Platelet Contraction Cytometry: A Biophysical, High-Throughput Research-Enabling and Diagnostic Platform

Background : During blood clot formation, platelets undergo muscle-like contraction with nascent fibrin polymers to mechanically stabilize the clot and stem hemorrhage, and pathological alterations in this biophysical process are associated with bleeding and thrombotic diseases (Collet, et al. 2006) (Hvas,et al. 2007). However, how platelets integrate the myriad of biochemical and biophysical microenvironmental signals to actuate contraction is poorly understood. Bulk assays are insufficient for these studies as individual platelets exhibit highly variable behavior that depends on the biochemical and mechanical microenvironment. To that end, we developed a platelet contraction cytometer (Fig 1) that allows for the high-throughput measurement of platelet contraction force at the single cell level in a variety of microenvironments. With this system, we demonstrated that the biochemical and mechanical cues of the platelet microenvironment synergistically mediate contraction force, and observed associations between low contraction forces and symptomatic bleeding in various platelet disorders, suggesting that the biophysical force of individual platelets may function as a clinical diagnostic biophysical biomarker (Myers, et al. 2017). Here, we demonstrate that our versatile system can be leveraged to understand platelet contraction in several new contexts including immune thrombocytopenia (ITP), animal models of hemostasis, and platelet mitochondrial function.

Platelet Contraction Cytometer Operation : Fibrinogen microdot pairs are patterned on the surface of polyacrylamide (PAA) hydrogels with a known stiffness. Thrombin-activated platelets adhere to a microdot, spread to the neighboring microdot, and contract, pulling the microdots closer together (Fig 1a). Using confocal microscopy, only microdot displacement is needed to calculate the contraction force.Each device features parallel arrays of PAA gels at varying stiffnesses with integrated microfluidics that enable precise control of the mechanical, biochemical, and shear microenvironment above the platelets (Fig 1b-c).

Results: A potential clinical application for our platelet contraction cytometer is the management of ITP, a disorder in which autoimmune destruction of platelets leads to risk of life-threatening hemorrhage. As bleeding risk and platelet count have only proven to be loosely correlated with some reports stating that ITP platelets may even be "hyperactive", a clinical need exists for a laboratory test to determine which patients are at risk for life-threatening hemorrhage and require immediate treatment and which require only monitoring. We observed that platelet contraction forces of 3 out of 4 ITP patients were similar to those of healthy controls, even in patients with platelet counts <10,000/uL. However, platelets from one patient who presented with active bruising and a platelet count of 30,000/uL exhibited impaired platelet contractility (Fig 2a).

Separately, while many animal models are used to study hemostasis and thrombosis, the relevance of data obtained with those models to human health is often questioned due to interspecies differences (Jagadeeswaran, et al. 2015). Accordingly, we adapted our system to examine potential biophysical differences in platelet contraction force between different species. Our initial studies show that hound dog platelets apply much more force than human platelets (Fig 2b), suggesting that the biophysical behavior of clots may dramatically differ between species.

Finally, we are exploring potential mechanisms for the high variability of platelet contractile force in identical mechanical and biochemical environments. Since the mitochondria of platelets have high inter- and intra-patient variability (Fig 2c), we hypothesized and confirmed that the mitochondrial number correlates with contractile force of individual platelets (Fig 2d). Here, mitochondrial fluorescence intensity from MitoTracker Red CMXRos acts as a surrogate for mitochondrial number. Moreover, since mitochondrial dysfunction in platelets is associated with various inflammatory diseases, such as asthma and sickle cell disease (Tomasiak-Lozowska, et al. 2016) (Cardenes, et al. 2014), more studies on mitochondrial function in the context of biophysical contraction may have important pathophysiologic and diagnostic implications.

Figure 1

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Figure 1

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