Heparin cofactor II(Oslo). Mutation of Arg-189 to His decreases the affinity for dermatan sulfate

M. A. Blinder, T. R. Andersson, U. Abildgaard, D. M. Tollefsen

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Abstract

Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) ~1000-fold by providing a catalytic template to which both the inhibitor and the proteinase bind. A variant form of HCII that binds heparin but not dermatan sulfate has been described recently in two heterozygous individuals (Andersson, T.R., Larsen, M.L., and Abildgaard, U. (1987) Thromb. Res. 47, 243-248). We have now purified the variant HCII (designated HCII(Oslo)) from the plasma of one of these individuals. HCII(Oslo) or normal HCII (11 nM) was incubated with thrombin (9 nM) for 1 min in the presence of heparin or dermatan sulfate. Fifty percent inhibition of thrombin occurred at 26 μg/ml dermatan sulfate with normal HCII and >1600 μg/ml dermatan sulfate with HCII(Oslo). In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 μg/ml) with both inhibitors. To identify the mutation in HCII(Oslo), DNA fragments encoding the N-terminal 220 amino acid residues of HCII were amplified from leukocyte DNA by the Taq DNA polymerase chain reaction and both alleles were cloned. A point mutation (G → A) resulting in substitution of His for Arg-189 was found in one allele. The same mutation was constructed in the cDNA of native HCII by oligonucleotide-directed mutagenesis and expressed in Escherichia coli. The recombinant HCII(His-189) reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCII(Oslo).

Original languageEnglish
Pages (from-to)5128-5133
Number of pages6
JournalJournal of Biological Chemistry
Volume264
Issue number9
StatePublished - 1989

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