Carolyn L. Orthner

Evidence that human α-thrombin is a monovalent cation-activated enzyme

Abstract The activity of human α-thrombin (EC 3.4.21.5) on small peptide substrates was enhanced by NaCl or KCl while tetramethylammonium chloride ((CH 3 ) 4 NCl) or choline chloride (HO(CH 2 ) 2 N(CH 3 ) 3 Cl) which were used as ionic strength controls were without effect. The steady-state kinetic parameters of thrombin amidolysis of several peptidyl p -nitroanilide substrates were measured. Na + enhanced thrombin activity by decreasing the K m,app (0.2 to 0.7-fold) of all substrates, as well as increasing thombin turnover (3.4 to 4.5-fold) of some substrates. The average K A for Na + for the four substrates examined was 3.5 × 10 −2 m . A comparison of the effects of Na + vs K + on thrombin hydrolysis of a single substrate indicated that both cations similarly decreased the K m,app (0.2 to 04.-fold) and increased the k cat,app (3.1 to 3.4-fold) except that higher K + concentrations ( K A = 2.8 × 10 −1 M) were required. The rate of inactivation of thrombin by the active site-directed inhibitor N-p -tosyl-lysine chloromethyl ketone under pseudo-first-order conditions was enhanced 3-fold by saturating NaCl. Also, the fibrinogen clotting activity of thrombin was enhanced by NaCl compared to the choline chloride control. Spectral studies demonstrated that thrombin titration by Na + caused a positive ultraviolet difference spectrum with maxima at 281.5 and 288.5 nm (Δϵ 288.5 = +1067). The K m for Na + was 2.3 × 10 −2 m which agrees with the kinetically determined K A for Na + . The results are consistent with Na + binding to thrombin causing a conformational change in the active site. It is concluded that human α-thrombin is a monovalent cation-activated enzyme.

The effect of metal ions on the amidolytic activity of human factor Xa (Activated Stuart-Prower Factor)☆

Abstract The effect of metal ions on human activated Factor X (Factor Xa) hydrolysis of the chromogenic substrate benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222) was studied utilizing initial rate enzyme kinetics. The divalent metal ions Ca2+, Mn2+, and Mg2+ enhanced Factor Xa amidolytic activity with Km values of 30 μ m , 20 μ m , and 1.4 m m , respectively. Na+ activation of Factor Xa amidolytic activity was also found. The Km for Na+ activation was 0.31 m . Both the divalent metal ions and Na+ increased the affinity of Factor Xa for S2222 and had no effect on the maximal velocity of the reaction. Other monovalent cations were unable to activate Factor Xa. However, K+ was a competitive inhibitor of the Na+ activation (Ki = 0.14 m ). Lanthanide ions inhibited Factor Xa amidolytic activity. Gd3+ inhibition of Factor Xa hydrolysis of S2222 was noncompetitive and had a Ki of 3 μ m . The lanthanide ion inhibition could not be reversed by Ca2+ even when Ca2+ was present in a 1000-fold excess over its Km indicating nonidentity of the Factor Xa lanthanide and Ca2+ binding sites. It is concluded that the Factor Xa Ca2+ binding sites have characteristics different from those previously described for the Factor X molecule and that Mg2+, Na+, and K+ may be physiological regulators of Factor Xa activity.

Large-Scale Production and Properties of Immunoaffinity-Purified Human Activated Protein C Concentrate

Activated protein C (APC) is a highly specific serine proteinase which functions as an important naturally occurring antithrombotic enzyme. APC also has antiinflammatory properties. We have developed a large‐scale process for the production of APC for therapeutic use starting with cryoprecipitate‐poor human plasma. This report describes the process, its performance at the pilot plant scale, and the characteristics of immunoaffinity‐purified human APC concentrate referred to as APC (human). The process consists of three chromatographic steps, an enzymatic conversion step, and incorporates a solvent/detergent treatment step for the inactivation of lipid‐enveloped viruses. Solvent/detergent was shown to rapidly inactivate spiked HIV‐1, as well as three marker viruses to nondetectable levels under process conditions. The immunoaffinity‐purified protein C (PC) intermediate was enriched 13,600‐fold over plasma and had a specific activity of 231 U/mg. The overall yield of the process following enzymatic conversion of the PC intermediate to APC and its processing by anion exchange chromatography was 36%. APC (human) was shown to be highly purified, functional and stable.

