The Behavior of Blood Platelets in the Context of Inflammation

Abstract

Platelets are known as central regulators of hemostasis and thrombosis. More recently, they have been revealed to play an active role in inflammation and immune defense. Beyond biochemical signaling, platelet function is strongly influenced by biomechanical cues, including substrate stiffness, thrombus structure and cytoskeletal organization. However, how these biochemical and biomechanical signals influence platelet behavior in an inflammatory context remains poorly understood. In this thesis, platelet behavior in the context of inflammation was investigated using high-resolution scanning-ion conductance microscopy (SICM), fluorescence microscopy, deep-learning-supported tracking, and biomechanical quantification. The work illustrates platelet behavior in inflammation in three distinct areas: the influence of (oxidized) lipids on platelet cellular stiffness and actin architecture, the mechanosensitive regulation of platelet migration by substrate stiffness, and the three-dimensional structure of thrombi regulated by cyclic guanosine monophosphate (cGMP). A short summary of the results from each area is given in each of the following paragraphs. An increased blood content of (oxidized) lipids is often a precursor of cardiovascular diseases, but the influence of those lipids on platelet biomechanics is poorly understood. Platelet biomechanics and morphology were investigated in the context of (oxidized) lipid exposure and atypical chemokine receptor 3 (ACKR3) signaling using SICM stiffness mapping. Pharmacological activation of ACKR3 significantly reduced platelet Young’s modulus. In addition, oxidized low-density lipoprotein (OxLDL), but not native LDL, softened platelet Young’s modulus. ACKR3 activation did not reverse platelet softening induced by OxLDL. Fluorescence microscopy revealed a distinct change in adhered platelet morphology and actin network structure upon (Ox)LDL addition, including filopodia formation and actin nodules. These results demonstrate that lipid uptake heavily influences platelet stiffness and illustrates platelet Young’s modulus as a biophysical marker of platelet activation under hyperlipidemic conditions. Inflamed tissue changes its stiffness. As platelet migration is an important part of the immune defense and plays a significant role in inflammation, the influence of this change in substrate stiffness on platelet haptotactic migration was investigated. Platelet haptotactic migration was tracked on three fibrinogen-coated polydimethylsiloxane (PDMS)-based substrates with defined Young’s moduli using light microscopy and deep-learning-based image analysis. Platelets migrated efficiently on stiff substrates, but migration speed and number of migrating platelets was significantly diminished on softer substrates. Platelet three-dimensional shape analysis revealed that platelets on soft substrates failed to polarize. The shape anisotropy typical for migrating platelets was also significantly reduced. Fluorescence microscopy of fluorescent fibrinogen revealed that platelets create a distinct fibrinogen gradient during migration, with the gradient generated by platelets migrating on soft substrate being significantly reduced. These findings demonstrate a dependency between the substrate stiffness and the ability of platelet to haptotactically migrate. If inflammation of the blood vessel wall is unimpeded, a vessel wall rupture and resulting thrombosis is inevitable. To regulate this build-up of a thrombus after vessel wall rupture, the body uses a nitric oxide (NO) and cGMP-based signaling cascade to inhibit platelet activation. The influence of this signaling cascade on thrombus architecture was investigated. Three-dimensional SICM imaging revealed that pharmacological stimulation of NO-sensitive soluble guanylyl cyclase (NO-GC) with riociguat and NO-donors reduced thrombus volume, without fully disrupting hemostasis. In addition, thrombus surface roughness was significantly increased, hinting at a reduced capacity for clot contraction. These results highlight the regulatory effect of the cGMP-based signal cascade on thrombus stability while preserving physiological hemostasis at clinically relevant levels.