Distinct platelet F-actin patterns and traction forces on von Willebrand factor versus fibrinogen

Upon vascular injury, platelets form a hemostatic plug by binding to the subendothelium and to each other. Platelet-to-matrix binding is initially mediated by von Willebrand factor (VWF) and platelet-to-platelet binding is mediated mainly by fibrinogen and VWF. After binding, the actin cytoskeleton of a platelet drives its contraction, generating traction forces that are important to the cessation of bleeding. Our understanding of the relationship between adhesive environment, F-actin morphology, and traction forces is limited. Here, we examined F-actin morphology of platelets attached to surfaces coated with fibrinogen and VWF. We identified distinct F-actin patterns induced by these protein coatings and found that these patterns were identifiable into three classifications via machine learning: solid, nodular, and hollow. We observed that traction forces for platelets were significantly higher on VWF than on fibrinogen coatings and these forces varied by F-actin pattern. In addition, we analyzed the F-actin orientation in platelets and noted that their filaments were more circumferential when on fibrinogen coatings and having a hollow F-actin pattern, while they were more radial on VWF and having a solid F-actin pattern. Finally, we noted that subcellular localization of traction forces corresponded to protein coating and F-actin pattern: VWF-bound, solid platelets had higher forces at their central region while fibrinogen-bound, hollow platelets had higher forces at their periphery. These distinct F-actin patterns on fibrinogen and VWF and their differences in F-actin orientation, force magnitude, and force localization could have implications in hemostasis, thrombus architecture, and venous versus arterial thrombosis.SIGNIFICANCE Impaired platelet contraction is associated with bleeding and enhanced platelet contraction may be associated with thrombosis, implicating platelet traction forces as an important component of hemostasis. F-actin drives the generation of traction forces, but a direct connection between F-actin structure and force localization has been limited by methodologies that do not permit the simultaneous measurement of subcellular forces and immunofluorescent stains. Here, we used our recently developed black dots technique to connect adhesive environment, platelet F-actin pattern, F-actin filament orientation, force magnitude, and force subcellular localization. Relationships between protein stimuli and F-actin structure and function may be important in hemostasis, thrombosis, and thrombus architecture.