“Self-Deposition” of Matrix Proteins from Platelet α-Granules Enable Extended Adhesion and Spreading on Micron/Submicron-Scale Fibrinogen and Collagen Substrates.

Introduction: While the platelet’s role in achieving hemostasis in the context of bleeding is well-characterized, how platelets support and maintain vascular integrity during homeostatic, non-thrombotic conditions remains unclear. Previous studies have shown that single platelets fill micron/submicron-sized “gaps” of small discontinuities in the endothelium. In this context, however, as the overall force of adhesion correlates with the area of exposed subendothelial matrix, how platelets establish sufficient adhesion to resist the dynamic shear forces of the circulation are unknown. In this study, we investigate, using protein microcontact printing, how platelets sense the geometry of the matrix micro/nano-environment and transduce those cues to affect platelet function. Our results reveal that platelets, upon sensing spatially-limited (<25 µm² area) micro/nanopatterns of fibrinogen or collagen, spatially regulate actin-rich filopodia extension, via Rac1, beyond the geometric constraints of the matrix pattern, redistribute α-granules to enable “self-deposition” of fibrinogen/fibronectin matrix on those regions, and spread onto those newly deposited matrix via integrin α_IIbβ₃-mediated interactions. Furthermore, this phenomenon is markedly attenuated in Gray Platelet Syndrome platelets, which lack α-granules, and Wiskott-Aldrich Syndrome platelets, which have impaired integrin α_IIbβ₃-mediated activation, decreased α-granules, and cytoskeletal defects.

Materials and Methods: Human blood was collected in ACD and platelets were isolated via two-step centrifugation or gel filtration. Platelets were suspended in Tyrode’s buffer containing 2 mM MgCl₂ and exposed to various pharmacologic agents such as eptifibatide, Y27632, NSC23766. Fibrinogen and collagen micro/nanostamps were prepared on glass coverslips after which platelets were incubated onto. A plasma membrane dye, phalloidin, anti-P-selectin, anti-fibronectin and PAC-1, were used to visualize platelet morphology, f-actin, α-granule secretion, matrix depositions, and activated integrin α_IIbβ₃, respectively.

Results and Discussion: Upon contact with the protein microstamps, platelets formed filopodia and lamellipodia and spread to conform to the micropattern boundaries with high fidelity. However, on micropatterned circles (microdots) of less than 25 µm² area, platelets spread beyond the geometric constraints (Fig.1A). Anti P-selectin staining revealed α-granule secretion at the extended lamellipodia (Fig.1B left), and interestingly, this phenomenon was not detected with GPS platelets. Also we observed direct deposition of fibronectin from the platelets granules via anti-fibronectin staining (Fig.1B center). In addition, immunostaining with PAC-1 revealed a predominance of activated α_IIbβ₃ on regions of platelets at and beyond the borders of the microdots (Fig.1B right). When exposed to eptifibatide, an α_IIbβ₃ antagonist, platelet spreading beyond the microdot boundaries was completely eliminated. Various cytoskeleton inhibition experiments provided insight into how actin rearrangement is involved in this processes of platelet extension beyond matrix boundaries. Interestingly, disruption of cytoplasmic stress fibers by inhibiting Rho kinase with Y25632 facilitated platelet extension beyond the microdot boundaries and strong actin polymerization at the margins. On the other hand, platelet extension beyond the microdot boundaries was completely inhibited with NSC23766, a Rac-1 inhibitor (Fig.1C). We also confirmed that WAS platelets had significantly less f-actin, α-granule secretion, and overall extension beyond microdot boundaries. Overall, our results indicate that the matrix geometry, in and of itself, dictates platelet α-granule secretion, α_IIbβ₃ distribution and activation, and Rac1 activation, all of which work in concert to extend adhesion and spreading on matrix substrates of <25 µm². In addition, we show that during this process, directionally directed α-granule secretion enables a platelet to “self-deposit” fibrinogen/fibronectin matrix to accommodate increased spreading. This phenomenon likely endows platelets a certain amount of “spatial flexibility” to establish firm adhesion upon contact with small sites of exposed subendothelium and may provide insight into how platelets help support vascular integrity.

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