Evidence that the platelet integrin alphaIIb beta3 is regulated by the integrin-linked kinase, ILK, in a PI3-kinase dependent pathway.
ABSTRACT Platelet aggregation is mediated by the integrin alphaIIb beta3 which is activated by intracellular signals during platelet activation. We have attempted to determine if ILK ("Integrin-Linked Kinase") is involved in the regulation of alphaIIb beta3 function. ILK co-immunoprecipitated with beta3 in stimulated platelets. Using confocal microscopy, ILK was detected in the cytoplasm of resting platelets. ADP or PMA stimulation led to its translocation to the plasma membrane. In parallel, there was a transient increase in ILK kinase activity, association with and phosphorylation of beta3. Inhibition of PI3-kinase by two unrelated inhibitors (wortmannin and LY294002) prevented ILK-related functions. However, it did not prevent the conformational change in alphaIIb beta3 (shown by PAC-1 binding), although integrin affinity for fibrinogen was decreased as measured using FITC-fibrinogen. Furthermore, aggregate formation was reduced. Thus ILK transiently associates with and phosphorylates beta3 in a PI3-kinase dependent manner suggesting that it participates at an intermediate stage in a critical mechanism for assuring large stable aggregates.
- [show abstract] [hide abstract]
ABSTRACT: Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease. A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied. Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation. Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.Rheumatology (Oxford, England) 07/2009; 48(9):1036-44. · 4.24 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Platelet adhesion and activation rates are frequently used to assess the thrombogenicity of biomaterials, which is a crucial step for the development of blood-contacting devices. Until now, electron and confocal microscopes have been used to investigate platelet activation but they failed to characterize this activation quantitatively and in real time. In order to overcome these limitations, quartz crystal microbalance with dissipation (QCM-D) was employed and an explicit time scale introduced in the dissipation versus frequency plots (Df-t) provided us with quantitative data at different stages of platelet activation. The QCM-D chips were coated with thrombogenic and non-thrombogenic model proteins to develop the methodology, further extended to investigate polymer thrombogenicity. Electron microscopy and immunofluorescence labelling were used to validate the QCM-D data and confirmed the relevance of Df-t plots to discriminate the activation rate among protein-modified surfaces. The responses showed the predominant role of surface hydrophobicity and roughness towards platelet activation and thereby towards polymer thrombogenicity. Modelling experimental data obtained with QCM-D with a Matlab code allowed us to define the rate at which mass change occurs (A/B), to obtain an A/B value for each polymer and correlate this value with polymer thrombogenicity.Journal of The Royal Society Interface 02/2011; 8(60):988-97. · 4.91 Impact Factor
Article: There's something about ILK.[show abstract] [hide abstract]
ABSTRACT: Integrin-Linked Kinase (ILK) is associated with integrin and growth factor receptor signalling. As both signalling pathways contribute to cancer cell resistance, ILK seems well suited as a promising tumour target. Data were obtained by performing a PubMed database search and summarised with a focus on the function of ILK in cancer biology. The findings on the catalytic function of ILK, on the putative substrates of ILK and on the expression of ILK in tumour and normal tissues are heterogeneous. In the context of cancer, two of these issues might be of importance. First, a variety of reports indicate a lack of ILK overexpression in tumours. Second, wild-type or overexpression of ILK has been found to considerably sensitise tumour cells to ionising irradiation as compared to ILK knockout or ILK knockdown conditions. In contrast, wild-type or overexpression of ILK has been shown to protect tumour cells from chemotherapy-induced cell death. Due to these conflicting data, it is difficult to evaluate if therapeutic targeting of ILK is a reasonable strategy in cancer therapy. A more comprehensive understanding of the molecular mechanisms controlled by ILK may help to answer this question.International Journal of Radiation Biology 11/2009; 85(11):929-36. · 1.90 Impact Factor