Use of Size-Exclusion High-Performance Liquid Chromatography for the Analysis of the Activation of Prothrombin,

Abstract Prothrombin is a single-chain protein ( M r 72,000) which can be converted to the two-chain coagulation enzyme thrombin ( M r 37,000) by a protein present in the venom of Echis carinatus . During the course of this reaction, several intermediates and products are produced. We have found that size-exclusion high-performance liquid chromatography is a useful method for identifying the various intermediates as well as for determining the rates at which they are produced. The intermediates were identified by comparison with either authentic proteins or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of duplicate samples. The separation of prothrombin, thrombin, and the other products was accomplished in less than 18 min using a TSK Type SW 3000 column equilibrated with 0.1 m Tris buffer, pH 7.2, containing 0.15 m NaCl. Estimates of the molecular weights of the products determined from calibration curves did not necessarily agree with actual values. Meizothrombin, the two-chain active form of prothrombin, has a longer elution time than prothrombin, although both molecules have the same molecular weight. Ca(II) increased the elution time of prothrombin and Fragment 1. The rate of disappearance of prothrombin as determined by peak-height analysis agreed with the rate of formation of active enzyme as determined by active site titration. Thus, high-performance liquid chromatography is a rapid analytical tool for the study of the proteolytic modification of proteins.

Critical role of the carbohydrate moiety in human von Willebrand factor protein for interactions with type I collagen

The ability of von Willebrand factor protein (vWF) to agglutinate platelets with ristocetin depends upon the presence of its highest molecular weight multimers (HMWM) and its intact carbohydrate structure. Previously we demonstrated that the HMWM are preferentially adsorbed to purified fibrillar type I collagen. The role of the carbohydrate structure of vWF in this function has not been established. In these studies complete desialylation (greater than 95%) of the intact protein by neuraminidase did not interfere with the normal adsorption of vWF activity to type I collagen. In contrast, modification of the penultimate galactose of the desialylated protein with galactose oxidase or beta-galactosidase markedly reduced adsorption of vWF activity by collagen. Subsequent reduction of the oxidized desialylated protein with potassium borohydride completely regenerated the normal adsorption of vWF activity by collagen. Enzymatic modification of the penultimate galactose moiety of vWF resulted in a loss of the HMWM, as observed following SDS-glyoxyl agarose electrophoresis. This was in contrast to desialylated vWF, which appeared intact structurally and which predictably lost its HMWM upon exposure to collagen in a manner similar to native vWF. Therefore, the carbohydrate structure of vWF and, in particular, the penultimate galactose moiety, may be critical for vWF-collagen interactions and for the mediation of primary hemostasis.

Inhibition of human coagulation factor Xa by thrombin substrates

Abstract The thrombin specific chromogenic substrates, Phe-Pipecolyl-Arg-p-nitroanilide (S-2238) and benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) are inhibitors of factor Xa amidolytic and proteolytic activity. The inhibition is of the partial noncompetitive type in that no change in the Km for the hydrolyzed substrate was observed and some factor Xa activity remains even at infinite inhibitor concentration. High performance liquid chromatographic analysis indicated that the inhibitors were essentially free of either p-nitroaniline or free peptide contamination. Neither p-nitroaniline nor acid hydrolyzed inhibitors were capable of inhibiting factor Xa demonstrating that a minimal peptide structure is necessary for inhibition. Our results indicate that factor Xa contains a regulatory site which, when occupied by certain peptides, causes a decrease in the rate of catalysis.