Contribution of Contractility to Platelet Biomechanical Functions in Haemostasis and Wound Healing

Re-organization of the actin cytoskeleton accompanies platelet activation and is a prerequisite for the development of platelet contractility. Defective platelet contractility can give rise to bleeding diathesis and increases the likelihood of thrombus embolization. Whether and if so so, how platelet contractility affects aspects of platelet function other than clot retraction is poorly understood. It is also unclear how physical cues of the local microenvironment at vascular injury sites, especially different adhesion proteins and the fibrous topography of the extracellular matrix inform functional responses through integrin-based substrate adhesions. These may contribute to the development of platelet traction forces, adhesion and spreading, but also to granule secretion, pro-coagulant activity, and ECM remodelling and thus, have significance for wound healing beyond haemostasis. By interrogating contractility at multiple stages of thrombus formation using a combination of myosin IIa inhibition, traction force microscopy, super-resolution microscopy, and in vitro assays, our data showed that impaired contractility affects traction force generation, actin bundling, vinculin morphology, GPVI-stimulated platelet aggregation, and single platelet contraction in aggregates formed by GPVI stimulation. Platelet adhesion to vWF under shear, and alpha granule secretion were independent of contractility. This data indicates that contractility contributes to platelet GPVI signalling, but doesn’t affect GPIb-mediated platelet adhesion. Further research is required to identify the downstream effectors involved in contractility-dependent GPVI signalling. Per-resolution microscopy complemented by biophysical methods revealed that adhesion protein identity directed platelet force generation and the dimensionality of formed fibronectin matrix. Platelets adhered to fibronectin developed high traction forces and pulled fibronectin fibrils along their apical membrane, while platelets on laminin developed lower traction forces and pulled fibrils along their basal membrane. Platelets assembled fibronectin nanofibrils using αIIbβ3 rather than α5β1 integrins. Our findings have potential implications for maintenance of mechanical thrombus integrity during remodeling and vascular repair. Using fibrinogen scaffolds of different topographies and quantitative microscopy methods, we observed that platelet interactions in the presence of plasma were greater on nanofibrous fibrinogen scaffolds compared to planar fibrinogen scaffolds. Our data show that nanofibers present a higher locally accessible surface area, effectively increasing the ligand density for integrin-mediated responses, which resulted in robust platelet adhesion and spreading, and alpha granule secretion while minimizing pro-coagulant activity.