Anticoagulant Activity Of Heparan Sulfate From Cerebral Microvascular Tissue

Abstract

Microvascular tissue (MT) was isolated by standard sieving techniques from the cerebral cortex of yearling calves. After proteolysis of MT, the cell debris was pelleted and the supernatant was charged to DEAE-Sepharose A-25 equilibrated with 0.1 M NaCl in 0.01 M Tris-HCl, pH 7.5. Uronic acid determination demonstrated that two peaks of mucopolysaccharides (MS) were eluted at an added salt concentration of 0.4 M NaCl and 0.8 M NaCl, respectively. Enzymatic and electrophoretic analyses of the first peak revealed the presence of a minimally sulfated MS with no ability to accelerate thrombin or factor Xa neutralization by antithrombin (AT). MS isolated from the second peak were degraded by chondroitin ABC lyase and Flavobacterium heparinase. This material migrated slightly behind NIH standard heparan sulfate (HS) and distinctly separately from NIH standard heparin (Hp) which had much greater electrophoretic mobility. Sequential degradation with chondroitinase and heparinase revealed that HS was responsible for the anticoagulant activity of the second peak. The antithrombin and anti-factor X activities of HS were 2.81 u/mg and 2.86 u/mg, respectively. Affinity fractionation of HS with AT resulted in an increase of antithrombin and anti-factor X activities to 36.6 u/mg (13 fold augmentation) and 41.3 u/mg (15 fold augmentation), respectively. Each of the biological activities required the presence of AT and disappeared upon exposure to heparinase. Electrophoretic analysis of the affinity fractionated material demonstrated that it migrated slightly behind NIH standard HS and 40% slower than NIH standard Hp. In conclusion, the ionic strength at which this affinity fractionated HS had been eluted from DEAE-Sepharose and its mobility during electrophoresis clearly distinguish it from classical Hp. These data demonstrate the existence of a specific species of HS from MT with significant anticoagulant activity. These components may in part, be responsible for the non- thrombogenic properties of vascular elements.