A Few Immobilized Thrombins Are Sufficient for Platelet Spreading

Abstract

Eukaryotic cells respond to signaling molecules with picomolar to nanomolar sensitivities. However, molar concentrations give no suggestion of the sufficient number of molecules per cell and are confusing when referring to physiological situations in which signaling molecules act in an immobilized state. Here, we studied platelet adhesion by thrombin, a key step in normal hemostasis and pathological arterial thrombosis. We generated a biofunctional nanosheet surface to mimic the in vivo solid-state interaction between platelets and thrombin at sites of injured tissues. We observed that <10 molecules readily activate platelets with high specificity, resulting in platelet adhesion and spreading. This number is much lower than expected from previous experiments in solution, in which the sole activation of platelets required a >1000-fold stoichiometric excess of thrombin. We conclude that immobilizing thrombin apposed to the membrane receptor allows platelets to respond with very high sensitivity. Moreover, we propose that irreversible cell activation may require several ligands to avoid activation by single, mislocalized signaling molecules.

Multicolor Fluorescence Nanoscopy by Photobleaching: Concept, Verification, and Its Application To Resolve Selective Storage of Proteins in Platelets

“…13 Due to their highly sensitive nature, platelets can be activated by many different molecules resulting in morphological changes followed by release or uptake of specific proteins. 14,15,16,17,18 Thereby platelets can release certain proteins where they are needed, such as fibrinogen at the location of a wound 19 or growth proteins such as VEGF at the location of rapid cell division. 20 Details of how proteins are stored in platelets thus carry a wealth of information regarding the status of our bodies and can potentially provide diagnostic indicators of different states and diseases, including cancer.…”

The role of fibrinogen spacing and patch size on platelet adhesion under flow

“…Likewise Kita et al,[16] used microcontact printing to generate micron sized lines of fibrinogen to demonstrate the effect of ligand patch geometry on platelet spreading at the single cell level, and the ability of platelets to extend filopodia to bridge and spread between fibrinogen patches. Finally Okamura et al,[17] developed a thrombin-coated nanosheet surface to show that < 10 thrombin molecules were sufficient to cause platelet adhesion and spreading.…”

Two-color nanoscopy of three-dimensional volumes by 4Pi detection of stochastically switched fluorophores

“…To explore the capacity of 4Pi-SMS microscopy for unambiguous imaging in samples thicker than λ/2, we first imaged the distribution of fibrinogen receptors on human platelets, placed onto coverslips carrying a thrombin-coated nanosheet 23 , which form clusters upon platelet activation 24 . We visualized the glycoprotein IIb/IIIa (GPIIb/IIIa) receptor complex by staining GPIIb using the photochromic Rhodamine S and the fluorescent dye Atto 532.…”

“…Platelets in HEPES-tyrode buffer were placed onto coverslips carrying a thrombin-coated nanosheet 23,33 (coating was performed with 10 U ml −1 thrombin overnight at 4 °C) and incubated for 10 min at 37 °C. After incubation, the non-adherent platelets were washed off.…”