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Several letters to the editor are presented in response to articles in previous issues including the review of the book "The Called and the Chosen One," by Monica Baldwin, the U.S. visit of Queen Elizabeth and Prince Phillip, and the International Industrial Development Conference.

A significant fraction (30%) of the genetically determined variance in plasma concentration of the von Willebrand factor antigen (vWf:Ag) has been shown to be related to ABH determinants. Individuals with blood group O, who have the highest amounts of blood group H substance, have the lowest concentration of vWf:Ag. The Lewis substances, Le(a) and Le(b), are biochemically closely related to the ABH substances as both can be produced from the same precursor substance. We studied the effect of the presence of the Lewis antigens on the plasma concentration of vWf:Ag and factor VIII antigen (VIII:Ag) in 323 individuals of different ABO groups from a series of twins and in 58 blood donors of blood group O. Among persons belonging to blood group O, those with the Le(a) antigen had a higher concentration of both vWf:Ag and VIII:Ag than individuals lacking Le(a). Le(a+b-) people are nonsecretors and Le(a-b+) people are secretors of ABH substance. Thus, the lowest concentration of vWf:Ag and VIII:Ag was found in group O secretors. The effect is most likely due to an effect of the secretor locus. This finding may be of importance for the detection of carriers of hemophilia A and for the diagnosis of type I von Willebrand disease.

We used recombinant DNA techniques to map a panel of six monoclonal antibodies (MoAbs) to regions of the human factor IX molecule. A-2 maps to 17 amino acids at the amino terminus of the heavy chain of IXa; 2D5, an inhibitor of clotting, is defined to 36 amino acids of the first EGF- like domain of human factor IX. A-4, A-5, C10D, and FXC008 all map to a region of the heavy chain containing amino acids 180 through 310, suggesting an immunodominant site. FXC008 has been reported to interfere with binding of factor IXa to factor VIII:Ca.

As an approach to the study of structure-function relationships in the normal and defective forms of human coagulation factor IX, we have begun to develop a series of monoclonal antibodies against specific sites on the protein. Zymogen and activated forms of normal factor IX were used initially as antigen for the preparation of monoclonal antibodies. Recombinant phage were prepared by cloning small (50- to 500-nucleotide) random DNA fragments from the coding region of a factor IX cDNA clone into the expression vector lambda gt11. Immunological screening of these recombinants with mixtures of monoclonal antibodies identified several immunoreactive phage. Further analysis showed that the monoclonal antibody designated IX-30 was generating the positive signals at a frequency of approximately 1/2,500 recombinants. Subcloning and sequence analysis of the inserted DNA in the immunoreactive phage revealed overlapping in-frame insertions, from which it could be inferred that the site in factor IX recognized by IX- 30 is confined to residues 111 through 132 in the light chain. Similar mapping with other monoclonal antibodies should provide additional probes for the protein structure of human factor IX.

We have used the polymerase chain reaction to amplify the entire coding region of canine factor IX from a hemophilia B animal. When the sequence was compared to that which codes for normal canine factor IX, a single missense mutation was identified. This mutation (G----A at nucleotide 1477) results in the substitution of glutamic acid for glycine-379 in the catalytic domain of the molecule. The mutation creates a new restriction site that allowed confirmation of the abnormal sequence in both hemophilic and carrier animals. Amino acid 379 in canine factor IX corresponds to position 381 in human factor IX, a location at which no human mutations have been described. Moreover, it occurs at one of the few amino acids that have been rigorously conserved among the trypsin-like serine proteases throughout evolution. The mutation responsible for canine hemophilia B results in a complete lack of circulating factor IX in the affected animals. As it is unusual for a missense mutation to result in a complete absence of protein product, structural modeling of the mutant and normal proteins was pursued. These studies suggest that the observed mutation would have major adverse effects on the tertiary structure of the aberrant factor IX molecule. The elucidation of this mutation sheds light on structure-function relationships in factor IX and should facilitate future experiments directed toward gene therapy of this disease.

Anti-factor VIII antibodies from three hemophilia A patients were selectively isolated by immune complex formation and secondary precipitation with polyethylene glycol. These antibodies were characterized as IgG, with only one type of light chains (kappa) and a mixture of IgG3 and IgG4 heavy chains. This finding indicated that stable complexes of factor VIII and homologous anti-factor VIII antibodies could be obtained, and that the immunologic response to the infusion of homologous factor VIII was oligoclonal rather than monoclonal in these patients.