The sequence HGLGHGHEQQHGLGHGH in the light chain of high molecular weight kininogen serves as a primary structural feature for zinc‐dependent binding to an anionic surface

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Abstract
The histidine-glycine-rich region of the light chain of cleaved high molecular weight kininogen (HK) is thought to be responsible for binding to negatively charged surfaces and initiation of the intrinsic coagulation, fibrinolytic, and kinin-forming systems. However, the specifically required amino acid sequences have not been delineated. An IgG fraction of a monoclonal antibody (MAb) C11C1 to the HK light chain was shown to inhibit by 66% the coagulant activity and by 57% the binding of HK to the anionic surface of kaolin at a concentration of 1.5 μM and 27 μM, respectively. Proteolytic fragments of HK were produced by successive digestion with human plasma kallikrein and factor XIa (FXIa). Those polypeptides that bound tightly (Kd = 0.77 nM) to a C11C1 affinity column were eluted at pH 3.0 and purified by membrane filtration. On 15% SDS polyacrylamide electrophoresis, the approximate M was 7.3 kDa (range 6.2–8.1 kDa). Based on N-terminal sequencing, this polypeptide (l2), which extends from the histidine residue 459 to a lysine at position 505, 509, 511, 512, 515, or 520, inhibits by 50% the coagulant activity expressed by HK at a concentration of 22 μM. The synthetic peptide HGLGHGH representing the N-terminal of the l2 fragment was synthesized, tested, and found at 4 mM to inhibit the procoagulant activity of HK 50%. A synthetic heptadecapeptide, HGLGHGHEQQHGLGHGH (residues 459–475) included within the l2 fragment, and with the ability to bind zinc, inhibited 50% of the HK coagulant activity at a concentration of 325 μM in the absence and presence of added Zn2+ (30 μM). The specific binding of 125I-HK to a negatively charged surface (kaolin) was inhibited 50% by unlabeled HK (5 μM). HGLGHGH, at a concentration of 7.0 mM, inhibited the binding to kaolin by 50%. The heptadecapeptide inhibited the specific binding of 125I-HK to kaolin by 50%, at a concentration of 2.3 mM, in the absence of Zn2+. In contrast, when Zn2+ was added, the concentration to achieve 50% inhibition decreased to 630 μM, indicating that Zn2+ was required to attain a favorable conformation for binding. Moreover, the l2 fragment was found to inhibit 50% of the 125I-HK binding to kaolin at a concentration of 380 μM. These results suggest that residues contained within the l2 fragment, notably HGLGHGHEQQHGLGHGH, serves as a primary structural feature for binding to a negatively charged surface.
Funding Information
  • National Institute of Health (HL07795)
  • Biomedical Research (S07RR05417)
  • Minority Investigator Research Supplement (HL36579)
  • HL SCOR in Thrombosis (HL45486)
  • American Federation for Clinical Research
  • Temple University (Philadelphia, Pennsylvania
  • Jefferson University
  • Biomedical Research Centre
  • University of British