New insights into the binding of PF4 to long heparin oligosaccharides in ultralarge complexes using mass spectrometry
Authors:
Deling Shi 1 2, Huimin Zhao 1, Changkai Bu 1, Keith Fraser 2 3, Haoran Wang 1, Jonathan S. Dordick 2 3 4 5, Robert J. Linhardt 2 3 4 6, Fuming Zhang 2 4, Feng Shi 7, Lianli Chi 1
Affiliation:
1National Glycoengineering Research Center, Shandong University, Qingdao, Shandong Province, China
2Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, USA
3Department of Biological Sciences, Rensselaer Polytechnic Institute, Troy, New York, USA
4Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA
5Department of Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York, USA
6Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York, USA
7Shandong Institute for Food and Drug Control, Jinan, Shandong Province, China
Description:
Background
Heparin-induced thrombocytopenia (HIT) is a serious complication caused by heparin drugs. The ultralarge complexes formed by platelet factor 4 (PF4) with heparin or low molecular weight heparins (LMWHs) are important participants in inducing the immune response and HIT.
Objectives
We aim at characterizing the interaction between PF4 and long-chain heparin oligosaccharides and providing robust analytical methods for the analysis of PF4–heparin complexes.
Methods
In this work, the characteristics of PF4–enoxaparin complexes after incubation in different molar ratios and concentrations were analyzed by multiple analytical methods, especially liquid chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry with multiple reaction monitoring were developed to qualitatively and quantitatively monitor heparin oligosaccharides and PF4 in HIT-inducing complexes.
Results
The results showed that the largest proportion of ultralarge complexes formed by PF4 and enoxaparin was at a specific molar ratio, ie, a PF4/enoxaparin ratio of 2:1, while the ultralarge complexes contained PF4 tetramer and enoxaparin at a molar ratio of approximately 2:1.
Conclusion
A binding model of PF4 and enoxaparin in ultralarge complexes is proposed with one heparin oligosaccharide chain (∼ dp18) bound to 2 PF4 tetramers in different morphologies to form ultralarge complexes, while PF4 tetramer is surrounded by multiple heparin chains in smaller complexes. Our study provides new insights into the structural mechanism of PF4–LMWH interaction, which help to further understand the mechanism of LMWH immunogenicity and develop safer heparin products.
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