(E-194) Distinct platelet F-actin patterns and traction forces on von Willebrand Factor versus fibrinogen

Introduction:: The first step in the repair of vascular injury is the formation of a hemostatic platelet plug that begins to form as platelets bind to the subendothelium and subsequently activate. Inactive platelets are usually round and about 2-5 μm in diameter and can spread up to 5 times that diameter when on a flat surface. The F-actin and myosin in platelets come together to form actomyosin which produces contractile forces that are thought to be imperative for blood clotting. The magnitude of these forces vary depending on the environment. Two main blood proteins that mediate platelet adhesion are (1) fibrinogen (FBN) that meditates platelet-to-platelet adhesion and (2) von Willebrand Factor (VWF) that mediates platelet-to-platelet and platelet-to-matrix adhesion. On FBN, F-actin morphology is characterized by punctate nodules early in activation that develop into filament networks that line the periphery. On VWF, F-actin morphology and contractile forces have not been explored as thoroughly as FBN. Previously, high F-actin intensity and distribution was correlated with high platelet forces. However, to our knowledge, there has been no detailed characterization of F-actin morphology and single-platelet forces on both FBN and VWF. In this investigation, we demonstrate that the F-actin morphology and force exerted by platelets varies depending on the matrix protein they are seeded on.


Materials and Methods:: Platelets were isolated from whole blood donated by informed and consenting healthy human subjects via density gradient centrifugation. FBN or VWF were seeded at physiological concentrations (1 mg/mL and 5 μg/mL respectively) on ethanol-cleaned glass coverslips or fluorescent PDMS substrates patterned with an orthogonal array of non-fluorescent circles 1 μm in diameter and 2 μm apart center-to-center (black dots). The surfaces were blocked with 1% BSA. Isolated platelets were seeded on top of these surfaces at physiological concentration and incubated for 10 minutes. Excess platelets were washed off and remaining platelets on the surface were allowed to spread for an additional 30 or 80 minutes in Tyrode buffer. Platelets were fixed using 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, and blocked with 10% goat serum. F-actin was stained using phalloidin-Alexafluor 488 and glycoprotein Ib with CD42b monoclonal antibody and an Alexa 647 goat anti-mouse IgG secondary antibody. The surfaces were mounted on glass slides and imaged using confocal microscopy. The morphology of each platelet’s individual F-actin structure was classified as nodular (punctate nodules), hollow (localized at the platelet’s periphery), solid (homogeneous over the whole area), or indeterminate using a convolutional neural network trained with 1597 manually classified images of platelets over 200 epochs and fine-tuned over an additional 200 epochs. Black dot displacement was calculated using Fourier Transform Traction Cytometry. The contraction force of a single platelet was the sum of the force calculated from the displacement of each dot under each platelet. Subcellular force distribution was also determined.




Results, Conclusions, and Discussions::
The morphology of spread platelets on glass coverslips was different when coated with FBN versus VWF. Of the platelets seeded on glass coverslips coated with (1) FBN: 54% were hollow, 24% nodular, 15% solid, and 3% indeterminate, and (2) VWF: 2% were hollow, 0% nodular, 95% solid, and 3% indeterminate. On black dots surfaces coated with (1) FBN, 52% were hollow, 40% nodular, 3% solid, and 5% indeterminate, and (2) VWF: 7.5% were hollow, 25% nodular, 74% solid, and 16% indeterminate. A Pearson’s Chi-squared test with p < 0.0001 indicated that the differences in these frequencies were significant for both glass coverslips and PDMS black dot surfaces. Single-platelet forces produced on VWF coated black dot surfaces were significantly greater than on FBN. Furthermore, the difference in force exerted by platelets with hollow morphology were significantly greater than those with a nodular morphology on FBN (p = 0.036). The traction force exerted by platelets with solid morphology on VWF was significantly greater than platelets with hollow morphology on FBN (p = 0.0296). Hollow platelets on VWF also exerted a significantly greater force than hollow platelets on FBN (p < 0.001). Additionally, indeterminate platelets on VWF produced more traction forces than indeterminate platelets on FBN (p < 0.0001). Significant difference was determined with an ANOVA and Tukey’s post hoc test. Subsequent analysis revealed that subcellular forces on VWF covered surfaces were more centrally localized than on FBN (p < 0.0001) and that subcellular forces on FBN covered surfaces were more peripherally localized than on VWF (p < 0.0001). Significant difference was determined with an ANOVA and Tukey’s post hoc test. These results indicate that forces are both protein- and F-actin pattern dependent.






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