Characterization Of Low Molecular Weight Heparin-Platelet Factor 4 Complex

Heparin is a mixture of negatively charged linear polysaccharides extracted from porcine small intestinal mucosa,consisting of repeating disaccharide units formed by sulfated hexuronic acid(HexA)and α-D-glucosamine(GlcN).As an anticoagulant drug,heparin is widely used in the prevention and treatment of thrombosis and embolism.Some adverse reactions such as bleeding and heparin induced thrombocytopenia(HIT)may occur when heparin is used to patients.HIT can further lead to deep venous thrombosis and pulmonary embolism,which may threaten the patients’ life.Low-molecular-weight heparins(LMWHs),as the products of heparin degradation by chemical or enzymatic method,are also used to prevent and treat thromboembolic diseases.Compared with heparin,LMWHs have the advantages of high bioavailability,long half-life and less side effects.However,HIT may still occur in the application of LMWHs and lead to serious clinical consequences.HIT induced by heparin and LMWHs is closely related to platelet factor 4(PF4),which is an alkaline protein synthesized and released by platelet α particles.PF4 generally exists in the form of tetramer in the body,and it can combine with heparin or LMWHs into the human body to form PF4-heparin complex,trigger the immune response of the body and then mediate occurrence of HIT.The characteristics of the complex formed by PF4 tetramer and LMWHs(including the size,amount and charge of the complex)are closely related to the subsequent initiation of immune response and the occurrence of HIT.Therefore,it is critical to study the characteristics of the complexes formed by the interaction of LMWHs and PF4 to evaluate the immunogenicity of LMWHs and ensure the safety of drug use.The complexes formed by PF4 and LMWHs were studied from various perspectives using various analytical methods.First,molecular docking technology was used to simulate the docking of PF4 protein and heparin oligosaccharide,and the structure of complex formation between PF4 protein and heparin drugs was predicted theoretically;Secondly,size exclusion chromatography(SEC)was used to study the proportion of complex formed by PF4 and major three LMWHs(enoxaparin sodium,nadroparin calcium and dalteparin sodium)at different concentrations.Compared with enoxaparin sodium and nadroparin calcium,a smaller molar amount of dalteparin sodium can form more complex with PF4.Then,the complex formed after incubation of enoxaparin sodium and PF4 were qualitative and quantified by reversed phaseliquid chromatography-multiple reaction monitoring(RP-LC-MRM),and the results showed that the composition ratio of enoxaparin sodium and PF4 tetramer in the complex formed by the incubation of enoxaparin sodium and PF4 was about 1:2.Finally,the Zeta potential and particle size of the complex formed by PF4 and enoxaparin sodium at different concentrations were analyzed by nanoparticle size-potentiometry,and the particle size was compared with size of the complex formed by PF4 and heparin sodium at different concentrations.The results showed that the size and Zeta potential of the complex formed by PF4 and enoxaparin sodium at different concentrations were different.The particle size of the complex formed by PF4 and heparin at low concentrations was larger than that of enoxaparin sodium.The results of this paper provide a scientific basis for further understanding the mechanism of LMWHs immunogenicity,and have certain value for improving the quality and safety of LMWHs drugs in China.