Human Gamma-thrombin Research Articles

Thrombin exosite for fibrinogen recognition is partially accessible in prothrombin

The procoagulant alpha-thrombin is produced by the proteolytic cleavages of a minimum of two peptide bonds Arg274-Thr275 and Arg323-Ile324 in prothrombin. The Arg323-Ile324 cleavage is required for the expression of the active site of thrombin (Morita, T., Iwanaga, S. Suzuki, T. (1976) J. Biochem. (Tokyo) 79, 1089-1108; Hibbard, L. S., Nesheim, M. E., and Mann, K. G. (1982) Biochemistry 21, 2285-2292). It is not yet clear to what extent the proteolytic events are responsible for exposing protein recognition exosites on thrombin. We employed high resolution NMR spectroscopy to examine interactions of prothrombin and thrombin with synthetic hirudin peptides targeted toward the fibrinogen recognition exosite of thrombin. The hirudin tail synthetic analogues (acetyl-Asp55-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln65/G ly65-OH) exhibited similar NMR relaxation enhancements (line broadening patterns and transferred nuclear Overhauser effects) with human prothrombin as with human alpha-thrombin, indicating that both proteins bind the peptide in a similar manner. The protein-induced relaxation enhancements are specific to the interaction of the hirudin peptides with the fibrinogen recognition exosite of thrombin since no significant effects were observed with either human serum albumin or with human gamma-thrombin, which has an impaired recognition exosite. The binding affinities were determined from NMR relaxation time measurements, which gave approximate Kd values of 500 microM and < 100 microM for prothrombin and alpha-thrombin, respectively. Since the hirudin tail fragment binds specifically to the fibrinogen recognition exosite in alpha-thrombin, this exosite appears to be partially accessible in prothrombin in a proenzyme form.

Linkage between proton binding and amidase activity in human gamma-thrombin.

The amidase activity of human gamma-thrombin has been studied in the pH range 6-10 as a function of NaCl concentration and temperature. As recently found for human alpha-thrombin [Di Cera, E., De Cristofaro, R., Albright, D.J., & Fenton, J.W., II (1991) Biochemistry 30, 7913-7924], the Michaelis-Menten constant, Km, shows a bell-shaped dependence over this pH range with a minimum around pH 7.9 in the presence of 0.1 M NaCl at 25 degrees C. The catalytic constant, kcat, has a bell-shaped pH dependence with a maximum around pH 8.6. A thermodynamic analysis of these parameters has enabled a characterization of the linkage between proton and substrate binding, its dependence on NaCl concentration, and the relevant entropic and enthalpic contributions to binding and catalytic events. Three groups seem to be responsible for the control of gamma-thrombin amidase activity as a function of pH. One of these groups has pK values that are significantly different from those found for alpha-thrombin, and all groups show slightly perturbed enthalpies of ionization. The dependence of gamma-thrombin amidase activity on NaCl concentration is different from that of alpha-thrombin. Increasing NaCl concentration always decreases the substrate affinity for the enzyme in the case of alpha-thrombin, regardless of pH. In the case of gamma-thrombin, such an effect is observed only in the pH range 7.5-9, and a reversed linkage is observed at pH less than 7 and greater than 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function.

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.

Human Gamma-Thrombin: Lack of Correlation Between a Platelet Functional Response and Glycoprotein V Hydrolysis

The ability of purified human gamma-thrombin to stimulate platelet function was related to its capacity to degrade GP V. Compared to alpha-thrombin, much greater amounts of gamma-thrombin were required to induce platelet aggregation; and this also applied to secretion from dense bodies, alpha-granules and lysosomal granules. Platelet stimulation by gamma-thrombin was additionally characterized by the presence of a lag-phase. Platelets with 3H-labelled surface glycoproteins showed the same functional response to both alpha- and gamma-thrombin as unlabelled platelets. But while threshold levels of alpha-thrombin induced little GP V hydrolysis confirming McGowan et al. (1), amounts of gamma-thrombin which induced substantial degradation (e.g. 8.3 nM degraded 36% of platelet GP V in 3 min) were unable to sustain either platelet aggregation or secretion. These results suggest that protein-binding regions remote from the catalytic site of alpha-thrombin are more important for platelet activation than GP V hydrolysis. They also provide further support to the argument that GP V hydrolysis may not be the essential trigger of platelet activation by thrombin.

Refolding of a three (noncovalently linked)-domain enzyme. Human gamma-thrombin.

Human gamma-thrombin is a three (noncovalently linked)-domain enzyme which contains the known serine protease catalytic triad, Asp-His-Ser, one on each of the three noncovalent domains (Asp 99 on the A-B3 chain). While protein-folding dogma does not necessarily predict that the denatured form of this enzyme could refold to the correct conformation, a monitor of the esterase activity (Tos-Arg-OMe) shows complete recovery of native catalytically active conformation. When compared with the covalently intact alpha form which refolds from urea in less than 2 min with complete return of both clotting and esterase activity, gamma-thrombin requires up to 90 min to regain full esterase activity. The gamma-thrombin reactivation data best fit a single first order rate constant, k = 0.03 +/- 0.005 min-1. It was suggested that the gamma-thrombin renaturation process might represent first the rapid refolding, then subsequent reassociation and reisomerization of the three noncovalent domains to yield a lower energy, fully active, conformation. This study represents the only example known of the refolding (reconstitution) of a three (noncovalent)-domain protein.