Abstract
Background: Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing. Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. Methods: We combined live cell imaging, quantitative interference reflection microscopy and cryo-electron tomography to characterize, at a single platelet level, the differential spatiotemporal dynamics of the adhesion process to fibrinogen- and collagen IV-functionalized surfaces. Results: Initially, platelets sense both substrates by transient rapid extensions of filopodia. On collagen IV, a short-term phase of filopodial extension is followed by lamellipodia-based spreading. This transition is preceded by the extension of a single or couple of microtubules into the platelet's periphery and their apparent insertion into the core of the filopodia. On fibrinogen surfaces, the filopodia-to-lamellipodia transition was partial and microtubule extension was not observed leading to limited spreading, which could be restored by manganese or thrombin. Conclusions: Based on these results, we propose that interaction with collagen IV stimulate platelets to extend microtubules to peripheral filopodia, which in turn, enhances filopodial-to-lamellipodial transition and overall lamellipodia-based spreading. Fibrinogen, on the other hand, fails to induce these early microtubule extensions, leading to full lamellipodia spreading in only a fraction of the seeded platelets. We further suggest that activation of integrin αIIbβ3is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen.
Highlights
Mammalian platelets are small, anucleated megakaryocytes fragments that circulate in the bloodstream as single, smoothsurfaced discoid structures
The delayed lamellipodial spreading on collagen IV was commonly preceded by an extension of a single or a couple of microtubules from the circumferential marginal band to the platelet periphery, that penetrated into the core of peripheral filopodia. These results suggest that microtubule extension to the periphery of collagen IV-adherent platelets might trigger filopodia-to-lamellipodia transition, and promote platelet spreading
We confirmed that some platelets extended filopodial extensions while being motile on collagen IV- or on fibrinogen-coated surfaces with chemical fixation and phase contrast microscopy imaging (Figure 1B–C, arrows)
Summary
Mammalian platelets are small, anucleated megakaryocytes fragments that circulate in the bloodstream as single, smoothsurfaced discoid structures. In addition to the molecular composition of the environment, its physical properties, such as rigidity, topography and shear forces generated by the blood flow, influence the platelets adhesion and spreading process These molecular interactions of the platelets with the microenvironment are governed by specific adhesion receptors, such as αIIbβ integrin, which bind to fibrinogen and participated in the formation of the fibrin clot, and α2β1 and GP-VI, that mediate platelet adhesion to collagen IV5–9. Upon wound formation, platelets adhere to the neighboring extracellular matrix and spread on it, a process which is critical for physiological wound healing Multiple external factors, such as the molecular composition of the environment and its mechanical properties, play a key role in this process and direct its speed and outcome. We further suggest that activation of integrin αIIbβ is essential for filopodial-to-lamellipodial transition, based on the capacity of integrin activators to enhance lamellipodia spreading on fibrinogen
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