Dissecting The Effects Of Isoprenoid Pathway Inhibition On Hemostasis and Thrombosis: Differential Effects Of Atorvastatin and Digeranyl Bisphosphonate In Hypercholesterolemic Mice

Statins inhibit HMG-CoA reductase, leading to decreased production of cholesterol and isoprenoid metabolites. There is growing evidence that statins have protective effects on arterial thrombosis through pleiotropic mechanisms that may be independent of their cholesterol lowering effects. The antithrombotic effects of statins have been proposed to be related in part to diminished synthesis of geranylgeranyl pyrophosphate (GGPP). To test this hypothesis, we determined the hemostatic and thrombotic effects of atorvastatin and digeranyl bisphosphonate (DGBP), a specific inhibitor of GGPP synthase, in apolipoprotein E-deficient (ApoE-/-) mice.

ApoE-/- mice were treated with either vehicle, atorvastatin (50 mg/kg/d), or DGBP (0.4 mg/kg/d) subcutaneously for 7 days. Tissue levels of GGPP and its precursor farnesyl pyrophosphate (FPP) were measured by HPLC. Plasma total cholesterol was measured enzymatically. Susceptibility to thrombotic occlusion of the carotid artery was measured in response to injury with FeCl₃. Platelet count, early platelet activation responses (surface expression of P-selectin and activation of integrin αIIbβ3), platelet clot retraction, and tail-transection bleeding times were also assessed.

Compared with vehicle or atorvastatin-treated mice, DGBP- treated mice had elevated levels of FPP in heart (P<0.001) and lung (P<0.001) and decreased levels of GGPP in heart (P<0.01) and liver (P<0.01). Plasma total cholesterol was decreased in atorvastatin-treated mice (199±14 mg/dL) compared with vehicle- (284±32 mg/dL) or DGBP- treated (321±21 mg/dL) mice (P<0.05). The platelet count was decreased in DGBP-treated mice (638,000±90,000/mL) compared with vehicle-treated mice (1,208,000±72,000/mL; P<0.01). The time to stable occlusion of the carotid artery was prolonged in atorvastatin-treated mice compared with vehicle-treated mice (8.6±2.4 vs. 5.3± 0.3 minutes; P<0.05). In contrast, DGBP-treated mice were not protected from carotid artery thrombosis (6.8±0.9 minutes). Bleeding times were markedly prolonged in DGBP-treated mice (502±68 seconds) compared with atorvastatin-treated (152±66 seconds) or vehicle-treated (132±72 seconds) mice (P<0.05). Early platelet activation responses (αIIbβ3 activation and surface P-selectin expression) after stimulation with thrombin (0.01, 0.05 and 0.1 units/mL) did not differ between vehicle-, atorvastatin- and DGBP-treated mice, but platelet clot retraction was significantly delayed in DGBP-treated mice compared with atorvastatin- or vehicle-treated mice (P<0.05).

The observed changes in isoprenoid metabolites were consistent with the known inhibitory effects of atorvastatin on the isoprenoid pathway upstream of cholesterol synthesis, and of DGBP downstream of FPP synthesis. In contrast to our hypothesis, inhibition of GGPP production with DGBP did not prevent arterial thrombosis despite causing modest thrombocytopenia, decreased platelet clot retraction, and impairment of primary hemostasis in hypercholesterolemic mice. The differential effects of DGBP and atorvastatin suggest that the antithrombotic effects of statins are largely independent of geranylgeranyl-mediated processes.

Hohl:Terpenoid Therapeutics: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Lentz:Novo Nordisk: Consultancy, Research Funding.