Mouse Carotid Artery Thrombosis Research Articles

The preparation and characterization of novel peptide antagonists to thrombin and factor VIIa and activation of protease-activated receptor 1.

Thrombin and protease-activated receptor 1 (PAR1) activation antagonists were prepared based upon the peptide RPPGF, the angiotensin-converting enzyme breakdown product of bradykinin. A library of 72 peptides consisting of d and/or synthetic amino acids was designed with various substitutions in positions 1 to 5 in Arg-Pro-Pro-Gly-Phe (RPPGF). Two compounds, rOicPGF (TH146) and betaAK2K-4(rOicPGF) (MAP4-TH146), were characterized further. TH146 or MAP4-TH146 completely inhibits threshold gamma-thrombin-induced platelet aggregation at a concentration of 142 +/- 0.05 or 19 +/- 0.06 microM, respectively. TH146 completely inhibits threshold alpha-thrombin-induced washed platelet aggregation at 444 +/- 0.04 microM. TH146 or MAP4-TH146 blocks 2 nM alpha-thrombin-induced fibroblast calcium mobilization with an IC(50) value of 110 or 18 microM, respectively. Furthermore, significant prolongation of the activated partial thromboplastin time, prothrombin time, or thrombin clotting time occurs at 31, 62, or 7.8 microM TH146 and 0.4, 6.25, or 1.56 microM MAP4-TH146, respectively. TH146 and MAP4-TH146 inhibit both alpha-thrombin with a K(i) value of 97 and 49 microM, respectively, and factor VIIa with a K(i) value of 44 and 5 microM, respectively. Both TH146 and MAP4-TH146 specifically bind to the exodomain of recombinant PAR1. MAP4-TH146 (200 microM) completely blocks thrombocytin, a PAR1-activating snake venom protease, without inhibiting the enzyme's active site. TH146 inhibits gamma-thrombin-induced aggregation of mouse platelets, prolongs mouse bleeding times, and delays the time to mouse carotid artery thrombosis. TH146 and MAP4-TH146 inhibit human and mouse platelet aggregation and mouse thrombosis. Analogs of RPPGF are model compounds to develop PAR1 activation antagonists as well as direct inhibitors to thrombin and factor VIIa.

Effect of mechanical ventilation on carotid artery thrombosis induced by photochemical injury in mice

Increasing use of transgenic and gene targeting techniques for the investigation of hemostasis and vascular biology has generated interest in experimental models of carotid artery thrombosis in mice. We tested the hypothesis that hypoventilation in anesthetized mice may cause hypercapnia, increased carotid artery blood flow, and altered thrombotic responses to photochemical injury of the carotid artery. Arterial blood gases and carotid artery blood flow were measured in pentobarbital-anesthetized BALB/c or C57BL/6 J mice with and without mechanical ventilation. Photochemical injury of the carotid artery was induced using the rose bengal method. Compared with ventilated mice, unventilated mice had a 45% increase in carotid artery blood flow (0.74 +/- 0.04 vs. 0.41 +/- 0.03 mL min-1; P < 0.001) that was associated with an elevation of arterial PCO2 (58 +/- 4 vs. 33 +/- 4 mmHg; P < 0.05) and a decrease in arterial pH (7.18 +/- 0.05 vs. 7.32 +/- 0.03; P < 0.05). Time to first occlusion of the carotid artery after photochemical injury was shorter in ventilated than in unventilated mice (29 +/- 6 vs. 73 +/- 9 min; P < 0.001). Time to stable occlusion was also shorter in ventilated mice (49 +/- 8 vs. 81 +/- 6 min; P < 0.05). Elevated carotid artery blood flow, hypercarbic acidosis, and prolonged occlusion times also were observed in mice ventilated with supplemental carbon dioxide. General anesthesia without mechanical ventilation has the potential to confound studies of experimental thrombosis in vivo by producing hypoventilation, hypercapnia, acidosis, and altered carotid artery blood flow. Mechanical ventilation with maintenance of normal blood gases may enhance the physiological insight gained from experimental models of carotid artery thrombosis in mice.

Function of Tissue-type Plasminogen Activator Releaser on Vascular Endothelial Cells and Thrombolysis In Vivo

The effect of monosodium[2-(6-hydroxynaphthalen-2-yl)-6-methylpyrimidin-4-yloxy]acetate dihydrate (JTV-926) on fibrinolysis was investigated in vitro and in vivo. JTV-926 released tissue-type plasminogen activator (t-PA) from human vascular endothelial cells in a dose-dependent manner. The thrombolytic effect of JTV-926 was studied using three animal thrombosis models; a photo-irradiation-induced mouse carotid artery thrombosis model, a photo-irradiation-induced rat femoral artery thrombosis model and a thrombin-induced rat venous thrombosis model. In the mouse thrombosis model, t-PA deficient mice (t-PA-/- mice) and their wild-type (t-PA+/+) were used. JTV-926 was injected as a bolus 30 min after the interruption of blood flow by an occlusion thrombi. Blood flow was continuously monitored for 180 min after intravenous administration of JTV-926 (1 mg/kg). Although the recanalization rate of the occluded artery was 37.5% in t-PA +/+ mice with the vehicle control, it increased to 75% in t-PA+/+ mice after JTV-926 administration. However, when JTV-926 was administrated in t-PA-/- mice, vascular recanalization was not observed in any arteries. In the photo-irradiation-induced rat femoral artery thrombosis model, intra-duodenal administration of JTV-926 induced thrombolysis. Moreover, in the thrombin-induced rat venous thrombosis model, the dose-dependent thrombolysis was also observed by oral administration of JTV-926. It was suggested that JTV-926 revealed a sufficient thrombolytic effect through the absorption from the intestine. Thus, a newly synthesized compound, JTV-926 induced t-PA release from vascular endothelial cells and effective thrombolysis in vivo.