Recombinant human protein C derivatives: altered response to calcium resulting in enhanced activation by thrombin.

Abstract

Calcium plays a dual role in the activation of protein C: it inhibits protein C activation by a-thrombin, whereas it is required for protein C activation by the thrombomodulin-thrombin complex. Available information suggests that these calcium effects are mediated through calcium induced structural changes in protein C. In this paper, we demonstrate that substitution of Aspl67 (located in the activation peptide of human protein C, occupying position P3 relative to the peptide bond Argl69-Leul7O which is susceptible to hydrolysis by thrombin) by either Gly or Phe results in protein C derivatives which are characterized by an altered response to calcium. At 3 mM calcium, a-thrombin activated the derivatives 5-to 8-fold faster compared with the wild-type, an effect which was shown to be caused by a decreased inhibitory effect of calcium on the reaction. These same single amino acid substitutions enhanced the affinity of the thrombomodulin-thrombin complex for the substrate at 3 mM calcium 3-(Glysubstitution) to 6-(Phe-substitution) fold, either without influencing kc.t (Gly-substitution) or with a 2.5-fold decrease of keat. For both derivatives, the calcium concentrations resulting in half maximal inhibition of activation by ct-thrombin and in half maximal stimulation of activation by the thrombomodulin -thrombin complex increased from 0.3 mM to 0.6 mM. It is concluded that Aspl67 is involved in the calcium induced inhibition of protein C activation by thrombin. Moreover, our studies demonstrate that it is feasible to enhance the efficiency of enzymatic reactions by introducing point mutations in the substrate.

Exposure of R169 controls protein C activation and autoactivation

“…A defect in protein C may compromise the value of k cat /K m independent of a defect in thrombin, so it is possible that the cofactor thrombomodulin corrects a defect in the substrate protein C rather than the enzyme thrombin. The proposal that thrombomodulin changes the conformation of the active site of thrombin is supported by spectroscopic 15 and mutagenesis [16][17][18][19] studies. Structural biology has remained inconclusive because the thrombinthrombomodulin complex has been crystallized with the active site occupied by an inhibitor 20 or crystal contacts.…”

“…on May 10, 2018. by guest www.bloodjournal.org From of the role of residues D167 and D172 in protein C activation, previously assumed to cause electrostatic clash with residues E192 and E39 of thrombin. [16][17][18][19]41 When a binding interaction depends on electrostatics, it is influenced significantly by changes in the salt concentration or ionic strength of the solution. An increase in the salt concentration decreases/increases the strength of interaction depending on whether electrostatic coupling/clash is involved.…”

“…27 The EDD mutant of protein C reported here removes all acidic residues in the anionic cage and is activated 63-fold more rapidly by thrombin in the absence, but not the presence of thrombomodulin. This is a very significant enhancement of protein C activation compared with existing mutations of thrombin or protein C. [16][17][18]25,26,28 In energetic terms, the 63-fold enhancement afforded by the EDD mutation accounts for more than half of the 1700-fold total effect of thrombomodulin, thereby making exposure of R169 an important molecular event linked to the thrombomodulin-induced enhancement of protein C by thrombin. The balance of the total effect of thrombomodulin could come from changes in the thrombin active site, 15 mapped by mutagenesis to the oxyanion hole, Y60a and residues of the protein C peptide (yellow sticks) dock into the active site of thrombin.…”

Charge reversal at the P3′ position in protein C optimally enhances thrombin affinity and activation rate

“…At physiological levels of calcium, protein C is a very poor substrate for free a-thrombin, however, it is a relatively good substrate for thrombin in the absence of calcium, or when thrombin is complexed with the integral membrane protein, thrombomodulin. Previously, we and others have shown by site-directed mutagenesis that neutralization of acidic residues near the thrombin cleavage site e.g., Asp at the P3 and P3' positions, increases activation by free a-thrombin due to a reduction in the inhibitory effect of calcium (Ehrlich et a]., 1990;Rezaie & Esmon, 1992;Richardson et al, 1992). When bound to thrombomodulin, the inhibitory influence of these acidic residues in protein C has Reprint requests to: Brian W. Grinnell, Cardiovascular Research, Lilly 46285-0434.…”

“…Of further interest, we combined the previously described change at the P3 position, D167F (Ehrlich et al, 1990), with each P3' derivative, and the resulting combination resulted in a synergistic increase in activation rate. Strikingly, D167F/D172K showed an activation rate approximately 100-fold higher than wild-type HPC in the presence of calcium, again due to a combination of decreased calcium inhibition and increased affinity observed even in the absence of calcium (data not shown).…”