To cite this article: Kim PY, Stewart RJ, Lipson SM, Nesheim ME. The relative kinetics of clotting and lysis provide a biochemical rationale for the correlation between elevated fibrinogen and cardiovascular disease. J Thromb Haemost 2007; 5: 1250-6. Summary. Background: Elevated plasma fibrinogen is a well known risk factor for cardiovascular disease. The mechanistic rationale for this is not known. Objectives: These studies were carried out to determine the fibrinogen concentration dependencies of clotting and lysis times and thereby determine whether these times rationalize the correlation between an increased risk of cardiovascular disease and elevated plasma fibrinogen. Methods: The time courses of clot formation and lysis were measured by turbidity in systems comprising a) fibrinogen, thrombin and plasmin, or b) fibrinogen, thrombin, plasminogen and t-PA, or c) plasma, thrombin and t-PA. From the lysis times, k cat and K m values for plasmin action on fibrin were determined. Results: The time to clot increased linearly from 2.9 to 5.6 minutes as the fibrinogen concentration increased from 1 to 9 lM and did not increase further as the fibrinogen concentration was raised to 20 lM. In contrast, the clot lysis time increased linearly over the input fibrinogen concentration range of 2 to 20 lM. A similar linear trend was found in the two systems with t-PA and plasminogen. Apparent K m and k cat values for plasmin were 1.1 ± 0.6 lM and 28 ± 2 min )1 , respectively. K m values for plasmin in experiments initiated with t-PA and plasminogen were 1.6 ± 0.2 lM in the purified system and 2.1 ± 0.9 lM in plasma. Conclusion: As the concentration of fibrinogen increases, especially above physiologic level, the balance between fibrinolysis and clotting shifts toward the latter, providing a rationale for the increased risk of cardiovascular disease associated with elevated fibrinogen.

Objective-Inhibition of 2,3-oxidosqualene:lanosterol cyclase (OSC), an enzyme in the cholesterol synthesis pathway, has the unique ability to inhibit cholesterol synthesis while simultaneously enhancing oxysterol synthesis. Our objectives were to determine, in vivo, if a novel OSC inhibitor reduced low-density lipoprotein (LDL) cholesterol and to define the mechanism(s) involved. Methods and Results-Miniature pigs received the OSC inhibitor RO0717625 or placebo and a diet containing fat (34% of energy) and 400 mg per day of cholesterol. Treatment decreased plasma total cholesterol (Ϫ20%) and LDL cholesterol (Ϫ29%). Apolipoprotein B (apoB) kinetic parameters were determined. Very low-density lipoprotein (VLDL) apoB pool size decreased 22% because of inhibition of VLDL production (Ϫ43%). LDL apoB pool size decreased 22% because of a 1.5-fold increase in fractional catabolic rate (FCR). The increased FCR was associated with a 2-fold increase in hepatic LDL receptor mRNA. Hepatic total and microsomal cholesterol were reduced by 16% and 27%, respectively. Plasma lathosterol concentrations decreased 57%, reflecting inhibition of hepatic cholesterol synthesis. Treatment reduced plasma plant sterols and decreased postprandial cholesterol transport in chylomicrons. Conclusions-A novel OSC inhibitor, RO0717625, decreased VLDL and LDL apoB100 through decreased VLDL production and enhanced LDL clearance. Thus, OSC represents a potential therapeutic target for dyslipidemia. Key Words: oxidosqualene:lanosterol cyclase Ⅲ apoB kinetics Ⅲ LDL receptor Ⅲ ABCG5 Ⅲ ABCG8 L andmark trials using 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) revealed reductions in cardiovascular mortality and morbidity associated with low-density lipoprotein (LDL) cholesterol lowering. 1 Statins are well tolerated, although reductions in nonsterol intermediates in the cholesterol-synthesis pathway, such as isoprenoids and coenzyme Q, may theoretically be associated with adverse clinical events. 2,3 This has stimulated the development of compounds that inhibit cholesterol biosynthesis yet act distal to the synthesis of these nonsterol intermediates. 2,3-oxidosqualene:lanosterol cyclase (OSC; enzyme collection 5.4.99.7, also known as lanosterol synthase), a microsomal enzyme, represents a unique target for cholesterol-lowering drugs. 4,5 OSC is downstream of isoprenoid synthesis. Furthermore, compounds that decrease plasma concentrations of atherogenic lipoproteins by more than 1 mechanism are likely to be more efficacious.OSC catalyzes the highly selective cyclization of 2,3-monoepoxysqualene (MOS) to lanosterol, the first sterol to be formed. 4 OSC also catalyzes cyclization of 2,3;22,23-diepoxysqualene (DOS), which itself is derived from MOS, to 24(S),25-epoxylanosterol, the immediate precursor of 24(S),25-epoxycholesterol. Synthesis of 24(S),25-epoxycholesterol is favored over cholesterol synthesis under conditions of partial OSC inhibition, whereas complete OSC inhibition results in decreased synthesis of choleste...

