The serine protease factor VIIa (FVIIa) in complex with its cellular cofactor tissue factor (TF) initiates the blood coagulation reactions. TF⅐FVIIa is also implicated in thrombosis-related disorders and constitutes an appealing therapeutic target for treatment of cardiovascular diseases. To this end, we generated the FVIIa active site inhibitor G17905, which displayed great potency toward TF⅐FVIIa (K i ‫؍‬ 0.35 ؎ 0.11 nM). G17905 did not appreciably inhibit 12 of the 14 examined trypsin-like serine proteases, consistent with its TF⅐FVIIa-specific activity in clotting assays. The crystal structure of the FVIIa⅐G17905 complex provides insight into the molecular basis of the high selectivity. It shows that, compared with other serine proteases, FVIIa is uniquely equipped to accommodate conformational disturbances in the Gln 217 -Gly 219 region caused by the ortho-hydroxy group of the inhibitor's aminobenzamidine moiety located in the S1 recognition pocket. Moreover, the structure revealed a novel, nonstandard conformation of FVIIa active site in the region of the oxyanion hole, a "flipped" Lys 192 -Gly 193 peptide bond. Macromolecular substrate activation assays demonstrated that G17905 is a noncompetitive, slow-binding inhibitor. Nevertheless, G17905 effectively inhibited thrombus formation in a baboon arterio-venous shunt model, reducing platelet and fibrin deposition by ϳ70% at 0.4 mg/kg ؉ 0.1 mg/kg/ min infusion. Therefore, the in vitro potency of G17905, characterized by slow binding kinetics, correlated with efficacious antithrombotic activity in vivo.

Hepatocyte growth factor (HGF) is a pluripotent mitogen thought to be involved in liver regeneration. It is synthesized as a single chain promitogen and requires proteolytic processing to a two-chain heterodimeric form for biological activity. The pharmacokinetics and tissue distribution of radioiodinated single chain recombinant human HGF ([125I]rhuHGF) were studied in male Sprague-Dawley rats after an iv bolus dose. Pharmacokinetic parameters were determined from trichloroacetic acid-precipitable radioactivity in serum samples. There was a rapid distribution phase (t1/2 alpha = 3.1 min) and a slower elimination phase (t1/2 beta = 114 min). Tissue distribution was assessed by whole body autoradiography 5, 60, and 1440 min after an iv bolus dose. rhuHGF rapidly distributed to the liver, kidney, adrenal gland, and spleen. The importance of the liver in the rapid clearance and subsequent conversion of single chain pro-rhuHGF to the mitogenically active two-chain form was demonstrated using an isolated rat liver perfusion system. TCA-precipitable radioactivity excreted into the bile (1.0 +/- 0.1%) and released into the venous effluent (38.9 +/- 6.1%) was monitored for 60 min after a portal vein injection. The appearance of radioactivity in both the bile and venous effluent was maximal between 20-35 min. Further characterization of the reduced samples by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that the two-chain form of [125I]rhuHGF was the predominant form after hepatic perfusion. These studies suggest that the liver plays a major role in the rapid clearance and subsequent activation of pro-rhuHGF in vivo.

Abstract-Vascular endothelial growth factor (VEGF) induces hypotension in normotensive subjects, which is considered to be a major side effect for treatment of ischemic diseases. However, the hypotensive effect of VEGF has not been investigated in the setting of hypertension. This study determined effects of VEGF on hemodynamics, pharmacokinetics, and release of NO and prostaglandin I 2 (PGI 2 ) in vivo and on vasorelaxation of mesentery artery rings in vitro in spontaneously hypertensive rats (SHR) compared with Wistar-Kyoto rats (WKY). Intravenous infusion of VEGF for 2 hours produced a dose-related decrease in arterial pressure, which was enhanced in conscious SHR compared with WKY (PϽ0.01), and an increase in heart rate in WKY but not in SHR. In response to similar doses of VEGF, compared with WKY, SHR had a higher plasma VEGF level and lower VEGF clearance (PϽ0.01). Circulating NO and PGI 2 levels after VEGF administration were not increased in SHR versus WKY, and VEGF-induced vasorelaxation was blunted in SHR versus WKY in vitro, suggesting endothelial dysfunction in SHR. One-week VEGF infusion also caused greater hypotension (PϽ0.05) in the absence of tachycardia in SHR compared with WKY controls. Thus, despite blunted vasorelaxation in vitro because of endothelial dysfunction, SHR exhibited exaggerated hypotension without tachycardia in response to VEGF, which was independent of NO and PGI 2 . The exaggerated hypotensive response to VEGF in SHR may be owing to impaired baroreflex function and reduced VEGF clearance. The data may also suggest that more caution should be taken when VEGF is administered in patients with hypertension. Key Words: growth substances Ⅲ rats, spontaneously hypertensive Ⅲ hemodynamics Ⅲ hypotension Ⅲ blood pressure V ascular endothelial growth factor (VEGF) is a unique mitogen specific for vascular endothelial cells. [1][2][3][4] As a fundamental mediator of normal and pathological angiogenesis, VEGF has been shown to induce a strong angiogenic response in vitro 5,6 and in vivo. 2,3,[7][8][9] Animal studies have demonstrated that administration of VEGF produces beneficial angiogenic effects in peripheral vascular ischemia 10 -12 and in coronary ischemia. [13][14][15][16] VEGF has been shown to induce endothelium-dependent vasorelaxation in normal animals in vitro. [17][18][19] Because of its vasodilatory effect, VEGF induces hypotensive and tachycardic responses in vivo in normotensive animals, including rats, rabbits, and pigs, 14,21 and the hypotensive effect is mediated, at least in part, by NO. 20,21 In humans, intracoronary administration of VEGF caused a hypotensive effect that was dependent on VEGF infusion rate. 22 Although hypotensive effects of VEGF have been characterized in normotensive subjects, these effects have not been investigated in the setting of hypertension.Our previous studies have demonstrated that VEGF induces endothelium-dependent vasorelaxation in aortic rings in vitro, which is diminished in spontaneously hypertensive rats (SHR) compared with normoten...

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