Behavior of Rabbit Antithrombin III at the Surface of the Normal and Deendothelialized Rabbit Thoracic Aorta in Vitro and in Vivo

  • M. W. C. Hatton
  • S. L. Moar
  • M. Richardson
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)


A study in vitro of the interaction between radiolabeled rabbit antithrombin III (AT-III) and the luminal surface of rabbit thoracic aorta segments revealed that AT-III does not bind significantly to the endothelium but. nevertheless, crosses the endothelium relatively rapidly (1.8 x 108 molecules/cm2 per minute using an initial [ 125I]AT-III concentration of 2.2 pmol/ml) and accumulates in the suben-dothelium. Removal of the aorta endothelium (by balloon catheter injury after exsanguination) allowed a more rapid uptake of [125T]AT-III (5.5 × 108 molecules/cm2 per min) by the denuded intima. Pretreatment of the deendothelialized aorta with purified glycosaminoglycan-specific enzymes decreased the uptake of [125I]AT-III either by 30–40% (chondroitinase AC or ABC) or by 80% (heparitinase) relative to uptake by buffer-treated, deendothelialized vessel segments. Furthermore, AT-III adsorbed to the deendothelialized intima reacted rapidly with, and was displaced by, thrombin to yield thrombin-AT-III: 50% of bound [125I]AT-IΠ (2.80 fmol/cm2) was dislodged by 5.0 nM thrombin in 5 min at 37°C.

The uptake of AT-III by aorta segments in vitro was then compared with results from in vivo studies. After i.v. injection of [125I]AT-III. the aorta endothelium was saturated rapidly (0.2 pmol/cm2); the permeability of the endothelium allowed [125I]AT-III to accumulate in the subendothelium at 3 × 109 molecules/cm2 per min. Balloon catherization led to a rapid uptake of [125I]AT-III by the denuded intimal surface, approaching a steady state (4.3 pmol bound/cm2) at approximately 80 min after injury. The distribution ratio of aorta-bound [125I]AT-III between the adherent platelet-white cell layer and the underlying deendothelialized intima varied from approximately 0.20 (at 30 min) to 0.03 (at 60 min) after balloon injury. Binding of [125I]AT-III by the adherent platelet-white cells at the subendothelial surface was negligible at 100 min after balloon injury. Uptake of [125I]AT-III injected at 40–80 min after balloon injury by the underlying intimal surface was not obstructed by the platelet layer; thus, the platelet layer allowed free passage of [125I]AT-III into the intima. Administration of heparin (i.v.; 1000 u/kg) at 20 min after [125I] AT-III injection to the balloon-injured rabbit caused a significant decrease (∼50%) of aorta-bound AT-III-during a 20-min circulation time. Furthermore, modification (carbamylation) of lysine residues of AT-III, which destroyed the heparin-binding property but retained the antithrombin activity, correlated with a substantially decreased uptake of the [125I]-labeled derivative by the subendothelium from experiments in vivo and in vitro.

The experiments in vitro suggest that proteoheparin sulfate binding sites of the subendothelium, which are exposed by a deendothelializing injury, attract AT-III for the purpose, presumably, of inactivating surplus thrombin (or factor Xa) generated during hemostatic imbalance; in contrast, AT-III is not bound by the uninjured endothelial surface. Our experiments indicate that similar events may also take place in vivo.


Luminal Surface Anticoagulant Activity Endothelial Surface Rabbit Aorta Balloon Injury 
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Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • M. W. C. Hatton
    • 1
  • S. L. Moar
    • 1
  • M. Richardson
    • 1
  1. 1.Department of PathologyMcMaster University Health Sciences CentreHamiltonCanada

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