Published Online
on April 29, 2002

A more recent version of this article appeared on May 14, 2002

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Submitted on January 7, 2002
Accepted on February 26, 2002

Inhibition of Tissue Factor Gene Induction and Activity Using a Hairpin Ribozyme

Erdal Cavusoglu MD, Irene Chen BS, Jay Rappaport PhD, and Jonathan D. Marmur MD^*

From the Department of Medicine, Bronx VA Medical Center, Bronx, NY (E.C.); the Department of Medicine, Mount Sinai School of Medicine, New York, NY (E.C., I.C., J.D.M.); and the Department of Medicine, Temple University School of Medicine, Philadelphia, Pa (J.R.).

^* To whom correspondence should be addressed. E-mail: jonathan{at}marmur.com.

Background—Tissue factor (TF) is a membrane-bound glycoprotein that initiates the clotting cascade. Inhibition of the TF pathway has been shown to prevent thrombosis and restenosis after arterial injury in a variety of animal models.

Methods and Results—We describe a novel approach to inhibiting the expression of the TF protein that involves the targeted destruction of cellular TF mRNA with the use of a tetraloop hairpin ribozyme. After construction of the ribozyme and determination of its optimal length and kinetic parameters, a ribozyme expression vector that used the retroviral vector pMV12 was constructed. The ability of this expression vector to generate anti-TF ribozyme was further augmented by positioning of the anti-TF ribozyme downstream of a rat tRNA val (RNA polymerase II) promoter. The resultant construct containing the anti-TF ribozyme was then used to transfect vascular smooth muscle cells and generate a variety of clonal cell lines. Northern blot analyses performed on 3 transfected and 3 untransfected clones demonstrated markedly reduced TF mRNA levels in the transfected clones both during quiescence and after serum stimulation. Cell lysates analyzed for total TF activity by monitoring factor Xa generation similarly demonstrated a statistically significant and concordant reduction in TF activity in smooth muscle cells transfected with the ribozyme expression vector compared with both untransfected clones and clones transfected with the empty vector.

Conclusions—These results demonstrate the feasibility of an antithrombotic strategy based on ribozyme technology.

Key words: restenosis • thrombosis • muscle, smooth

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