RESUMO
Platelet function testing is essential for the diagnosis of patients with bleeding disorders. Specifically, there is a need for a whole blood assay that is capable of analysing platelet behaviour in contact with a patient-specific autologous von Willebrand factor (vWF), under physiologically relevant conditions. The creation of surface topography capable of entrapping and uncoiling vWF for the support of subsequent platelet adhesion within the same blood sample offers a potential basis for such an assay. In this study, spin coating of polystyrene/poly (methyl methacrylate) (PS/PMMA) demixed solutions onto glass substrates in air has been used to attain surfaces with well-defined topographical features. The effect of augmenting the PS/PMMA solution with uniform 50 µm PS microspheres that can moderate the demixing process on the resultant surface features has also been investigated. The topographical features created here by spin coating under ambient air pressure conditions, rather than in nitrogen, which previous work reports, produces substrate surfaces with the ability to entrap vWF from flowing blood and facilitate platelet adhesion. The direct optical visualisation of fluorescently-labelled platelets indicates that topography resulting from inclusion of PS microspheres in the PS/PMMA spin coating solution increases the total number of platelets that adhere to the substrate surface over the period of the microfluidic assay. However, a detailed analysis of the adhesion rate, mean translocating velocity, mean translocation distance, and fraction of the stably adhered platelets measured during blood flow under arterial equivalent mechanical shear conditions indicates no significant difference for topographies created with or without inclusion of the PS microspheres.
RESUMO
Human platelets play a vital role in haemostasis, pathological bleeding and thrombosis. The haemostatic mechanism is concerned with the control of bleeding from injured blood vessels, whereby platelets interact with the damaged inner vessel wall to form a clot (thrombus) at the site of injury. This adhesion of platelets and their subsequent aggregation is dependent on the presence of the blood protein von Willebrand Factor (vWF). It is proposed here that the entrapment of vWF on a substrate surface offers the opportunity to assess an individual's platelet function in a clinical diagnostic context. Spin coating from demixed solutions of polystyrene (PS) and poly(methyl methacrylate) (PMMA) onto glass slides has been shown previously to support platelet adhesion but the mechanism by which this interaction occurs, including the role of vWF, is not fully understood. In this work, we report a study of the interaction of platelets in whole blood with surfaces produced by spin coating from a solution of a weight/weight mixture of a 25% PS and 75% PMMA (25PS/75PMMA) in chloroform in the context of the properties required for their use as a Dynamic Platelet Function Assay (DPFA) substrate. Atomic Force Microscopy (AFM) indicates the presence of topographical features on the polymer demixed surfaces in the sub-micron to nanometer range. X-ray Photoelectron Spectroscopy (XPS) analysis confirms that the uppermost surface chemistry of the coatings is solely that of PMMA. The deliberate addition of various amounts of 50 µm diameter PS microspheres to the 25PS/75PMMA system has been shown to maintain the PMMA chemistry, but to significantly change the surface topography and to subsequently effect the scale of the resultant platelet interactions. By blocking specific platelet binding sites, it has been shown that their interaction with these surfaces is a consequence of the entrapment and build-up of vWF from the same whole blood sample.