Immobilization of aprotinin to fibrinogen as a novel method for controlling degradation of fibrin gels

Bioconjug Chem. 2007 May-Jun;18(3):695-701. doi: 10.1021/bc060265o. Epub 2007 Apr 14.

Abstract

The goal of this work was to demonstrate that aprotinin conjugated to fibrinogen could (1) maintain its function and (2) control fibrin degradation. Using the chick chorioallantoic membrane (CAM) assay, we found that blood vessels did not directly invade fibrin constructs containing immobilized fibroblast growth factor-2. Because the fibrin quickly degraded within approximately 5 days, we hypothesized that controlling fibrinolysis may improve direct blood vessel invasion. Aprotinin, a protease inhibitor typically added to slow fibrinolysis, is a small protein and can diffuse out of the gel resulting in the loss of fibrinolysis protection. Therefore, using a novel synthesis strategy, aprotinin and a fluorescent reporter, Cy3, were chemically conjugated to fibrinogen. In vitro microplate absorbance assays showed that the conjugated aprotinin was able to inhibit plasmin-mediated fibrin degradation and that its activity was comparable to equimolar levels of soluble, nonconjugated aprotinin. Additionally, we found that fibrinolysis rates could be tuned by varying the level of conjugated aprotinin within the gel. The conjugated aprotinin also demonstrated functionality in vivo. In the chick CAM assay, fibrin gels containing conjugated aprotinin were approximately 5 times larger than gels containing soluble aprotinin after 4 days. Also, in support of our hypothesis, we found that immobilized aprotinin within fibrin gels demonstrated substantial blood vessel invasion.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Aprotinin / chemical synthesis
  • Aprotinin / chemistry*
  • Blood Vessels / drug effects
  • Carbocyanines / chemical synthesis
  • Carbocyanines / chemistry*
  • Chick Embryo
  • Chorioallantoic Membrane / blood supply
  • Chorioallantoic Membrane / chemistry
  • Fibrin / chemistry*
  • Fibrinogen / analogs & derivatives*
  • Fibrinogen / chemical synthesis
  • Fibrinogen / chemistry
  • Fibrinolysin / antagonists & inhibitors
  • Fibrinolysis
  • Gels / chemistry
  • Gels / pharmacology
  • Serine Proteinase Inhibitors / chemistry*
  • Tissue Engineering / methods*

Substances

  • Carbocyanines
  • Gels
  • Serine Proteinase Inhibitors
  • Fibrin
  • Fibrinogen
  • Aprotinin
  • Fibrinolysin