Microenvironmental Geometry Guides Platelet Adhesion and Spreading: A Quantitative Analysis at the Single Cell Level

Ashley Kita,Yumiko Sakurai,James N Huang,Ross Rounsevell,David R Myers,Daniel A Fletcher,Wilbur A Lam,Tae Joon Seok,Ming C Wu,Kyoungsik Yu,Daniel J Muller

doi:10.1371/journal.pone.0026437

Abstract

To activate clot formation and maintain hemostasis, platelets adhere and spread onto sites of vascular injury. Although this process is well-characterized biochemically, how the physical and spatial cues in the microenvironment affect platelet adhesion and spreading remain unclear. In this study, we applied deep UV photolithography and protein micro/nanostamping to quantitatively investigate and characterize the spatial guidance of platelet spreading at the single cell level and with nanoscale resolution. Platelets adhered to and spread only onto micropatterned collagen or fibrinogen surfaces and followed the microenvironmental geometry with high fidelity and with single micron precision. Using micropatterned lines of different widths, we determined that platelets are able to conform to micropatterned stripes as thin as 0.6 µm and adopt a maximum aspect ratio of 19 on those protein patterns. Interestingly, platelets were also able to span and spread over non-patterned regions of up to 5 µm, a length consistent with that of maximally extended filopodia. This process appears to be mediated by platelet filopodia that are sensitive to spatial cues. Finally, we observed that microenvironmental geometry directly affects platelet biology, such as the spatial organization and distribution of the platelet actin cytoskeleton. Our data demonstrate that platelet spreading is a finely-tuned and spatially-guided process in which spatial cues directly influence the biological aspects of how clot formation is regulated.

Highlights

At sites of vascular injury, platelets initiate the formation of the hemostatic plug
The platelets followed the geometric boundaries of the micropatterns with high fidelity and took on the morphology of the protein micropatterns themselves
Our study of platelet spreading on protein micro/nanopatterns shows that platelets regulate their spreading and morphology in response to geometrical constraints of the microenvironment, which is consistent with recent research [5]

Summary

Introduction

At sites of vascular injury, platelets initiate the formation of the hemostatic plug. Microcontact printing techniques have recently been applied to platelet adhesion [5,6], but there currently is no published data that systemically characterizes and quantifies, at the single platelet level, how the microenvironmental geometry influences platelet spreading, and what the physical limitations and constraints are. This concept of physical and microenvironmental control of platelet function is important to our understanding of platelet physiology and clot formation. To quantitatively investigate how single platelets guide spreading based on the geometry of their microenvironment, we combined standard platelet adhesion techniques with deep UV photolithography and micro/nanocontact protein printing using fibrinogen and collagen, which mediate platelet adhesion at sites of vascular injury [8,9]

Methods

Results

Conclusion