A Comparative Medicine Study of Platelet Biophysics Among Hemostasis Models of Different Species
Authors: Copeland Renee , Oshinowo Oluwamayokun T. , Sakurai Yumiko , Fay Meredith Ellen , Petrich Brian G. , Brainard Benjamin , Lam Wilbur A , Myers David R. 
Published: October 2019

Abstract
Background: Animal models of hemostasis play a crucial role in understanding hemostasis and developing novel therapeutic treatments. Hemostasis is a mechanical phenomenon and recent studies have demonstrated that biophysical parameters, such as platelet margination (Walton et al. Blood 2017), thrombus porosity (Welsh et al. Blood 2014), shear forces (Nesbitt et al. Nat Med 2009), compression forces (Ju et al. Nat Comm 2018), and single platelet forces, as measured by platelet contraction cytometry, developed by our laboratory, (Myers et al. Nat Mat. 2017) affect hemostatic processes and may be pathologically altered in disease states. However, little is known about the most basic biophysical interspecies differences between platelets of various animal hemostasis models, which could provide critical insight into interpreting data and results. Here, we report the results of our initial study on platelet biophysics in humans (n=5), mice (n=8), pigs (n=5), and dogs (n=6), which are commonly used for hemostasis models due to anatomical similarities to human vessels, organs, and cell function (Siller-Matula et al. Thromb & Haem 2008) (Jagadeeswaran et al. Circ Res 2015). We focus on understanding differences in platelet adhesion on collagen and fibrinogen, spreading area, singe cell contraction forces, and bulk contraction, which all may influence the initiation and stability of blood clots (Fig. 1). Our data demonstrates striking differences between each species, especially concerning adhesion, spreading area, and single platelet force. Surprisingly, measurements of volumetric bulk contraction, the most commonly used biophysical assay in hemostasis research, appear to be quite consistent between species.
Methods: We performed a comprehensive study of platelet biophysics using adhesion assays, bulk contraction, and single platelet contractile forces on humans, mice, pigs, and dogs' platelets. For the adhesion assays, spreading area, and platelet contractile forces, platelets were gel-filtered as described previously (Myers et al. 2017). Cell Adhesion & Spreading Assay: Gel filtered platelets were diluted to 20M/mL, incubated on coverslips that had been incubated with type 1 collagen (100 µg/mL) or fibrinogen (100 µg/mL), allowed to adhere for 2 hours, counted, and measured. Platelet Contraction Cytometry: Polyacrylamide hydrogels with pairs of fibrinogen microdots are fabricated at physiologically relevant stiffnesses. Gel-filtered thrombin-activated platelets (1 U/mL) are then plated to attach, spread, and contract the microdots together. Individual platelet force is directly proportional to the microdot displacement measured with fluorescent imaging. Bulk Clot Contraction Assay: Platelets are washed as described previously (Lam et al. Nat Mat 2011) and then diluted to 200M/mL in a solution of fibrinogen (2 mg/mL), CaCl2 (5 mM), and thrombin (1 U/mL). Clots are left to form in cuvettes lined with gridded paper and measured every 30 mins for 2.5 hours. All assays were performed on at least 3 subjects from each species and in triplicate when possible.
Results: We found both significant biophysical differences and similarities between platelets of different species (Fig. 2). Overall, murine platelets have the highest biophysical similarity to human platelets regarding adhesion, volumetric contraction, and single platelet contractile force despite substantial size differences. Porcine platelets exhibited enhanced adhesion on collagen with little adhesion to fibrinogen; while canine platelets showed the opposite. Such differences could have significant implications on the biophysical initiation of blood hemostasis and thrombosis, as well as the thrombogenicity tests of new materials. In bulk and single platelet contraction, we noted that similarities between most species except for canine platelets which produced forces nearly triple that of humans, the highest contractile forces of any platelets measured to date. As recent work has shown that forces are linked to clot porosity and therefore activation (Mirramezani et al. JTH 2018), such high forces could influence the clot hierarchical structure and subsequent lysis. Taken together, these results demonstrate how the biophysical properties of platelets differ across species and may be another reason why animal studies of hemostasis do not always translate to humans.

Disclosures
No relevant conflicts of interest to declare.