Yumiko Sakai

Flavocetin-A and -B, two high molecular mass glycoprotein Ib binding proteins with high affinity purified from Trimeresurus flavoviridis venom, inhibit platelet aggregation at high shear stress

Two high molecular mass proteins, flavocetin-A and flavocetin-B, were purified from Trimeresurus flavoviridis venom. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the apparent molecular mass of flavocetin-A and -B were 149 and 139 kDa, respectively, under nonreducing conditions. On reduction, flavocetin-A showed two distinct subunits (17 and 14 kDa), and flavocetin-B three distinct subunits (17, 15 and 14 kDa). At 1 microgram/ml, flavocetin-A and -B (flavocetins) inhibited the von Willebrand factor (vWF)-dependent aggregation of fixed human platelets. However, flavocetins (10 micrograms/ml) had no effect on ADP- and collagen-induced platelet aggregation in PRP. Flavocetins (3 micrograms/ml) also inhibited shear-induced platelet aggregation at high shear stress. Furthermore, flavocetin-A completely inhibited the aggregation of and ATP release from washed platelets stimulated with a low concentration of thrombin. Flavocetin-A specifically bound to platelet with high affinity (Kd = 0.35 +/- 0.13 nM) at 21,500 +/- 1760 binding sites per platelet. The N-terminal amino acid sequences of the subunits of flavocetin-A show a high degree of homology with those of echicetin, botrocetin, alboaggregin-B and factor IX/factor X-binding protein. These results suggest that flavocetins may be a useful tool for further investigation of the GPIb-vWF interaction.

Isolation and Amino Acid Sequence of Flavostatin, a Novel Disintegrin From the Venom of Trimeresurus flavoviridis

Flavostatin, a novel disintegrin purified from the venom of Trimeresurus flavoviridis, consists of 68 amino acids, including an Arg-Gly-Asp sequence and 12 Cys residues at positions highly conserved among disintegrins. The N-terminal sequence of flavostatin was identical to those of triflavin and flavoridin, previously reported disintegrins from the Trimeresurus flavoviridis venom. Differences among the C-terminal sequences of these disintegrins are considered to affect their biological potencies. Isolated flavostatin inhibited ADP collage, and thrombin receptor agonist peptide-induced platelet aggregation in human platelet-rich plasma with an IC50 range of 59 to 98 nM. Contrary to expectations, these values were similar to those for triflavin.

Comparison of the antiplatelet effect of YM337 and abciximab in rhesus monkeys

We directly compared the effects of YM337, the Fab fragment of the humanized monoclonal antibody C4G1, on platelet aggregation and template bleeding time with those of abciximab, the Fab fragment of the human/murine chimeric monoclonal antibody 7E3, in rhesus monkeys. The duration of inhibition of platelet aggregation by abciximab after i.v. bolus injection was much longer than that by YM337. Although YM337 significantly prolonged template bleeding time at 5 min after i.v. bolus injection, this action recovered within 1 h after injection. In contrast, although abciximab also prolonged template bleeding time, the duration of this effect was sustained. In a dose-escalating continuous infusion study, we evaluated the relationship between inhibition of platelet aggregation and prolongation of template bleeding time. Platelet aggregation was inhibited by over 80% by both agents at 3 microg/kg per min, and template bleeding time was prolonged to about 30 min at 30 microg/kg per min for YM337 and 10 microg/kg per min for abciximab. Interestingly, plasma concentrations between inhibition of platelet aggregation and prolongation of template bleeding time did not overlap with YM337, but did overlap with abciximab. These results suggest that YM337 allows easier control of antiplatelet activity with less effect on bleeding time than abciximab, and has a wider therapeutic window than abciximab.

Comparison of the antiplatelet agent potential of the whole molecule, F(ab)2 and Fab fragments of humanized anti-GPIIb/IIIa monoclonal antibody in monkeys.

1. The potential antiplatelet agent use of the whole molecule, F(ab)2 and Fab fragments of humanized antiglycoprotein (GP) IIb/IIIa monoclonal antibody, hC4G1, were investigated in rhesus monkeys. 2. Fab completely inhibited platelet aggregation 1 hr after an i.v. bolus administration of 1 mg/kg without a decrease in platelet count or prolongation of bleeding time, and the duration of inhibition was much shorter than that of F(ab)2. 3. These results suggest that the Fab fragment of hC4G1 may be a more useful antiplatelet agent in patients with acute thromboembolic diseases than the whole molecule or F(ab)2 fragments.

Synergistic effect of aurintricarboxylic acid and triflavin in a photochemically induced thrombosis model in rats.

We report here the synergistic antithrombotic effect of aurintricarboxylic acid in combination with a snake venom-derived disintegrin, triflavin, in a photochemically induced thrombosis model in rats. The time to initiation of thrombus was prolonged by i.v. bolus injection of aurintricarboxylic acid at 10 mg/kg. In contrast, time to occlusion was dose-dependently prolonged by both agents, this prolongation being significant with aurintricarboxylic acid at 10 mg/kg i.v. and with triflavin at more than 3 mg/kg i.v. Interestingly, the combination of aurintricarboxylic acid at 3 mg/kg i.v. and triflavin at 1 mg/kg i.v. prolonged not only the initiation of thrombus, but also the time to occlusion.