Conversion of thrombin into an anticoagulant by protein engineering.

At sites of vascular injury, thrombin interacts with multiple procoagulant substrates, to mediate both fibrin clotting and platelet aggregation. But upon binding to thrombomodulin on the vascular endothelium, thrombin instead activates protein C, thereby functioning as an anticoagulant and attenuating clot formation. Upon infusion in vivo, both the procoagulant and anticoagulant effects of thrombin were observed. Preliminary studies indicating that thrombin's protein C activating and fibrinogen clotting activities could be dissociated by mutagenesis suggested to us that a thrombin variant that lacked procoagulant activity while retaining anticoagulant function might be an attractive antithrombotic agent. Using protein engineering, we introduced a single substitution, E229A, that substantially shifted thrombin's specificity in favour of the anticoagulant substrate, protein C. In monkeys, this modified thrombin functioned as an endogenous protein C activator demonstrating dose-dependent, reversible anticoagulation without any indication of procoagulant activity. Notably, template bleeding times were not prolonged, suggesting a reduced potential for bleeding complications.

Plant regeneration from isolated microspores of Triticum aestivum.

Wheat microspores were isolated, without prior anther culture, from a range of genotypes and cultured to regenerate self-fertile plants. Microspores were isolated using a microblender and competent microspores were enriched by gradient centrifugation. The use of maltose as the sole carbohydrate in the culture medium and co-culture of microspores with wheat or barley ovaries were critical for development of microspore-derived embryos. Results were also improved when spikes were pretreated by emersion of the basal ends of detached heads in water at 25°C for 2d. This procedure leads to highly reproducible production of plants.