Identification of protein C epitopes altered during its nanoencapsulation.
Protein C is a plasmatic inhibitor which regulates the blood coagulation mechanism by modulating the anticoagulant response. The improvement of its bioavailability would be beneficial for the treatment of the disorders caused by its homozygous deficiency or by an other plasmatic inhibitor deficiency. In this context, the protein C encapsulation into biodegradable nanoparticles could be used to approach the problem. However, the method used to prepare the nanoparticles requires the use of ultrasonication and of an organic solvent such as methylene chloride which interferes with protein activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that neither ultrasonication nor methylene chloride, singly or in combination, led to protein C aggregation or cleavage. Thus, a binding study using an ELISA assay with four characterized monoclonal antibodies was carried out to identify the epitopes damaged by these experimental constraints. The correlation between the immunological assay and a functional one i.e. by the means of the assay of its anticoagulant activity (activated partial thromboplastin time) made it possible to show that protein C amino acids 166-169 of the activation peptide were probably altered after ultrasonication and methylene chloride treatment. Indeed, it is likely that these residues were no longer surface-exposed but had moved inside the protein core.
Chognot
D
D
Laboratoire d'Hématologie-Physiologie, Faculté de Pharmacie, Nancy, France.
Zambaux
M F
MF
Bonneaux
F
F
Gaussem
P
P
Pittet
J L
JL
Aiach
M
M
Vigneron
C
C
D000925:Anticoagulants / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics; D001682:Biological Availability; D002942:Circular Dichroism; D004339:Drug Compounding; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry*; D008752:Methylene Chloride / Q000737:chemistry; D011486:Protein C / Q000008:administration & dosage / Q000737:chemistry* / Q000493:pharmacokinetics; D011487:Protein Conformation; D014465:Ultrasonics
D000925:Anticoagulants; D000939:Epitopes; D011486:Protein C; D008752:Methylene Chloride