Platelets play a major role in hemostasis and thrombosis, by binding to the underlying extracellular matrix around injured blood vessels, via integrin receptors. In this study, we investigated the effects of adhesive ligand spacing on the stability of platelets’ adhesion and the mode of their spreading on extracellular surfaces. Toward this end, we have examined the differential adhesion and spreading of human platelets onto nanogold-patterned surfaces, functionalized with the αIIbβ3 integrin ligand, SN528. Combining light- and scanning electron-microscopy, we found that interaction of platelets with surfaces coated with SN528 at spacing of 30–60 nm induces the extension of filopodia through which the platelets stably attach to the nanopatterned surface and spread on it. Increasing the nanopattern-gold spacing to 80–100 nm resulted in a dramatic reduction (>95%) in the number of adhering platelets. Surprisingly, a further increase in ligand spacing to 120 nm resulted in platelet binding to the surface at substantially larger numbers, yet these platelets remained discoid and were essentially devoid of filopodia and lamellipodia. These results indicate that the stimulation of filopodia extension by adhering platelets, and the consequent spreading on these surfaces depend on different ligand densities. Thus, the extension of filopodia occurs on surfaces with a ligand spacing of 100 nm or less, while the sustainability and growth of these initial adhesions and induction of extensive platelet adhesion and spreading requires lower ligand-to-ligand spacing (≤60 nm). The mechanisms underlying this differential ligand-density sensing by platelets, as well as the unexpected retention of discoid platelets on surfaces with even larger spacing (120 nm) are discussed.
The primary physiological function of blood platelets is to seal vascular lesions after injury and form hemostatic thrombi in order to prevent blood loss. This task relies on the formation of strong cellular-extracellular matrix interactions in the subendothelial lesions. The cytoskeleton of a platelet is key to all of its functions: its ability to spread, adhere and contract. Despite the medical significance of platelets, there is still no high-resolution structural information of their cytoskeleton. Here, we discuss and present 3-dimensional (3D) structural analysis of intact platelets by using cryoelectron tomography (cryo-ET) and atomic force microscopy (AFM). Cryo-ET provides in situ structural analysis and AFM gives stiffness maps of the platelets. In the future, combining highresolution structural and mechanical techniques will bring new understanding of how structural changes modulate platelet stiffness during activation and adhesion.
Both JAK2V617F and calreticulin (CALR) mutated essential thrombocythemia (ET) patients have different clinical characteristics, with lower thrombosis risk in patients with CALR mutations. To elucidate the mechanism for this lower risk we studied platelet function in ET patients with either JAK2V617F or a CALR mutation. Platelet activation state was similar in ET and healthy controls at baseline using P‐selectin and PAC1 flow cytometry analysis. However, CALR mutated platelets were significantly less activated following ADP stimulation, compared to control or JAK2 mutated platelets (P < .001). In live‐cell imaging of platelet attachment to immobilized fibrinogen by Interference Reflection Microscopy (IRM), the number of attached CALR mutated platelets was lower compared to control and JAK2 mutated platelets, with lower fractions of platelets achieving the fully spread state (90%, 78% and 54% of adherent cells for control, JAK2 and CALR mutated subjects, respectively). Compared to controls, ET patients, regardless of the mutation type, had increased numbers of immature platelets (IP) and leukocyte platelet aggregates (LPA), as well as plasma sP‐selectin. These were all correlated with the platelet count and not to the state of platelet activation. We also found that intracellular free Ca2+ was increased in resting ET compared to control platelets. Note, CALR had a more dispersed localization in activated ET platelets compared to healthy controls, and mutated CALR interact physically with TpoR in CALR mutated platelets. We hypothesize that defects in platelet activation and spreading in CALR mutated patients can explain, at least in part, the lower thrombotic tendency in CALR mutated ET patients.
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