The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells
✍ Scribed by Justinian R. Ngaiza; Geoffrey Manley; Jürgen Grulich-Henn; John L. Krstenansky; Eric A. Jaffe
- Publisher
- John Wiley and Sons
- Year
- 1992
- Tongue
- English
- Weight
- 719 KB
- Volume
- 151
- Category
- Article
- ISSN
- 0021-9541
No coin nor oath required. For personal study only.
✦ Synopsis
Thrombin stimulation of prostacyclin (PGI,) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [CaL+],. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recogni-Lion of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in 1Ca2+], and PCI, production. Thrombin variants which lack either the catalytic site (DIP-u-thrombin) or anion-binding exosite (y-thrombin) either alone or in combination failed to induce rises in [Ca2 ' li or PGI, production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N*-acetyldesulfato-hirudin,,,, or Hir45-65) inhibited thrombin-induced (0.5
Uirnl) rises in [Ca2'li and PGI, production with I C, , of 0.13 and 0.71 FM, respectively. Similar results were obtained using shorter hirudin-derived peptides.
Thus, the fibrinogen anion-binding exosite of thrombin is required for u-thrombin-induced rises in [CaL+], and PGI, production in HUVEC.