Isolation of endogenous anticoagulant N-sulfated glycosaminoglycans in human plasma from healthy subjects

• Published in: Pathophysiology of haemostasis and thrombosis Vol. 32; no. 1; p. 44
• Main Authors: Ruggiero, Marco, Melli, Michele, Parma, Bruna, Bianchini, Pietro, Vannucchi, Simonetta
• Format: Journal Article
• Language: English
• Published: Switzerland 01.01.2002
• Subjects: Animals; Anticoagulants - blood; Anticoagulants - isolation & purification; Anticoagulants - standards; Biological Factors - blood; Biological Factors - isolation & purification; Biological Factors - standards; Blood Coagulation Tests; Cattle; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Fibrinolytic Agents - blood; Fibrinolytic Agents - isolation & purification; Fibrinolytic Agents - standards; Glycosaminoglycans - blood; Glycosaminoglycans - isolation & purification; Glycosaminoglycans - standards; Heparin - blood; Heparin - isolation & purification; Heparin - standards; Humans; Methods; Molecular Weight; Nitrous Acid - chemistry; Proteins - metabolism; Reference Standards
• ISSN: 1424-8832
• DOI: 10.1159/000057288

Endogenous N-sulfated glycosaminoglycans (GAGs) comigrating with standard heparin and sensitive to nitrous acid treatment were isolated from plasma of healthy donors. The amount of these compounds was 7-10 microg/ml, and activated partial thromboplastin time, anti-Xa and anti-IIa activities were similar to those of standard heparin of high molecular mass. Analysis with gradient PAGE of the putative endogenous heparin showed a mean molecular mass of 12 kD. These N-sulfated GAGs could be isolated only after removal of binding peptides that impaired purification by ion-exchange chromatography. We used SDS-PAGE as a tool to separate peptides from endogenous GAGs. N-sulfated GAGs exited the gel before peptides when the electrophoresis was overrun. Endogenous GAGs could be recovered by ion-exchange chromatography of the SDS-PAGE buffer, 'free' from associating peptides. These results strongly support the hypothesis that endogenous heparin is associated in vitro with a variety of proteins and that this association could be responsible for modification of both heparin and protein activities.