The ability to promote or inhibit specific platelet–surface interactions in well-controlled environments is crucial to studying fundamental adhesion and activation mechanisms. Here, microcontact printing was used to immobilize human fibrinogen covalently in the form of randomly placed, micrometer-sized islands at an overall surface coverage of 20, 50, or 85%. The nonprinted background region was blocked with covalently immobilized human albumin. Platelet adhesion and morphology on each substrate were assessed using combined differential interference and fluorescence microscopy. At 20% coverage, most of the fibrinogen surface features were small round islands, and platelet adhesion and spreading areas were limited by the position and the size of the islands. Platelet circularity, indicated the morphology was mostly rounded. At 50% coverage, some fibrinogen islands coalesced and platelet adhesion and spreading areas increased. Platelet morphology was controlled by the shape of underlying fibrinogen islands, leading to more irregular spreading. At 85% coverage, the fibrinogen pattern was completely interconnected and both platelet adhesion and the spreading area were significantly higher than at lower coverage. In addition, platelets also spread over the albumin regions, suggesting that after a critical surface density of fibrinogen ligands is reached, platelet spreading is no longer inhibited by albumin. Increasing the overall fibrinogen coverage resulted in higher activation levels defined by key morphological characteristics of the spreading platelet.

A new polymer brush chemistry containing sulfonated carbohydrate repeat units has been synthesized from silicon substrates using ATRP methods and characterized both in bulk and using surface analysis. The polymer brush was designed to act as a mimic for the naturally occurring sulfonated glycosaminoglycan, heparin, commonly used for modifying blood-contacting surfaces both in vitro and in vivo. Surface analysis showed conversion of brush saccharide precursor chemistry to the desired sulfonated polymer product. The sulfonated polymer brush surface was further analyzed using three conventional in vitro tests for blood compatibility -- plasma recalcification times, complement activation, and thrombin generation. The sulfonated polymer brush films on silicon oxide wafers exhibited better assay performance in these blood component assays than the unsulfonated sugar functionalized polymer brush in all tests performed.

Negative surface charge density gradients were prepared on fused silica slides using selective oxidation of a 3-mercaptopropyltrimethoxysilane (MTS) monolayer converting surface thiol groups (-SH) into negatively charged sulfonate (-SO3-) groups. The sulfonate-to-thiol gradient samples were characterized by water contact angle and electron spectroscopy for chemical analysis (ESCA). Gradients were pre-adsorbed with proteins from three different solutions: platelet free plasma (PFP), fibrinogen, or albumin in phosphate buffered saline (PBS). Washed platelets were perfused over gradient samples in a parallel plate flow chamber and platelet adhesion was measured across the gradients using differential interference contrast (DIC) microscopy. Gradients pre-adsorbed with PFP showed adhesion contrast inversely related to the negative surface charge density. The magnitude of the adhesion contrast along the gradient was also dependent on PFP concentration. Gradients pre-adsorbed with fibrinogen showed an adhesion maximum in the center of the gradient region. Albumin coating of the gradients resulted in low overall platelet adhesion with increased adhesion in regions of high negative charge density. The effect of gradient orientation with respect to the flow was also investigated. Gradients pre-adsorbed with 10% PFP showed different adhesion contrast when the platelets were perfused in opposite directions. This suggests that platelet adhesion is, in addition to responding to the local surface properties, also dependent on the upstream conditions.

Circulating activated platelets roll and make transient contacts before ultimately adhering to a substrate. However, despite the dynamic nature of platelet adhesion, most in vitro adhesion and activation studies have focused on establishing local cause and effect relationships. Here, we determined the effect of exposing platelets to immobilized upstream human fibrinogen on downstream adhesion and activation. Microcontact printing was used to prepare substrates that contained well defined fibrinogen priming regions. Washed platelets were perfused over the substrates and adhesion and activation in a downstream capture region were compared with samples that did not contain a fibrinogen priming region. It was found that samples containing an upstream priming region resulted in higher adhesion, platelet spreading areas and aggregation than samples that lacked the priming region. Also, when the priming region was selectively blocked with a polyclonal anti-fibrinogen antibody, the platelet response was attenuated. To characterize this phenomenon further, flow cytometry was used to assess bulk platelet activation following fibrinogen priming. The expression of two activation markers, PAC-1 and P-selectin were quantified. Expression of both activation markers was found to be higher after perfusion over fibrinogen versus albumin-coated substrates.

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