Two-dimensional affinity resolution electrophoresis demonstrates that three distinct heparin populations interact with antithrombin III.
Heparin is a polydisperse, highly sulfated polysaccharide consisting of repeating 1-->4 linked uronic acid and glucosamine sugar residues that binds to coagulation proteins, complement proteins, and growth factors to regulate a variety of biological activities. Heparin is best known as an anticoagulant, an activity that results largely from a specific pentasaccharide sequence in heparin that interacts with a unique site in antithrombin III. Little is known about additional structures within heparin that might interact with antithrombin III or the heparin structures that interact with the myriad of other heparin-binding proteins and peptides. Unfractionated glycosaminoglycan heparin that had been prepared from porcine intestinal mucosa was examined for its capacity to bind antithrombin III using a new technique developed to quantitate that interaction. Two-dimensional affinity resolution electrophoresis is a powerful method that allows assessment of unique species of heparin molecules that bind to protein, allowing determination of heparin molecular weight for each protein-binding heparin species as well as the dissociation constant of each interaction. This study provides the first definitive evidence that glycosaminoglycan heparin contains at least three populations of molecules with affinity for antithrombin III. Furthermore, the affinity of each heparin species for antithrombin III appears to vary inversely with the size of the heparin chain, with some smaller oligosaccharides having greater affinity for antithrombin III than larger oligosaccharides.
Edens
R E
RE
Iowa City VA Medical Center, Iowa.
Fromm
J R
JR
Fromm
S J
SJ
Linhardt
R J
RJ
Weiler
J M
JM
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism*; D001665:Binding Sites; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D006025:Glycosaminoglycans / Q000737:chemistry; D006493:Heparin / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D066298:In Vitro Techniques; D007413:Intestinal Mucosa / Q000737:chemistry; D015394:Molecular Structure; D013552:Swine
D006025:Glycosaminoglycans; D000990:Antithrombin III; D006493:Heparin