A biofunctionalized material with wound-like features to rapidly measure platelet function under flow

摘要

Blood platelet dysfunction is a strong predictor of blood loss and poor outcomes after injury. Generation of adequate contractile force by blood platelets is critical to produce a cohesive hemostatic plug capable of stopping bleeding. Platelets are activated to forcefully contract at wound sites by exposure adhesive proteins and high shear forces. We developed a microfluidic device that measures platelet function by reproducing the high-shear forces found at wounds and measures the resulting platelet forces. Whole human blood is flowed through a microfabricated chip with arrays of protein-coated microscale blocks and microposts arranged along the bottom of a microchannel. The surface of the blocks and microposts are coated with adhesive von Willebrand factor and type Ⅱ collagen typically present at wounds to encourage rapid platelet adhesion. As blood is passes over and around a block, its shape induces a high shear force that causes platelets to aggregate on its surface. A single micropost is situated behind each block. Within seconds after platelets aggregate between the block and micropost, their contractile forces cause the micropost to bend toward the block. The deflection of the post is recorded under fluorescence microscopy and analyzed using quantitative image analysis of the videos. Since microposts bend like cantilever beams, their deflection behaves according to Hooke's law and can be used to calculate platelet forces directly. Blebbistatin, a cytoskeletal myosin inhibitor, was used to confirm that deflection of the microposts was due to cytoskeletal contraction in the platelets. When blood was incubated with 2-MeSAMP, a P2Y12 activation pathway antagonist, platelets were able to aggregate, but their ability to generate contractile forces was reduced. Likewise, incubating blood with acetyl salicylic acid inhibited platelet force generation. Platelet aggregation and contractile forces were also reduced when glycoprotein Ib-Ⅴ-Ⅸ complex and integrin allbβ3 were inhibited with antibody AK2 and antibody fragment c7E3 Fab, respectively. When blood was incubated with tissue plasminogen activator, platelets aggregated and produced contractile forces that increased steadily within the first ten minutes, but then decreased substantially as the platelet microaggregates fractured and dissolved. These results indicate that platelets can produce measureable contractile forces that are affected by multiple platelet activation pathways. Measuring platelet forces under wound-like conditions may be a useful and novel way to rapidly identify platelet dysfunction in bleeding situations.