hirudin and Carotid-Artery-Diseases

To understand the role of epidermal growth factor receptor (EGFR) transactivation in G protein-coupled receptor (GPCR) agonist-induced signaling events, we have studied the capacity of thrombin in the activation of Gab1-SHP2 in vascular smooth muscle cells (VSMCs). Thrombin activated both Gab1 and SHP2 in EGFR-dependent manner. Similarly, thrombin induced Rac1 and Cdc42 activation, and these responses were suppressed when either Gab1 or SHP2 stimulation is blocked. Thrombin also induced PAK1 activation in a time- and EGFR-Gab1-SHP2-Rac1/Cdc42-dependent manner. Inhibition of activation of EGFR, Gab1, SHP2, Rac1, Cdc42, or PAK1 by pharmacological or genetic approaches attenuated thrombin-induced VSMC stress fiber formation and motility. Thrombin activated RhoA in a time-dependent manner in VSMCs. LARG, a RhoA-specific GEF (guanine nucleotide exchange factor), was found to be associated with Gab1 and siRNA-mediated depletion of its levels suppressed RhoA, Rac1 and PAK1 activation. Dominant negative mutant-mediated interference of RhoA activation inhibited thrombin-induced Rac1 and PAK1 stimulation in VSMCs and their stress fiber formation and migration. Balloon injury induced PAK1 activity and interference with its activation led to attenuation of SMC migration from media to intima, resulting in reduced neointima formation and increased lumen size. Inhibition of thrombin signaling by recombinant hirudin also blocked balloon injury-induced EGFR tyrosine phosphorylation and PAK1 activity. These results show that thrombin-mediated PAK1 activation plays a crucial role in vascular wall remodeling and it could be a potential target for drug development against these vascular lesions.

Topics: Angioplasty, Balloon; Animals; Carotid Artery Diseases; Carotid Stenosis; cdc42 GTP-Binding Protein; Cell Movement; Cells, Cultured; Disease Models, Animal; ErbB Receptors; Fibrinolytic Agents; Gene Transfer Techniques; Genetic Therapy; Guanine Nucleotide Exchange Factors; Hirudins; Humans; Muscle, Smooth, Vascular; Mutation; p21-Activated Kinases; Phosphoproteins; Phosphorylation; Protein Kinase Inhibitors; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Quinazolines; rac1 GTP-Binding Protein; Rats; Rho Guanine Nucleotide Exchange Factors; rhoA GTP-Binding Protein; RNA Interference; RNA, Small Interfering; Stress Fibers; Thrombin; Time Factors; Transfection; Tyrphostins

Myointimal hyperplasia is the condition usually responsible for recurrent stenosis (restenosis) after endarterectomy, bypass grafting and angioplasty. Its cause is still not known. The present study examined whether inhibition of thrombin by tissue plasminogen activator (r-TPA) or polyethylene glycol recombinant hirudin (PEG-hirudin) could reduce restenosis in an animal model. Restenosis was induced in 20 cholesterol-fed rabbits. The right carotid artery underwent a double-balloon injury while left carotid artery acted as a control. Recombinant tissue plasminogen activator (1 mg kg(-1) s.c.) and PEG-hirudin (0.7 mg kg(-1) s.c.) were given subcutaneously with normal saline acting as a control. Blood levels of PEG-hirudin were measured by both ELISA and an Ecarin (activity) assay. Vessel dimensions were measured in histological sections, obtained from perfusion-fixed tissue, using computerised planimetry. The model reproduced many of the histological changes found in human restenosis, such as intramural thrombus, rupture of the elastic lamina, macrophage infiltration and smooth muscle migration. Reinjury caused an almost three-fold reduction in the area of the lumen (median 0.25 mm(2)) compared with uninjured vessels (median 0.72 mm(2)). The mean plasma levels of PEG-hirudin and r-tPA achieved were 291 ng/ml (S.E.M. 28 ng/ml) and 34 IU/ml (S.E.M. 12 IU/ml), respectively. PEG-hirudin significantly inhibited the effect of balloon injury on luminal area compared with saline-treated controls (0.21 versus 0.44 mm(2), respectively, P<0.05). Recombinant tPA also had a similar inhibitory affect, but this did not reach statistical significance (0.16 versus 0.44 mm(2), respectively, P>0.05). The magnitude of luminal narrowing was significantly reduced by subcutaneous injection of PEG-hirudin. Further studies are required to determine whether this effect can be enhanced by other antithrombins or improved methods of delivery.

Topics: Animals; Anticoagulants; Carotid Arteries; Carotid Artery Diseases; Catheterization; Constriction, Pathologic; Disease Models, Animal; Hirudins; Rabbits; Recombinant Proteins; Recurrence; Tissue Plasminogen Activator

Further to characterize the processes involved in the FeCl3-induced thrombosis model, we determined the effect of aspirin, heparin, hirudin, trans-4-(aminomethyl) cyclohexane carboxylic acid (AMCHA), thrombocytopenia, and flow modifications on time to occlusion (TTO) and thrombus weight (TW) in the rat carotid artery. Aspirin, from 3 to 100 mg/kg, showed no dose-response relation for either TTO or TW and did not significantly affect ex vivo platelet aggregation. Heparin, at doses that significantly increased the activated partial thromboplastin time (APTT), dose-dependently increased the TTO of animals that showed an occlusion during the monitoring period and also reduced the TW. Hirudin required constant infusion to prevent occlusion and reduce the TW, when the APTT was also significantly increased. AMCHA did not affect the TW but reduced the TTO. Animals made thrombocytopenic by the use of antiplatelet serum did not occlude during the monitoring period, and the TW was significantly reduced. Changes in flow showed that the TTO was not affected, but the TW showed an inverse correlation with average flow. The results obtained for platelet depletion and flow modifications expand on previous findings with this model and support the physiological relevance of the model.

Topics: Animals; Antifibrinolytic Agents; Antithrombins; Arterial Occlusive Diseases; Aspirin; Blood Platelets; Carotid Artery Diseases; Carotid Artery, Common; Chlorides; Ferric Compounds; Fibrinolytic Agents; Heparin; Hirudins; Immune Sera; Ligation; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Thrombosis; Tranexamic Acid