Isolation of endogenous anticoagulant N-sulfated glycosaminoglycans in human plasma from healthy subjects.
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.
Ruggiero
Marco
M
Department of Experimental Pathology and Oncology, University of Florence, Italy.
Melli
Michele
M
Parma
Bruna
B
Bianchini
Pietro
P
Vannucchi
Simonetta
S
D000818:Animals; D000925:Anticoagulants / Q000097:blood / Q000302:isolation & purification* / Q000592:standards; D001685:Biological Factors / Q000097:blood / Q000302:isolation & purification / Q000592:standards; D001780:Blood Coagulation Tests; D002417:Cattle; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005343:Fibrinolytic Agents / Q000097:blood / Q000302:isolation & purification* / Q000592:standards; D006025:Glycosaminoglycans / Q000097:blood / Q000302:isolation & purification* / Q000592:standards; D006493:Heparin / Q000097:blood / Q000302:isolation & purification / Q000592:standards; D006801:Humans; D008722:Methods; D008970:Molecular Weight; D009608:Nitrous Acid / Q000737:chemistry; D011506:Proteins / Q000378:metabolism; D012015:Reference Standards
D000925:Anticoagulants; D001685:Biological Factors; D005343:Fibrinolytic Agents; D006025:Glycosaminoglycans; D011506:Proteins; D006493:Heparin; D009608:Nitrous Acid