Discovery of the ileal apical sodium-dependent bile acid transporter (ASBT) permitted development of specific inhibitors of bile acid reabsorption, potentially a new class of cholesterol-lowering agents. In the present study, we tested the hypothesis that combining the novel ASBT inhibitor, SC-435, with the HMG-CoA reductase inhibitor, atorvastatin, would potentiate reductions in LDL cholesterol (LDL-C) and LDL apolipoprotein B (apoB). ApoB kinetic studies were performed in miniature pigs fed a typical human diet and treated with the combination of SC-435 (5 mg/kg/day) plus atorvastatin (3 mg/kg/day) (SC-435 ؉ A) or a placebo. SC-435 ؉ A decreased plasma total cholesterol by 23% and LDL-C by 40%. Multicompartmental analysis (SAAM II) demonstrated that LDL apoB significantly decreased by 35% due primarily to a 45% increase in the LDL apoB fractional catabolic rate (FCR). SC-435 ؉ A significantly decreased hepatic concentrations of free cholesterol and cholesteryl ester, and increased hepatic LDL receptor mRNA consequent to increased cholesterol 7 ␣ -hydroxylase expression and activity. In comparison, SC-435 (10 mg/kg/day) monotherapy decreased LDL apoB by 10% due entirely to an 18% increase in LDL apoB FCR, whereas atorvastatin monotherapy (3 mg/kg/day) decreased LDL apoB by 30% due primarily to a 22% reduction in LDL apoB production.We conclude that SC-435 ؉ A potentiates the reduction of LDL-C and LDL apoB due to complementary mechanisms of action. Cloning of the ileal apical sodium-dependent bile acid transporter (ASBT) has identified a new pharmacologic target for modulation of plasma lipoproteins (1-4). SC-435 is a potent specific inhibitor of the ASBT, resulting in interruption of the enterohepatic circulation of bile acids and ultimately leading to decreased LDL cholesterol (LDL-C) in a variety of animal models (5-7). In vitro, SC-435 inhibits uptake of bile acids in BHK cells transfected with the human ASBT gene with an IC 50 of 1.5 nM (6). In vivo, we recently demonstrated that, in miniature pigs fed normal amounts of fat and cholesterol, SC-435 enhanced bile acid excretion, leading to a significant 20% reduction in LDL-C (5). Apolipoprotein B (apoB) kinetic studies revealed that SC-435 treatment increased LDL apoB clearance, mediated by enhanced hepatic expression of LDL receptors secondary to upregulation of hepatic cholesterol 7 ␣ -hydroxylase ( CYP7A1 ) expression. These effects are consistent with the molecular basis for CYP7A1 regulation, recently elucidated by Lu et al. (8). Depletion of bile acids due to a decrease in their return to the liver reduces the farnesoid X receptor (FXR)-mediated activation of the short heterodimer partner (SHP) gene. Reduction in SHP allows for the induction of the liver receptor homolog-1 (LRH-1). LRH-1 is a positive transacting factor for the mouse ASBT gene ( slc10a2 ) (9) and the CYP7A1 gene (10, 11). Cholesterol 7 ␣ -hydroxylase, the product of CYP7A1 , is the rate-limiting enzyme for the conversion of cholesterol to bile acids [as reviewed in ref. (12)]...