Thrombomodulin (TM) increases the catalytic efficiency of thrombin (IIa)-mediated activation of thrombin-activable fibrinolysis inhibitor (TAFI) 1250-fold. Negatively charged residues of the C-loop of TM-EGF-like domain 3 are required for TAFI activation. Molecular models suggested several positively charged residues of TAFI with which the C-loop residues could interact. Seven TAFI mutants were constructed to determine if these residues are required for efficient TAFI activation. TAFI wild-type or mutants were activated in the presence or absence of TM and the kinetic parameters of TAFI activation were determined. When the three consecutive lysine residues in the activation peptide of TAFI were substituted with alanine (K42/43/44A), the catalytic efficiencies for TAFI activation with TM decreased 8-fold. When other positively charged surface residues of TAFI (Lys-133, Lys-211, Lys-212, Arg-220, Lys-240, or Arg-275) were mutated to alanine, the catalytic efficiencies for TAFI activation with TM decreased by 1.7–2.7-fold. All decreases were highly statistically significant. In the absence of TM, catalytic efficiencies ranged from 2.8-fold lower to 1.24-fold higher than wild-type. None of these, except the 2.8-fold lower value, was statistically significant. The average half-life of the TAFIa mutants was 8.1 ± 0.6 min, and that of wild type was 8.4 ± 0.3 min at 37 °C. Our data show that these residues are important in the activation of TAFI by IIa, especially in the presence of TM. Whether the mutated residues promote a TAFI-TM or TAFI-IIa interaction remains to be determined. In addition, these residues do not influence spontaneous inactivation of TAFIa.

Mn³⁺ distribution at octahedral M1, M2, and M3 sites of piemontite and its effect on structural changes were investigated by X-ray Rietveld analysis of synthetic Ca₂Al_3-pMn³⁺ _pSi₃O₁₂(OH) piemontites. The material studied was synthesized from starting materials of Ca₂Al_3-qMn³⁺_qSi₃O_12.5 + H₂O in hydrothermal experiments with P_fluid of 200 and 350 MPa and temperature of 500 °C. Piemontites crystallized as single phases from q = 0.5, 0.75, 1.0, and 1.1 starting materials, whereas minor amounts of bixbyite and parawollastonite were associated with those synthesized from q = 1.5 and 1.75 starting materials. EPMA analyses of synthetic piemontites showed that the maximum Mn³⁺ content was 1.3(1) apfu. Site preference of Mn³⁺ at the octahedral sites is M3 > M1 ≫ M2. Mn³⁺ occupancies (g) at the M3 and M1 sites are correlated with the p-value in piemontite as g^M3 = −0.20p² + 1.00p and g^M1 = 0.23p² − 0.06p, respectively, where 0.0 ≤ p ≤ 1.3. With increasing p-value, the mean M3-O and M1-O distances of piemontites increase, but the M1-O1 distance and the O5-Si3-O6 angle change nonlinearly due to the Jahn-Teller effect. The nonlinear variations of the a and c parameters with increasing p are caused by changes in the M1-O1 distance and the O5-Si3-O6 angle, respectively. The M3 octahedra, which are more distorted than the M1 octahedra, become more tetragonally compressed with increasing Mn³⁺ at the M3 site, due to the substantial increase of the M3-O1 distance.