Anuradha Kumar

The dysfunction of coagulation factor VIIPadua results from substitution of arginine-304 by glutamine

This study addresses whether a mutation in the factor VIIPadua gene could explain the reduced activity of the inherited variant protein. All nine exons of the normal and Padua factor VII gene were amplified using the polymerase chain reaction, cloned into pUC19 and sequenced. A point mutation (G to A at nucleotide position 10828) was found which results in the substitution of a glutamine (CAG) for arginine (CGG) at amino acid position 304. This substitution creates a PvuII restriction site useful in screening for the defect and in demonstrating homozygosity. This substitution involves an arginine residue in the catalytic domain within a Leu*****Pro******Cys motif which occurs in conserved region 5 in up to 16 coagulation and other serine proteinases. On the basis of conformational homology among serine proteinases, it is suggested that the observed amino acid substitution in factor VIIPadua could cause structural changes affecting its activation and/or catalytic activity.

Inhibition of factor Xa-mediated procoagulant activity of human lung fibroblasts and pleural mesothelial cells.

Extravascular fibrin deposition characterizes diverse forms of lung and pleural injury. Fibrin formation in these compartments is locally potentiated by the assembly and expression of the prothrombinase procoagulant complex (factors Xa, Va and II) at the surface of human lung fibroblasts and pleural mesothelial cells. We sought to identify structural domains on factor Xa that mediate expression of prothrombinase activity by these cells. In order to accomplish this objective, we used panels of monoclonal antibodies (MoAbs) to factor X to block prothrombinase assembly and function on the surface of cultured human lung fibroblasts and pleural mesothelial cells. Of 30 factor X MoAbs that recognized native factors X and Xa, 10 completely inhibited factor Xa function (prothrombin activation), and five others neutralized Xa function without affecting cell-binding, presumably by blocking the prothrombin binding site. Western blots showed that these inhibitory MoAbs reacted with the Xa heavy-chain. One MoAb that recognized the factor Xa light-chain blocked prothrombin activation at the factor Va binding site. Our results indicate that prothrombinase activity at the surface of lung parachymal or pleural cells can be blocked by MoAbs that interact with either the heavy- or light-chain of factors X. Antibodies that neutralize cell surface-expressed prothrombin activation offer a potential means to arrest pericellular fibrin formation in the lung and pleural space.