R. Zingali - Academia.edu (original) (raw)
Papers by R. Zingali
Archives of Biochemistry and Biophysics, 2009
alphaIIbbeta3 is an integrin that is involved in platelet adhesion and aggregation. This receptor... more alphaIIbbeta3 is an integrin that is involved in platelet adhesion and aggregation. This receptor may be inhibited by cysteine-rich peptides known as disintegrins. We isolated two disintegrins from Bothrops jararaca venom called jarastatin and jararacin. We evaluated the structural characteristics and the effects on human platelet aggregation of these disintegrins. Inhibitory profiles were compared to six distinct peptides synthesized based on their RGD hairpin loop primary sequences. Both jarastatin and jararacin inhibited ADP and thrombin induction. Conversely, none of the cyclic peptides showed high-quality activity in assays induced by ADP or thrombin. We constructed homology models for all of these molecules, and theoretically evaluated their interaction with the alphaIIbbeta3 crystal structure using a molecular modeling approach. These results support the observations that the cyclic peptides had little effects, and also reinforce the observation that residues outside the disintegrin RGD sequence are required for interactions with receptor.
Biochemistry, 1993
A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the... more A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.
Biochemistry, 1996
The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., &a... more The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], is a 27 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or prothrombin fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.
Archives of Biochemistry and Biophysics, 2009
alphaIIbbeta3 is an integrin that is involved in platelet adhesion and aggregation. This receptor... more alphaIIbbeta3 is an integrin that is involved in platelet adhesion and aggregation. This receptor may be inhibited by cysteine-rich peptides known as disintegrins. We isolated two disintegrins from Bothrops jararaca venom called jarastatin and jararacin. We evaluated the structural characteristics and the effects on human platelet aggregation of these disintegrins. Inhibitory profiles were compared to six distinct peptides synthesized based on their RGD hairpin loop primary sequences. Both jarastatin and jararacin inhibited ADP and thrombin induction. Conversely, none of the cyclic peptides showed high-quality activity in assays induced by ADP or thrombin. We constructed homology models for all of these molecules, and theoretically evaluated their interaction with the alphaIIbbeta3 crystal structure using a molecular modeling approach. These results support the observations that the cyclic peptides had little effects, and also reinforce the observation that residues outside the disintegrin RGD sequence are required for interactions with receptor.
Biochemistry, 1993
A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the... more A new thrombin inhibitor, bothrojaracin, has been identified and purified to homogeneity from the venom of Bothrops jararaca, the most common venomous snake of South America. Bothrojaracin has an isoelectric point of 4.2 and a molecular mass of 27 kDa and is made of two distinct polypeptide chains of 15 and 13 kDa, linked by disulfide bridges. Purified bothrojaracin is devoid of phospholipase A2, amidolytic, or fibrino (geno)lytic activity. Bothrojaracin forms a noncovalent complex with alpha-thrombin, without changing its catalytic activity on small peptide substrates. Bothrojaracin behaves as a potent and specific antagonist of thrombin-induced platelet aggregation and secretion, characterized by an IC50 ranging from 1 to 20 nM depending on the alpha-thrombin concentration. Bothrojaracin prolongs fibrinogen clotting time, and this effect is related to a competitive inhibition of the binding of alpha-thrombin to fibrin(ogen) (Ki 15 nM). Binding of alpha-thrombin to thrombomodulin is inhibited up to 87% by bothrojaracin, and the rate of protein C activation by alpha-thrombin is also decreased. Bothrojaracin antagonizes the inhibition of thrombin amidolytic activity by hirudin. These results indicate that bothrojaracin acts as a very potent ligand of the exosite of alpha-thrombin.
Biochemistry, 1996
The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., &a... more The thrombin inhibitor, bothrojaracin [Zingali, R. B., Jandrot-Perrus, M., Guillin, M. C., & Bon, C. (1993) Biochemistry 32, 10794-10802], is a 27 kDa protein isolated from the venom of Bothrops jararaca that blocks several thrombin functions, including fibrinogen clotting, platelet activation, and fibrin and thrombomodulin binding, but does not interact with the catalytic site. In the present report, we show that the high affinity binding of alpha-thrombin to immobilized bothrojaracin (Kd = 0.6 nM) is inhibited by the C-terminal peptide of hirudin and that the gamma-cleavage within exosite 1 reduces the affinity of bothrojaracin for thrombin (Kd = 0.3 microM), indicating that bothrojaracin binding to exosite 1 is a major determinant of the thrombin-bothrojaracin interaction. In addition, we show that bothrojaracin decreases the rate of inhibition of alpha- and gamma-thrombin by the antithrombin III-heparin complex. Competition of bothrojaracin with heparin or prothrombin fragment 2 for binding to thrombin indicates that bothrojaracin not only binds exosite 1 but also binds exosite 2 or in close proximity. Bothrojaracin binds to the thrombin precursor, prothrombin. This interaction is calcium-independent and is prevented by heparin, suggesting that it is mediated by exosite 2. Bothrojaracin inhibits platelet activation induced by clot-bound thrombin and slowly dissociates thrombin from the fibrin clots. Altogether, our results indicate that the high affinity of bothrojaracin for thrombin is supported by a double-site interaction and results in an efficient inhibition of both soluble and clot-bound thrombin.