Regular ArticleThrombin regulates vascular smooth muscle cell proteoglycan synthesis via PAR-1 and multiple downstream signalling pathways
Introduction
Thrombin is a serine protease that is generated by cleavage of its inactive precursor, prothrombin, and has been intensively studied due to its essential role in hemostasis [1]. In the coagulation cascade, thrombin converts the monomer fibrinogen to insoluble fibrin, in addition to activating other clotting factors V, VIII and XIII, thus facilitating thrombus formation. The discovery of seven transmembrane G protein coupled receptors 7TM GPCR, that are activated by proteolytic cleavage [2], demonstrated that thrombin is able to regulate vascular smooth muscle cell (VSMC)s independent of its effects on hemostasis. VSMCs predominantly express protease activated receptor 1 (PAR-1) and PAR-4 [3], although certain VSMC types have been shown to express other PARs [4]. G protein pathways coupled to the PAR-1 receptor include the Gαq/11 regulated phospholipase Cβ/DAG/IP3 pathway [5], the Gα12/13 guanine exchange factor regulated Rho/Rho kinase pathway [6] and Gαi/o mediated pathways [7]. Activation of PAR-1 by thrombin causes cellular effects associated with activation of these pathways, including calcium signalling, proliferation [8], [9], cytoskeletal rearrangement, contraction [10] and extracellular matrix synthesis [11].
Proteoglycans are macromolecules that are composed of a core protein to which sulfated glycosaminoglycan (GAG) chains are covalently attached [12]. These molecules form part of the extracellular matrix of a blood vessel where they play a role in physiological regulation, but also participate in pathological processes including the development of atherosclerotic plaques [13]. Their major role in plaque development occurs very early through binding and retention of lipoproteins in the neointima [13], [14]. Nakashima et al. recently demonstrated that accumulation of lipid in the outer layer of the diffuse intimal thickening of human coronary arteries is the earliest step in the formation of an atherosclerotic lesion. This lipid retention is very closely associated with expression of the proteoglycan, biglycan [14]. Furthermore, O'Brien and colleagues have very recently shown that the accelerated atherosclerosis of diabetes is associated with the enhanced expression of GAGs and proteoglycans [15].
The synthesis and hence structure of the GAG chains on proteoglycans is able to be modified by extracellular mediators. Angiotensin II and transforming growth factor β (TGFβ) both stimulate the synthesis of proteoglycans that have longer GAG chains, a property which causes enhanced proteoglycan binding to LDL [16], [17]. Both agonists stimulate an increase in the expression of chondroitin sulfate (CS) containing proteoglycans, versican and biglycan [18], [19], [20], which has implications for atherogenesis as CS proteoglycans bind LDL with high affinity, whereas heparan sulfate proteoglycans do not demonstrate binding to LDL [21].
Thrombin stimulates the synthesis of proteoglycans from VSMC, particularly, the synthesis of a large heparan sulfate containing proteoglycan, identified as perlecan [22], however nothing is currently known about the effect of thrombin on CS/DS proteoglycans, or the signalling pathways used by thrombin to mediate its effects on proteoglycans. We have investigated the effect of thrombin on the synthesis and structure of CS/DS proteoglycans versican, biglycan and decorin, from human VSMC, and have determined the signalling pathways and mechanisms by which thrombin is able to mediate its effects on the synthesis and structure of proteoglycans.
Section snippets
Materials
Chemicals and reagents were purchased from suppliers as follows: endothelin-1, thrombin, EGF, AG1478, 6-amino caproic acid, benzamidine hydrochloride, methyl-β-D-xylopyranoside (xyloside), DEAE-Sephacel, Sepharose CL-6B, proteinase K, sodium borohydride, chondroitin sulfate and GF109203X (Sigma, St Louis, MO, USA); forskolin (EMD Biosciences, Darmstadt, Germany). Catalytically inactive thrombin (human α thrombin-phenylalanine-proline-arginine-chloromethylketone) (Haemtech, Essex Junction VT,
Thrombin stimulates an increase in the synthesis and size of CS/DS proteoglycans secreted by human VSMC
Quiescent human VSMC were treated with thrombin (0 – 10 or 30 U/mL) for 6 hours prior to addition of either [35S]sulfate (25 μCi/mL) or [3H]glucosamine (20 μCi/ml) for a further 18 hours. Thrombin stimulated a concentration dependent increase in [35S]-sulfate incorporation into secreted proteoglycans (Fig. 1) causing a 190% increase (p < 0.01) at the maximal concentration used (10 U/mL). As an alternative measure of proteoglycan synthesis we assessed the incorporation of [3H]glucosamine into
Discussion
The pro-coagulant serine protease, thrombin, is implicated in the vascular biology of atherosclerosis due to effects on VSMC proliferation and migration and also potentially via effects on secretion of matrix molecules [33]. Proteoglycans have recently been strongly implicated in atherosclerosis but the effect of thrombin on the synthesis and structure of the lipid binding CS/DS proteoglycans synthesized and secreted by VSMCs is unknown. We report that thrombin stimulation of VSMC caused an
Conclusion
The crucial role of proteoglycans in the initiation of atherosclerosis has been supported by extensive data over the last decade and most recently by definitive studies in human pathology [14], [15]. Multiple vasoactive agonists have been identified which regulate GAG synthesis consistent with increasing the atherogenicity of the proteoglycans. In terms of therapeutics it is unlikely that the enzymes or processes of GAG elongation would be suitable targets because these would most likely have
Acknowledgements
This work was supported by the Diabetes Australia Research Trust grants (PJL) and a GlaxoSmithKline Australia post-graduate student award to PJL to support the work of MEI.
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