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.

Depositing multiple proteins on the same substrate in positions similar to the natural cellular environment is essential to tissue engineering and regenerative medicine. In this study, the development and verification of a multi-protein micro-contact printing (μCP) technique is described. It is shown that patterns of multiple proteins can be created by the sequential printing of proteins with μm precision in registration using an inverted microscope. Soft polymeric stamps were fabricated and were mounted on a microscope stage while the substrate to be stamped was placed on a microscope objective and kept at its focal distance. This geometry allowed for visualization of patterns during the multiple stamping events and facilitated the alignment of multiple stamped patterns. Astrocytes were cultured over stamped lane patterns and were seen to interact and align with the underlying protein patterns.

A synthetic small-diameter vascular graft can often become stenotic due to intimal fibrous hyperplasia, either generally along the inside of the graft or at the anastomotic regions, leading to an increased shear force on flowing platelets. Our lab is studying how the upstream platelet preactivation (aka "priming") in flowing blood affects their downstream adhesion and activation. This manuscript describes a study in which priming of platelets is achieved by upstream stenotic narrowing in a microfluidic flow chamber. Such experimental design was intended to mimic a vascular implant with stenotic upstream anastomosis and downstream exposed platelet protein agonists. Understanding how the pre-activated platelets respond to imperfect vascular implant surfaces downstream is an important factor in designing better vascular implants.

A novel functional assay of antiplatelet drug efficacy was designed by utilizing the phenomena of platelet margination in flowing blood and transient platelet contacts with surface-immobilized platelet agonists. Flow margination enhances transient contacts of platelets with the walls of flow chambers covered with surface-immobilized proteins. Depending on the type and the surface density of the immobilized agonists, such transient interactions could "prime" the marginated platelet subpopulation for enhanced activation and adhesion downstream. By creating an upstream surface patch with an immobilized platelet agonist, platelet flow margination was used to test how effective antiplatelet drugs are in suppressing downstream platelet activation and adhesion. The platelet adhesion downstream was measured by a so-called "capture" patch region close to the distal end of the flow chamber. Platelet adhesion downstream was found to be dose-dependent on the upstream surface coverage of the "priming" patch, with immobilized fibrinogen acting as a platelet agonist. Several antiplatelet agents (acetylsalicylic acid, eptifibatide, and tirofiban) were evaluated for their efficacy in attenuating downstream adhesion after upstream platelet priming. The activation of the platelet population was found to be dependent on both the extent of the upstream agonist stimulus and the antiplatelet drug concentration. Such a relationship provides an opportunity to measure the efficacy of specific antiplatelet agents against the type and concentration of upstream platelet agonists.

As platelets encounter damaged vessels or biomaterials, they interact with a complex milieu of surface-bound agonists, from exposed subendothelium to adsorbed plasma proteins. It has been shown that an upstream, surface-immobilized agonist is capable of priming platelets for enhanced adhesion downstream. In this study, binary agonists were integrated into the upstream position of flow cells and the platelet priming response was measured by downstream adhesion in flowing whole blood. A nonadditive response was observed in which platelets transiently exposed to two agonists exhibited greater activation and downstream adhesion than that from the sum of either agonist alone. Antibody blocking of one of the two upstream agonists eliminated nonadditive activation and downstream adhesion. Crosstalk between platelet activation pathways likely led to a synergistic effect which created an enhanced activation response in the platelet population. The existence of synergy between platelet priming pathways is a concept that has broad implications for the field of biomaterials hemocompatibility and platelet activity testing.