Biochemistry of the vascular and respiratory system

Platelet biochemistry

Team members: Mauro Torti (full professor), Ilaria Canobbio (assistant professor), Gianni Guidetti (assistant professor), Caterina Visconte (PhD student), Marta Zarà (PhD student).

Research activity encompasses fundamental aspects of transmembrane signalling in platelets. Circulating blood platelets have a crucial role in haemostasis, thrombosis, and in many other processes, such as inflammation, neurodegeneration, and cancer metastasis. Understanding the exact contribution of platelets to these events is a big challenge for the future and may provide promising opportunities in biomedicine.

Analysis of platelet signal transduction pathways in thrombosis and haemostasis

The understanding of the mechanisms supporting platelet activation is critical for the identification of novel pharmacological targets for thrombotic diseases, which represent a major cause of death in western countries. In this context, our research is focused on the following aspects:

role of tyrosine kinases (Src, Syk, Pyk2, FAK) and protein phosphorylation in platelet adhesion, activation and aggregation

contribution of different phosphatidylinositol 3-kinase (PI3K) isoforms in platelet activation and analysis of their regulation

signal transduction pathways downstream of integrins, G protein-coupled receptors and ITAM-bearing receptors

mechanisms of platelet activation induced by environmental and engineered nanoparticles

 

Analysis of amyloid precursor protein (APP) metabolism and function in platelets: a novel link between hemostasis, thrombosis and Alzheimer’s disease

Alzheimer’s disease (AD) is the most invalidating dementia in the elderly and is characterized by the accumulation of amyloid peptides in brain parenchyma and cerebral vessel walls. Amyloid peptides derive from the metabolism of amyloid precursor protein APP. Platelets express high level of APP and release amyloid peptides in the circulation upon stimulation. Our research is mainly focused on:

define the effects of amyloid peptides in plasma. We have demonstrated that amyloid peptides are able to activate platelets and to promote a chronic inflammation state. A collaboration with Pula Lab, University of Bath (UK) is ongoing to study the possible effects of amyloid peptides on endothelial cell activation

define the role of platelet-derived amyloid peptides in AD onset and progression. We will use transgenic mice that overexpress APP and amyloid peptides (3xTgAD) and will evaluate the level of platelet activation and chronic inflammation. In addition, we will analyse the ability of platelet-derived amyloid peptides to cross the brain blood barrier and deposit in the brain

investigate the role of APP in platelet physiology and hemostasis. Using platelets isolated from APPKO mice (that do not express APP) and we have demonstrated that APP plays a crucial role in platelet adhesion and thrombus formation.

 

Analysis of the contribution of platelets and platelet-derived microparticles to cancer metastasis

Metastasis is strongly influenced by host-cancer interactions and it has been demonstrated that platelets are key players in cancer spread as their depletion, in thrombocytopenic patients or animal models, is associated to reduced metastasis. This suggests that also the pharmacological inhibition of platelet activation could represent a novel approach to control metastatic diffusion.

The goal of our study is the understanding of the molecular mechanisms behind the contribution of platelets to cancer spread. In particular, our attention is focused on the role of platelet-derived microparticles (PMPs), which are small vesicles released upon platelet activation and represent important carriers of biological signals. With this research we aim to:

  • analyse the ability of PMPs to alter the invasive potential of cancer cells
  • investigate the molecular mechanisms for PMPs modulation of the metastatic properties of cancer cells
  • analyse the effect of platelet pharmacological inhibition on the pro-metastatic properties of PMPs
  • evaluate the pro-metastatic effects of PMPs in vivo

Research group collaborations

Mitsuhiko Okigaki (Department of Cardiovascular Medicine,Kyoto Prefectural University of Medicine, Kyoto, Japan )

Emilio Hirsch (Molecular Biotechnology Center-MBC, University of Turin, Italy)

Carlo Balduini, Patrizia Noris, Alessandro Pecci (IRCCS Policlinico San Matteo, Pavia, Italy)

Maria Enrica Tira (Department of Biology and Biotechnology, University of Pavia, Italy)

Giampaolo Minetti (Department of Biology and Biotechnology, University of Pavia, Italy)

Giordano Pula (Department of Pharmacy and Pharmacology, University of Bath, UK)

Luciaanna Stivala, Ornella Cazzalini, Paola Perucca (Department of Molecular Medicine, University of Pavia, Italy)

