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RESEARCH PROGRAM

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Keywords
THROMBIN; GROWTH FACTORS; ANGIOGENESIS; VASCULAR PERMEABILITY; ALZHEIMER DISEASE

NON HAEMOSTATIC EFFECTS OF THROMBIN: INTERACTION WITH GROWTH FACTORS AND ITS ROLE IN CELL PROLIFERATION, IN NEURODEGENERATIVE DISORDERS, and IN CONDITIONS OF ALTERED VESSEL PERMEABILITY

Università Cattolica del Sacro Cuore
Abstract
The research project is aimed at studying non hemostatic roles of thrombin in processes of endothelial activation, angiogenesis, in phenomena of increased vascular permeability, and in neurodegenerative processes, particularly in the Alzheimer's disease.
The specific aims of he present study will be:
1) study of the proliferation and angiogenuc effects of trombin on coltured cell lines (endothelial cells [HUVEC], and melanoma
cells) in presence and absence of both growth factors and thrombin.
2) identification of new substrates of trombin among the most common
growth factors: FGF-2, PDGF-BB, NGF, by means of western-blotting techniques and HPLC.
3) study of the proteolytic effect of thrombin on FGF-2, PDGF-BB, PDGF-AA, and NGF to determine the steady-state kinetic
parameters of their hydrolysis. The cleavage sites will be determined by N-terminal sequence and mass spectrometry.
4) production of molecular models of interations between thrombin and FGF-2, by means of computer modelling techniques.
5) synthesis of peptides, that, on the basis of the molecular simulations studies, could inhibit the thrombin-FGF-2 interaction. The binding of these peptides with thrombin could then studied by the "surface plasmon resonance" (SPR) technique.
6) to obtain and compare the dosage of coagulation parameters, thrombin, prothrombi fragment, thrombin-prothrombin complex in plasma, urine and spinal fluid of controls and >>>

Principal Investigator
Raffaele LANDOLFI Università Cattolica del Sacro Cuore
Research Objectives
The findings that the present study could obtain may offer new insights into the mechanisms of non hemostatic effects of thrombin in vivo. In particular the study will deal with thrombin-linked cellular mechanisms of proliferation, angiogenesis, vascular permeability, and in processes involved in the pathogenesis of neurodegenerative disorders, and particularly in the Alzheimer disease. Very common clinical observations indicate that thrombin is involved in relevant cellular functions, such as angiogenesis and proliferation. For instance, it is commonly observed that after thrombosis in a large vein, the thrombus is recanalized with new vessels seen with angiography. It is also known that while thrombin in the plasma is rapidly inactivated by antithrombin, the thrombin molecules trapped within the thrombi are protected and slowly released during thrombolysis. Most likely, this trapped thrombin acts as a factor controlling angiogenesis in concerted action with growth factors, by attracting endothelial cells, mediating their angiogenic phenotype. In addition, thrombin is also known to interact with various constituents of the ECM. ECM-immobilized thrombin is protected from inactivation by its circulating inhibitors and induces many cellular responses. Binding of thrombin to the subendothelial ECM through a short anchorage binding site leaves the majority of the molecule functional and available for cellular interaction. Finally, there are two fundamental scientific knowledge >>>

Timescale
24 months
National and international background
THROMBIN-INDUCED CELLULAR EFFECTS: ENDOTHELIAL CELLS
Long recognized for its role in the coagulation cascade, thrombin is now known to regulate its target cells, such as platelets and endothelial cells, in part via activation of a G-Protein coupled recepror (1-3). The novel mechanism whereby thrombin activates its receptors involves the proteolytic unmasking of a cryptic NH2-terminal receptor sequence that, remaining tethered, binds to and triggers receptor function (1). In addition, short (5 to 6 amino acids) synthetic peptides, based on the proteolytically revealed motif, can activate the thrombin receptor without the unmasking of the tethered ligand (1). Since the cloning of the first receptor for thrombin (now termed "proteinase-activated receptor-1", or PAR-1), two more family members cleaved by thrombin (PAR-3 and -4) have been cloned (4-6), along with an additional PAR triggered by trypsin, but not thrombin (now termed PAR-2) (7). Key distinguishing features of the PAR family are 1) their selective sensitivity to serine proteinases (e.g., PAR-1 and -4 can be activated by both thrombin and trypsin, whereas PAR-2 is activated by trypsin and other serine proteinases, but not by thrombin); and 2) their distinct proteolytically revealed tethered activating sequences (e.g., SFFLRN for rat PAR-1, SLIGRL for rat PAR-2, and GFPGKP for rat PAR-4). Receptor-selective activating peptides (TRAPs) have proved of enormous utility to assess the potential effects of receptor >>>