Blinder M A, Andersson T R, Abildgaard U, Tollefsen D M
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1989 Mar 25;264(9):5128-33.
Heparin and dermatan sulfate increase the rate of inhibition of thrombin by heparin cofactor II (HCII) approximately 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 HCIIOslo) from the plasma of ne of these individuals. HCIIOslo 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 micrograms/ml dermatan sulfate with normal HCII and greater than 1600 micrograms/ml dermatan sulfate with HCIIOslo. In contrast, inhibition of thrombin occurred at a similar concentration of heparin (1.0-1.5 micrograms/ml) with both inhibitors. To identify the mutation in HCIIOslo, 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 HCIIHis-189 reacted with thrombin in the presence of heparin but not dermatan sulfate, confirming that this mutation is responsible for the functional abnormality in HCIIOslo.
肝素和硫酸皮肤素通过提供一个抑制剂和蛋白酶均可结合的催化模板，使肝素辅因子II（HCII）对凝血酶的抑制速率提高约1000倍。最近在两名杂合个体中描述了一种结合肝素但不结合硫酸皮肤素的HCII变异形式（安德森，T.R.，拉森，M.L.，和阿比尔德加德，U.（1987年）《血栓形成研究》47卷，243 - 248页）。我们现在已从其中一名个体的血浆中纯化出了变异的HCII（命名为HCII奥斯陆）。将HCII奥斯陆或正常HCII（11纳摩尔）与凝血酶（9纳摩尔）在肝素或硫酸皮肤素存在的情况下孵育1分钟。正常HCII在26微克/毫升硫酸皮肤素时可使凝血酶抑制50%，而HCII奥斯陆则需大于1600微克/毫升硫酸皮肤素。相反，两种抑制剂在相似浓度的肝素（1.0 - 1.5微克/毫升）下均可抑制凝血酶。为了鉴定HCII奥斯陆中的突变，通过Taq DNA聚合酶链反应从白细胞DNA中扩增出编码HCII N端220个氨基酸残基的DNA片段，并将两个等位基因进行克隆。在一个等位基因中发现了一个导致His替代Arg - 189的点突变（G→A）。通过寡核苷酸定向诱变在天然HCII的cDNA中构建了相同的突变，并在大肠杆菌中表达。重组的HCIIHis - 189在肝素存在下可与凝血酶反应，但在硫酸皮肤素存在下则不能，证实该突变是HCII奥斯陆功能异常的原因。
