces. VWF normally functions to initiate platelet attachment at sites of vessel injury. Thus, its adsorption to artificial surfaces under shear is likely to initiate platelet binding and the adsorption of other plasma proteins, including clotting proteins. We sought to create a surface that achieves ultra-low (< 5 ng/cm2) VWF adsorption under shear, which we anticipate will prevent further protein adsorption and solve the problem of blood and platelet deposition at its initiation.
To achieve this objective, we synthesized polymers from the zwitterion sulfobetaine methacrylate (polySBMA) based on published methods. Dopamine was used as the linker between polySBMA and the commonly used materials for devices, resulting in the direct attachment of the polymer to the surface in one step. A more uniform coating of the polymer on certain hydrophobic surfaces was achieved using a mixed solvent of methanol and water. Various coating conditions for polySBMA were tested, including variations in buffer type, salt concentration, polymer concentration, polymer/dopamine ratio, and methanol/water ratio. Eppendorf polypropylene tubes were incubated overnight with polySBMA solution, washed, and then exposed to a VWF solution under shear in a vortexer (3000 rpm, at RT for 90 min). We compared VWF adsorption in the polySBMA-coated PP tubes to adsorption in uncoated tubes. VWF adsorption to tubes coated under optimal conditions was 4% of the adsorption to uncoated tubes, with a VWF surface density below 2 ng/cm2. This coating condition was used for subsequent studies.
We next tested the stability of the polySBMA coating after long-term storage. Coated tubes were stored in PBS at 4°C for 1, 4, 7, 28, and 42 days and the integrity of the surface-bound polymers was then examined by evaluating the ability of the stored tubes to resist VWF deposition and comparing their performance to that of freshly coated tubes. Coated tubes stored up to 6 weeks retained their resistance to VWF adsorption under shear (less than 5 ng/cm2), indicating the long-term resilience of the polySBMA coating.
To mimic shear stress conditions of blood vessels in capillary, venous, and arterial flow, we used a perfusion system in which a pump (ibidi) generates different flow patterns (continuous unidirectional, oscillating, or pulsatile flow) and shear stresses. Surfaces within the perfusion system were coated with polySBMA to minimize VWF loss. Additional polypropylene test tubing (50 cm long), uncoated or polySBMA-coated, was connected to the perfusion system between two connecters. Purified VWF (2.5 µg/mL in PBS) was perfused through this system for 1 hr. The VWF remaining in the solution was measured by ELISA after the prescribed perfusion period, and multimer composition was compared before and after perfusion. The VWF concentration decreased by 74.1% with uncoated tubing after 1 hr at 20 dyn/cm2, with selective depletion of large VWF multimers. By contrast, in the polySBMA-coated tubing the concentration and multimer composition of VWF was unchanged under identical conditions.
In summary, polySBMA effectively prevented VWF adsorption under shear in two types of experiments, and was stable during storage. We expect this coating to significantly enhance the lifetime of blood-contacting devices and reduce both costs and medical complications.

Disclosures
No relevant conflicts of interest to declare.