Platelet subsets and priming: functional determinants of haemostasis and vascular protection (LSBR 1711)
Project leader: Prof. Dr. Johan W.M. Heemskerk / Paola J.E. van der Meijden (Biochemistry CARIM Maastricht University)
PhD student: Alicia Veninga (May 2018 – November 2022)
Technician: Stella Thomassen (January 2019 – January 2021 (0,4 fte))
Technician: Simone Wielders (January 2021 – November 2022 (0,4 fte))
Platelets are essential players in thrombosis and haemostasis. However, not all platelets are exact copies of each other, and distinct platelets in a subject can respond differently to the same inhibitors or activators. This results in the formation of different populations of activated platelets. The overall aims of this project were to further characterise the various platelet populations using multicolour flow cytometry and to provide new insights into the mechanisms how intrinsic and extrinsic factors influence platelet heterogeneity and functioning.
The expansion of platelet progenitor cells in the bone marrow due to somatic mutations will lead to an additional form of platelet heterogeneity. This process is referred to as clonal hematopoiesis of indeterminate potential (CHIP). Based on existing literature, we identified mutations in seven CHIP-related genes that are linked to an elevated platelet count and increased risk of thrombosis, of which only three mutations are related to a hyperreactive platelet phenotype. Also, we identified nine CHIP-related genes that associated with a reduced platelet count and an increased bleeding risk.
We introduced a multicolour flow cytometry tool with automated cell clustering analysis to allow improved identification and characterisation of platelet populations following priming and activation. Addition of the primers did not change the distribution of most of the populations, but it affected the fractions of resting and fully activated platelets. Regardless of stimulus strength or presence of priming substances, a common phenotype was observed in a subset of the platelets, suggesting a pre-programmed activation response that differs between platelet subsets.
We assessed platelet reactivity in size-separated platelet fractions from healthy individuals. Using our multicolour flow cytometry tool, we were able to link high glycoprotein VI (GPVI) expression to the highly reactive, juvenile platelet population, which was enriched in the large platelet fraction. This finding was confirmed by using ageing platelet concentrates and platelet samples from immune thrombocytopaenia patients.
Next, we provided an in-depth investigation of the antiplatelet properties of a class of anticancer therapeutics, commonly referred to as tyrosine kinase inhibitors (TKIs). As expected, we found the strongest platelet inhibiting effects for those TKIs with high affinities for molecular targets present in platelets. Interestingly, in vitro treatment of platelets with TKIs reduced the size of the fully activated platelet population upon stimulation but did not change the distribution profiles of the partly activated populations.
The environment of circulating platelets can be altered in certain disease states, which can affect platelet function and populations. We performed a pilot study to investigate this in a small cohort of patients with cerebral small vessel disease, who are at increased risk of ischaemic or hemorrhagic stroke. Despite the use of antiplatelet drugs in the investigated patients, thrombus formation was found to be similar in the patients and healthy controls. This suggested a high on-treatment platelet reactivity, possibly due to positive priming. Clustering analysis of the multicolour flow cytometric data indicated a higher abundancy of an activated platelet population with shedding of receptors, when compared to healthy controls. We also found evidence for an inflammatory response in the patients.
Vascular injury or atherosclerotic plaque erosion can result in VSMC exposure and platelet contact. We examined how VSMC phenotypes affected platelets under flow conditions. By assessing whole-blood thrombus and fibrin formation, we revealed that more thrombi and fibrin were formed on the synthetic VSMC, when compared to the contractile VSMC, and we identified several interaction mechanisms between platelets and VSMC. On the other hand, platelet exposure to endothelial cells provides an acute and long-term persistent inhibitory effect on functional responses. These changes are reflected by defined profiles of phosphorylation changes in platelet proteins.
By optimising the tools to distinguish and investigate platelet heterogeneity and by extending our knowledge, this project has provided evidence for the existence of preferentially aggregating, secreting, and shedding platelets. We foresee that more knowledge of these phenotypes will provide novel biomarker or diagnostic information, and likely also can assist in optimising the treatment efficacy of antiplatelet drugs.