Reprint requests to: Ernest Beutler, M.D., Department of Molecular and Experimental Medicine, Scripps Research Institute, 10666 N. Torrey Pines, La Jolla, CA 92037.

ABSTRACT. Among glycolytic enzyme defects, hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1; HK) deficiency is a very rare disease where the predominant clinical effect is nonspherocytic hemolytic anemia. Here we report the characterization at molecular level of the HK type I cDNA from a patient with hemolytic anemia due to hexokinase deficiency. PCR amplification and sequence of the cDNA revealed the presence of a deletion and of a single nucleotide substitution, both in heterozygous form. In particular, the deletion, 96 bp long, concerns nucleotides 577 to 672 in the HK cDNA sequence and was never found in the cDNAs of 14 unrelated normal subjects. The sequence of the HK allele without deletion showed a single nucleotide substitution from T to C at position 1667 which causes the amino acid change from Leu529 to Ser. This heterozygous mutation at nt 1667 was confirmed by direct sequencing of the patient genomic DNA, but when DNAs from 10 normal controls were examined by this technique the substitution at nt 1667 was never found. From these results we concluded that the patient is carrying a point mutation at nt 1667 of one HK allele and a 96 nt deletion in the other allele. In normal subjects two differences from the published cDNA sequence were documented.

Reprint requests to: Professor M. Magnani, Instituto Chimica Biologica, "G. Fornaini," Università degli Studi Urbino, Via Saffi 2, 61029-Urbino, ITALY; phone +39-722-305211, fax +39-722-320188.

ABSTRACT. Adherence and aggregation of leukocytes within the vessels of a prepared skin site has been shown to be essential for the pathogenesis of the local Shwartzman reaction (LSR)(Argenbright and Barton: J Clin Invest 89: 259, 1992). We have now performed experiments in rabbits to evaluate whether coagulation within the vessels of a prepared site is a second requirement for the reaction. Skin sites were prepared by an intradermal injection of endotoxin 24 hours before a provoking intravenous injection of endotoxin. Thirteen control rabbits all developed the LSR. Seven of 12 rabbits given warfarin to achieve anticoagulation approximating that used therapeutically in humans before the provoking injection were protected against the LSR (p=0.003). Five of nine rabbits given anti-rabbit factor X IgG before the provoking injection to yield mean values in individual rabbits of between 7% and 18% plasma factor X activity were protected against the LSR (p=0.009). Six of 11 rabbits given anti-rabbit factor VII IgG before the provoking injection to yield mean values in individual rabbits of between <0.5% and 2.2% were protected against the LSR (p=0.007). Four rabbits failed to develop the LSR at an endotoxin-prepared skin site when an infusion of tissue factor (TF) causing substantial intravascular coagulation was substituted for a provoking injection of endotoxin. It would appear that two events are required for the pathogenesis of the LSR provoked by endotoxin: formation of aggregated masses of WBC in the prepared skin vessels and deposition of fibrin due to TF-initiated coagulation.

Reprint requests to:  Samuel I. Rapaport, M.D., UCSD Medical Center, Mail Code 8423, 200 West Arbor Drive, San Diego, CA 92103; phone (619) 543-3552, fax (619) 543-3231.