Satya P. Kunapuli (Departments of Physiology and Pharmacology and Sol Sherry Thrombosis Research Center, Temple University, School of Medicine, Philadelphia, Pennsylvania)

Maries Van den Broek (Institute of Experimental Immunology, University of Zurich, Switzerland)

 

Methods and Techniques

Blood cell purification (platelets and neutrophils). Isolation and characterization of microparticles and microvesicles. Protein analysis (SDS-PAGE, western blotting, chemiluminescence, spectrophotometry, spectrofluorometric analysis, flow cytometry). Adhesion assays under static conditions and under flow. Microscopy (phase contrast and fluorescence microscopy). Molecular biology techniques, DNA and RNA extraction and analysis, PCR. Protein purification (expression, ion-exchange, affinity and size-exclusion chromatography, HPLC, FPLC). Cell culture and transfection, analysis of cell migration, invasion, proliferation, metalloproteinases release. Genetically modified animal models (Mus musculus): Pyk2-KO, PI3Kb-KD, APPKO, 3xTgAD, PLCg2-KO


Red blood cell biochemistry

 Group leader: Prof. Giampaolo Minetti

Collaborators: Dr Cesare Achilli, Post-doc; Dr Annarita Ciana, Post-Doc; Anjali Gaur, PhD student of the RELEVANCE project, Marie Skłodowska-Curie Innovative Training Networks, Horizon 2020.

Red blood cell biochemistry

The scientific interests of the research group deal mainly with the basic properties of biological membranes, focusing on the erythrocyte membrane as a classical model. The biochemical properties of normal, pathologic, in vivo aged and stored erythrocytes and of erythrocytes produced under conditions of neocytolysis are investigated. Erythrocyte membrane proteins and lipids are studied using biochemical and biophysical methods (electrophoresis, western blotting, fluorescence microscopy, electron paramagnetic resonance, omega-alkynyl fatty acid analogs and “click” chemistry to study protein acylation) to elucidate the structure and function of various membrane domains, such as the membrane skeleton, lipid rafts, membrane vesicles, and their interplay. High-resolution separation of erythrocytes of different age is implemented in his lab to study in vivo aged erythrocytes.

Methionine sulfoxide reductases

The oxidation of methionine residues often results in the loss of biological activity of the affected protein. Methionine sulfoxide reductase (Msr) enzymes are an important repair system for such oxidative damage. While studying Msr activity in human neutrophils, we observed a stereospecific reduction of only one of the two sulfoxides of L-Met. Our original suspect that two classes of Msr with opposite stereospecificity might exist in nature is now confirmed by several reports in the recent literature. In that study, a new method was proposed to assay Msr activity, which has been successfully adopted by several authors ever since. Neutrophils are cells of the innate immune system that engulf and kill invading pathogens by producing reactive oxygen species (ROS). ROS may induce auto-oxidation of the neutrophil structures. Msr are highly expressed in neutrophils, probably as a protective and/or repair system, in particular the selenoprotein MsrB1 whose activity and properties are actively investigated in his research group.

Blood cells as tools for assessing the toxicology of nanomaterials

A new research line involves the use of blood cells as tools for toxicological studies of nanoparticles.


Proteomics/Metabolomics in the investigation of medical disorders

Group leader: Prof. Paolo Iadarola

With the advent of proteomics, the screening of proteins as potential biomarkers has achieved important progresses. Detection and identification of proteins in different organs/tissues, with the aim of understanding whether they represent an attractive tool for monitoring alterations in these districts, is currently an area of increasing interest.

Clinical proteomics involves the application of proteomic technologies on clinical specimens such as biological fluids. In this context the current work of our laboratory is focused on the identification in biological fluids (sputum, exhaled breath condensate, broncoalveolar lavage fluid), lymphoblastoid cells and cellular extracts of biomarkers of disorders of different origin. In particular the following three lines of investigation are carried out:

  1. Study of proteomics/metabolomics of lung diseases (chronic obstructive pulmonary diseases, α1-antitrypsin deficiency, sarcoidosis, bronchiolitis obliterans syndrome)
  2. Proteomic analysis of lymphoblastoid cells from patients affected by a rare neurodegenerative disorder (Nasu-Hakola disease).
  3. Proteomic analysis of salivary glands and ovaries from the tick Ixodes ricinus.