Thrombin and Factor Xa Uptake and Inhibition by Cultured Bovine Aortic Endothelial Cells, Smooth Muscle Cells, and Rat Hepatoma Cells

  • Maciej Dryjski
  • Thorir D. Bjornsson
Part of the GWUMC Department of Biochemistry Annual Spring Symposia book series (GWUN)


The inhibition of thrombin as well as of factor Xa has been thought to occur in plasma primarily through the neutralizing action of the serine protease inhibitor antithrombin III (AT-III). However, inhibition of thrombin and Xa by this mechanism may not be sufficient for effective elimination of these clotting factors in states of increased coagulation activity. The potential role of the vascular endothelium in the inhibition of clotting factor activities has therefore received attention in recent years. In the case of thrombin, it has been postulated that this occurs through binding to the endothelial cells, at least in part through surface glycosaminoglycans (GAGs) such as heparin sulfate, which could serve as AT-III cofactor on the endothelial surface.

The aims of the present investigation were to characterize the binding and inhibition of thrombin and Xa to EC and SMC in vitro as well as to evaluate the role of plasma constituents on the inhibition of these clotting factors. The RHC was studied to determine whether uptake and inhibition of these factors is specific for cells derived from the vascular wall.

Purified bovine thrombin and factor Xa were used. The enzymatic activities of both factors were assayed amidolytically using the chromogenic substrates S-2222 and S-2238, respectively. The cells were grown in DMEM containing 10% calf serum. The cells were exposed to 5 U/ml thrombin and 0.5 U/ml factor Xa. After 10 min of incubation, the initial thrombin activity in the solution had decreased by about 20%. Thrombin activity recovered from the cell surfaces amounted to 0.02 U/cm2. When the cells were incubated for 30 sec with AT-III, 28–34% of the surface-related activity could be recovered. When the cells were incubated briefly with defibrinogenated plasma, however, only 10–15% of the cell surface activity was found. In similar experiments with factor Xa, after 10 min of incubation, the initial activity in the solution had decreased by 10%. Factor Xa activity recovered from the cell surfaces was 0.001 U/cm2. After 30 sec exposure for AT-III, no cell surface-related Xa activity was recovered, whereas 10% of the cell surface activity was still recovered after incubation with defibrinogenated plasma.

It is concluded that thrombin and factor Xa are taken up by EC, SMC, and RHC cell surfaces in similar ratios. These three different cell types inactivated thrombin and Xa activities to a similar degree, suggesting that cell surface-mediated inactivation of activated clotting factors is not restricted to vascular wall cells. The inactivation of factor Xa was dependent on AT-III; however, the inactivation of thrombin was further promoted by an additional unidentified plasma constituent.


Chromogenic Substrate Bovine Aortic Endothelial Cell Thrombin Activity Bovine Thrombin Amidolytic Activity 


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Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • Maciej Dryjski
    • 1
  • Thorir D. Bjornsson
    • 1
  1. 1.Division of Clinical Pharmacology, Department of MedicineDuke University Medical CenterDurhamUSA

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