Development of Coagulation Factor XII Antibodies for Inhibiting Vascular Device-Related Thrombosis.

INTRODUCTION: Vascular devices such as stents, hemodialyzers, and membrane oxygenators can activate blood coagulation and often require the use of systemic anticoagulants to selectively prevent intravascular thrombotic/embolic events or extracorporeal device failure. Coagulation factor (F)XII of the contact activation system has been shown to play an important role in initiating vascular device surface-initiated thrombus formation. As FXII is dispensable for hemostasis, targeting the contact activation system holds promise as a significantly safer strategy than traditional antithrombotics for preventing vascular device-associated thrombosis. OBJECTIVE: Generate and characterize anti-FXII monoclonal antibodies that inhibit FXII activation or activity. METHODS: Monoclonal antibodies against FXII were generated in FXII-deficient mice and evaluated for their binding and anticoagulant properties in purified and plasma systems, in whole blood flow-based assays, and in an in vivo non-human primate model of vascular device-initiated thrombus formation. RESULTS: A FXII antibody screen identified over 400 candidates, which were evaluated in binding studies and clotting assays. One non-inhibitor and six inhibitor antibodies were selected for characterization in functional assays. The most potent inhibitory antibody, 1B2, was found to prolong clotting times, inhibit fibrin generation on collagen under shear, and inhibit platelet deposition and fibrin formation in an extracorporeal membrane oxygenator deployed in a non-human primate. CONCLUSION: Selective contact activation inhibitors hold potential as useful tools for research applications as well as safe and effective inhibitors of vascular device-related thrombosis.

WEDGE: an anticoagulant thrombin mutant produced by autoactivation.

BACKGROUND: The production of therapeutically relevant proteases typically involves activation of a zymogen precursor by external enzymes, which may raise regulatory issues about availability and purity. Recent studies of thrombin precursors have shown how to engineer constructs that spontaneously convert to the mature protease by autoactivation, without the need for external enzymes. OBJECTIVES: Autoactivation is an innovative strategy that promises to simplify the production of proteases of therapeutic relevance, but has not been tested in practical applications. The aim of this study was to provide a direct test of this strategy. METHODS: An autoactivating version of the thrombin mutant W215A/E217A (WE), which is currently in preclinical development as an anticoagulant, was engineered. RESULTS AND CONCLUSIONS: The autoactivating version of WE can be produced in large quantities, like WE made in BHK cells or Escherichia coli, and retains all significant functional properties in vitro and in vivo. The results serve as proof of principle that autoactivation is an innovative and effective strategy for the production of trypsin-like proteases of therapeutic relevance.

Termination of mammalian rDNA replication: polar arrest of replication fork movement by transcription termination factor TTF-I.

A replication fork barrier (RFB) at the 3' end of eukaryotic ribosomal RNA genes blocks bidirectional fork progression and limits DNA replication to the same direction as transcription. We have reproduced the RFB in vitro in HeLa cell extracts using 3' terminal murine rDNA fused to an SV40 origin-based vector. The RFB is polar and modularly organized, requiring both the Sal box transcription terminator and specific flanking sequences. Mutations within the terminator element, depletion of the RNA polymerase I-specific transcription termination factor TTF-I, or deletion of the termination domain of TTF-I abolishes RFB activity. Thus, the same factor that blocks elongating RNA polymerase I prevents head-on collision between the DNA replication apparatus and the transcription machinery.

RNA polymerase I transcription termination: similar mechanisms are employed by yeast and mammals.

Termination of RNA polymerase I (Pol I) transcription requires the interaction of a specific DNA binding factor with terminator elements downstream of the pre-rRNA coding region. Both the terminator elements and the respective termination factors are distinct in yeast and mammals, and differences in the mechanism of transcription termination have been postulated. We have compared in vitro transcription termination of yeast and mouse Pol I using both the murine factor TTF-I, and the yeast homolog Reb1p. We show that, similar to TTF-I, Reb1p was sufficient for pausing of Pol I from either species, but was unable to cause release of the nascent transcripts from the paused ternary complex. The deficiency of Reb1p to mediate transcript release from Pol I of either species was complemented by the recently characterized murine release factor. Thus, both yeast and mouse Pol I termination requires a trans-acting factor that, in conjunction with the T-rich flanking sequence, releases the transcripts and Pol I from the template. The observation that the murine factor causes dissociation of ternary transcription complexes arrested by Reb1p suggests that the mechanism of Pol I termination is highly conserved from yeast to mammals.