Characterization of a genetically engineered inactivation-resistant coagulation factor VIIIa

Individuals with hemophilia A require frequent infusion of preparations of coagulation factor VIII. The activity of factor VIII (FVIII) as a cofactor for factor IXa in the coagulation cascade is limited by its instability after activation by thrombin. Activation of FVIII occurs through proteolytic cleavage and generates an unstable FVIII heterotrimer that is subject to rapid dissociation of its subunits. In addition, further proteolytic cleavage by thrombin, factor Xa, factor IXa, and activated protein C can lead to inactivation. We have engineered and characterized a FVIII protein, IR8, that has enhanced in vitro stability of FVIII activity due to resistance to subunit dissociation and proteolytic inactivation. FVIII was genetically engineered by deletion of residues 794-1689 so that the A2 domain is covalently attached to the light chain. Missense mutations at thrombin and activated protein C inactivation cleavage sites provided resistance to proteolysis, resulting in a single-chain protein that has maximal activity after a single cleavage after arginine-372. The specific activity of partially purified protein produced in transfected COS-1 monkey cells was 5-fold higher than wild-type (WT) FVIII. Whereas WT FVIII was inactivated by thrombin after 10 min in vitro, IR8 still retained 38% of peak activity after 4 hr. Whereas binding of IR8 to von Willebrand factor (vWF) was reduced 10-fold compared with WT FVIII, in the presence of an anti-light chain antibody, ESH8, binding of IR8 to vWF increased 5-fold. These results demonstrate that residues 1690–2332 of FVIII are sufficient to support high-affinity vWF binding. Whereas ESH8 inhibited WT factor VIII activity, IR8 retained its activity in the presence of ESH8. We propose that resistance to A2 subunit dissociation abrogates inhibition by the ESH8 antibody. The stable FVIIIa described here provides the opportunity to study the activated form of this critical coagulation factor and demonstrates that proteins can be improved by rationale design through genetic engineering technology.

The influence of the flow of the reacting gas on the conditions for a thermal explosion

The classical problem of thermal explosion is modified so that the chemically active gas is not at rest but is flowing in a long cylindrical pipe. Up to a certain section the heat-conducting walls of the pipe are held at low temperature so that the reaction rate is small and there is no heat release; at that section the ambient temperature is increased and an exothermic reaction begins. The question is whether a slow reaction regime will be established or a thermal explosion will occur. The mathematical formulation of the problem is presented. It is shown that when the pipe radius is larger than a critical value, the solution of the new problem exists only up to a certain distance along the axis. The critical radius is determined by conditions in a problem with a uniform axial temperature. The loss of existence is interpreted as a thermal explosion; the critical distance is the safe reactor’s length. Both laminar and developed turbulent flow regimes are considered. In a computational experiment the loss of the existence appears as a divergence of a numerical procedure; numerical calculations reveal asymptotic scaling laws with simple powers for the critical distance.

Activation of a protease cascade involved in patterning the Drosophila embryo

Dorsoventral patterning of the Drosophila embryo is initiated by a ventralizing signal. Production of this signal requires the serine proteases Gastrulation Defective (GD), Snake, and Easter, which genetic studies suggest act sequentially in a cascade that is activated locally in response to a ventral cue provided by the pipe gene. Here, we demonstrate biochemically that GD activates Snake, which in turn activates Easter. We also provide evidence that GD zymogen cleavage is important for triggering this cascade but is not spatially localized by pipe. Our results suggest that a broadly, rather than locally, activated protease cascade produces the ventralizing signal, so a distinct downstream step in this cascade must be spatially regulated to restrict signaling to the ventral side of the embryo.