(Invited) High-Throughput Single Cell Nanomechanical Measurements

Abstract

Microsystems have the potential to make an enormous contribution to biomedical and clinical settings. Fully realizing the capabilities of this established technology lies in designing new robust microsystems capable of answering clinically relevant problems. Here, I discuss the creation of the platelet contraction cytometer, a tool that has led to important insights into our understanding of the biomechanical process of clotting and may even represent a new type of diagnostic based on biophysics. Platelets constantly circulate in the bloodstream and respond to injury to prevent excessive blood loss. At a wound, platelets will aggregate on exposed collagen and undergo muscle-like contraction with nascent fibrin polymers to mechanically stabilize the clot and stem hemorrhage. During clot contraction, platelets can dramatically shrink the fibrin mesh and increase the clot stiffness by an order of magnitude. Previous studies have established that the clot stiffness is linked to disease, as overly softened and stiffened clots are associated with bleeding and thrombotic disorders, respectively. However, defining the reasons that clot mechanical properties change in disease has remained difficult, as clots are inherently complex and heterogeneous, and all existing bulk measurements depend on contributions from fibrin stiffness, fibrin architecture, red blood cell incorporation, and platelet contraction. Moreover, changes to the microenvironmental stiffness, biochemical concentration, and shear are known to alter platelet force or behavior at the single cell level. To that end, we developed a “platelet contraction cytometer (PCC)” that simultaneously measures the contractile force of hundreds of individual adherent platelets across the physiological range of mechanical microenvironments within a clot. Within the PCC, a single platelet attaches, spreads, and applies contractile force to a pair of fibrinogen microdots that are attached to a moveable, spring-like, surface. Since the applied platelet contractile force is directly proportional to the microdot displacement, the force may be calculated from a single fluorescent image of the platelet. As this device is microfabricated, thousands of microdots may be created on a single device to enable high-throughput measurements that are performed in a highly controlled mechanical, biochemical, and shear environment. Using this device, we have defined the range of forces that platelets apply within a clot and shown that mechanical and biochemical cues can synergistically combine to create highly contractile platelets. Moreover, we have been able to probe the pathways involved in contraction and shown which pathways control how platelets apply force in response to local stiffness. This device has even enabled new comparative studies that examine how platelet force varies between humans and other animal species, and may lead to new biological insights into how contractile force is generated and regulated. In addition to enabling mechanistic studies, we have collected data showing that platelet contraction cytometry may represent a new class diagnostic that is based on biophysics. In particular, our studies have focused on immune thrombocytopenia (ITP), a disease which is characterized by low platelet counts in the absence of any other cause. Approximately 10% of these patients will have major bleeding issues, but no existing biomarker can determine which patients are at risk for bleeding and whether the disease will self-resolve or become chronic. In a small cohort of patients, we have found that all patients with bleeding and/or bruising symptoms also have significantly lower platelet contractile forces. By defining an average force cutoff value of 26nN, we found that low forces identified bleeding in ITP with 100% sensitivity and 89.4% specificity. Importantly, since the PCC measures single platelets in high throughput, it also provides information on the distribution of forces applied by platelets and has helped to identify unique subpopulations of platelets in ITP. Healthy individuals have a normal distribution of contractile forces with a single peak, while ITP patients tend to have a bimodal distribution with two prominent peaks, one with a lower contractile force and one with a higher contractile force. Surprisingly, patients with bleeding symptoms are missing the highly contractile peak and suggests that the lack of highly contractile platelets may contribute to bleeding. Taken together, this work highlights the incredible potential for microsystems in solving clinical challenges. Designing a device capable of measuring single platelet forces led to new insights into the basic biophysics of platelets and clotting and may also represent a new type of diagnostic.