In Vitro Treatment of Extracorporeal Membrane Oxygenation Coagulopathy with Recombinant von Willebrand Factor or Lyophilized Platelets.

OBJECTIVES: The objective was to compare primary hemostasis between adult ECMO patients and cardiac surgical patients before heparinization and cardiopulmonary bypass. Furthermore, the authors explored whether in vitro treatment of ECMO patient blood samples with recombinant von Willebrand Factor (vWF) or lyophilized platelets improved primary hemostasis in vitro. DESIGN: Prospective cohort study. SETTING: Single academic medical center. PARTICIPANTS: Ten cardiac surgical patients and 8 adult ECMO patients. INTERVENTIONS: Cardiac surgical patients and ECMO patients had blood samples collected, and in vitro platelet thrombus formation was assessed using the ATLAS PST device. The ECMO patients had platelet thrombus formation evaluated at baseline and after in vitro treatment with recombinant vWF or lyophilized platelets, whereas cardiac surgical patients had a single blood sample obtained before heparinization and cardiopulmonary bypass run. MEASUREMENTS AND MAIN RESULTS: Median maximum force (39.7 v 260.2 nN) and thrombus area (0.05 v 0.11) at 5 minutes were lower in untreated ECMO patient samples compared with cardiac surgical patients (p = 0.008 and p < 0.001, respectively). The ECMO patient samples treated with recombinant vWF demonstrated an increase in both platelet maximum force (median value of 222.1 v 39.7 nN) (p = 0.01) and platelet thrombus area (median value of 0.16 v 0.05; p = 0.001). The ECMO patient samples treated with lyophilized platelets demonstrated no increase in platelet maximum force (median value of 193.3 v 39.7 nN; p = 0.18); however, there was a significant increase in platelet thrombus area (median value of 0.13 v 0.05; p = 0.04). CONCLUSIONS: Recombinant vWF and lyophilized platelets may help to restore primary hemostasis in ECMO patients. Future studies should further evaluate the safety and efficacy of these potential therapeutics in ECMO patients.

Decoupling substrate stiffness, spread area, and micropost density: a close spatial relationship between traction forces and focal adhesions.

Mechanical cues can influence the manner in which cells generate traction forces and form focal adhesions. The stiffness of a cell's substrate and the available area on which it can spread can influence its generation of traction forces, but to what extent these factors are intertwined is unclear. In this study, we used microcontact printing and micropost arrays to control cell spreading, substrate stiffness, and post density to assess their effect on traction forces and focal adhesions. We find that both the spread area and the substrate stiffness influence traction forces in an independent manner, but these factors have opposite effects: cells on stiffer substrates produce higher average forces, whereas cells with larger spread areas generate lower average forces. We show that post density influences the generation of traction forces in a manner that is more dominant than the effect of spread area. Additionally, we observe that focal adhesions respond to spread area, substrate stiffness, and post density in a manner that closely matches the trends seen for traction forces. This work supports the notion that traction forces and focal adhesions have a close relationship in their response to mechanical cues.

Flow mechanotransduction regulates traction forces, intercellular forces, and adherens junctions.

Endothelial cells respond to fluid shear stress through mechanotransduction responses that affect their cytoskeleton and cell-cell contacts. Here, endothelial cells were grown as monolayers on arrays of microposts and exposed to laminar or disturbed flow to examine the relationship among traction forces, intercellular forces, and cell-cell junctions. Cells under laminar flow had traction forces that were higher than those under static conditions, whereas cells under disturbed flow had lower traction forces. The response in adhesion junction assembly matched closely with changes in traction forces since adherens junctions were larger in size for laminar flow and smaller for disturbed flow. Treating the cells with calyculin-A to increase myosin phosphorylation and traction forces caused an increase in adherens junction size, whereas Y-27362 cause a decrease in their size. Since tugging forces across cell-cell junctions can promote junctional assembly, we developed a novel approach to measure intercellular forces and found that these forces were higher for laminar flow than for static or disturbed flow. The size of adherens junctions and tight junctions matched closely with intercellular forces for these flow conditions. These results indicate that laminar flow can increase cytoskeletal tension while disturbed flow decreases cytoskeletal tension. Consequently, we found that changes in cytoskeletal tension in response to shear flow conditions can affect intercellular tension, which in turn regulates the assembly of cell-cell junctions.

Contractile forces in platelet aggregates under microfluidic shear gradients reflect platelet inhibition and bleeding risk.

Platelets contract forcefully after their activation, contributing to the strength and stability of platelet aggregates and fibrin clots during blood coagulation. Viscoelastic approaches can be used to assess platelet-induced clot strengthening, but they require thrombin and fibrin generation and are unable to measure platelet forces directly. Here, we report a rapid, microfluidic approach for measuring the contractile force of platelet aggregates for the detection of platelet dysfunction. We find that platelet forces are significantly reduced when blood samples are treated with inhibitors of myosin, GPIb-IX-V, integrin αIIbß3, P2Y12, or thromboxane generation. Clinically, we find that platelet forces are measurably lower in cardiology patients taking aspirin. We also find that measuring platelet forces can identify Emergency Department trauma patients who subsequently require blood transfusions. Together, these findings indicate that microfluidic quantification of platelet forces may be a rapid and useful approach for monitoring both antiplatelet therapy and traumatic bleeding risk.

Micropatterning on micropost arrays.

Micropatterning of cells can be used in combination with microposts to control cell shape or cell-to-cell interaction while measuring cellular forces. The protocols in this chapter describe how to make SU8 masters for stamps and microposts, how to use soft lithography to replicate these structures in polydimethylsiloxane, and how to functionalize the surface of the microposts for cell attachment.