Dual inhibition of factor XIIa and factor XIa as a therapeutic approach for safe thromboprotection

Human plasma kallikrein, a product of contact-activated plasma proteolysis, is moderately inhibited by aprotinin, a small polypeptide from bovine lung that has been used as an experimental drug in human disease states. Aprotinin has a Lys residue in the P1 (reactive center) position occupying residue 15. Since kallikrein is an arginine-directed serine protease, we hypothesized that an altered form of aprotinin, Arg15-aprotinin, might be a better inhibitor. Kinetic evaluations were performed in 96-well microplates. We found that the KL (loose or Michaelis-Menten complex) was unchanged by the modification. However, the association rate constant was increased from 1.14 X 10(4) (mol/L)- 1s-1 to 1.5 X 10(5) (mol/L)-1s1, thus indicating that the inhibition rate was increased 14-fold for the modified protein. The Ki (at equilibrium) was decreased from 3.2 X 10(-7) mol/L to 1.5 X 10(-8) mol/L after substituting Arg for Lys in the P1 position. Therefore, the modified inhibitor binds to plasma kallikrein more tightly than the natural protein. We also investigated the effect of Arg15-aprotinin on tissue kallikrein, plasmin, factor XIIa, factor XIa, and thrombin and found that the Ki slightly decreased from 5.1 X 10(-7) mol/L to 1.2 X 10(-7) mol/L for tissue kallikrein and slightly decreased from 2 X 10(- 8) mol/L to 1 X 10(-8) mol/L for plasmin. Arg15-aprotinin did not inhibit thrombin or factor XIIa, even though both enzymes are arginine- directed serine proteases. However, factor XIa, although it was not inhibited by aprotinin, had a Ki of 3.4 X 10(-8) mol/L for Arg15- aprotinin. Therefore, Arg15-aprotinin is a more effective inhibitor of plasma kallikrein as well as factor XIa but shows minimal preference for plasmin and tissue kallikrein. This study also indicates that it is possible and practical to perform kinetic analyses directly in microplates.

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Dual inhibition of angiotensin-converting enzyme and neutral endopeptidase by the orally active inhibitor mixanpril: a potential therapeutic approach in hypertension.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.9.4072 ◽

1994 ◽

Vol 91 (9) ◽

pp. 4072-4076 ◽

Cited By ~ 40

Author(s):

M. C. Fournie-Zaluski ◽

W. Gonzalez ◽

S. Turcaud ◽

I. Pham ◽

B. P. Roques ◽

...

Keyword(s):

Angiotensin Converting Enzyme ◽

Therapeutic Approach ◽

Neutral Endopeptidase ◽

Converting Enzyme ◽

Active Inhibitor ◽

Dual Inhibition ◽

Orally Active

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The synthesis of arginylfluoroalkanes, their inhibition of trypsin and blood-coagulation serine proteinases and their anticoagulant activity

Biochemical Journal ◽

10.1042/bj2650539 ◽

1990 ◽

Vol 265 (2) ◽

pp. 539-545 ◽

Cited By ~ 18

Author(s):

T Ueda ◽

C M Kam ◽

J C Powers

Keyword(s):

Blood Coagulation ◽

Human Factor ◽

Anticoagulant Activity ◽

Factor Xa ◽

Activated Partial Thromboplastin Time ◽

Serine Proteinases ◽

Bovine Thrombin ◽

Bovine Trypsin ◽

Factor Xiia ◽

Factor Xia

Seven arginylfluoroalkanes (‘arginine fluoroalkyl ketones’) were synthesized by using a modified Dakin-West procedure. The structure of benzoyl-Arg-CF2CF3 was analysed by 19F-n.m.r. spectroscopy and m.s. and the compound was shown to exist primarily as a hydrate or cyclic carbinolamine. Arginylfluoroalkanes are good inhibitors of blood-coagulation serine proteinases and were found to be slow-binding inhibitors for bovine trypsin with Ki values of 0.2-56 microM. Benzoyl-Arg-CF2CF3 was the best inhibitor for bovine thrombin and human Factor XIa, and inhibited thrombin and Factor XIa competitively with Ki values of 13 microM and 62 microM respectively. The best inhibitor for pig pancreatic kallikrein was p-toluoyl-Arg-CF3, with a Ki value of 35 microM. Benzoyl-Arg-CF3 and benzoyl-Arg-CF2CF3 inhibited human plasma kallikrein competitively, with Ki values of 50 microM. None of the seven arginylfluoroalkanes was a good inhibitor of human factor Xa or of Factor XIIa. The arginylfluoroalkanes were tested in the prothrombin time (PT) and activated partial thromboplastin time (APTT) coagulant assays. Two fluoroketones, benzoyl-Arg-CF2CF3 and 1-naphthoyl-Arg-CF3, had significant anticoagulant activity. Benzoyl-Arg-CF2CF3 was found to prolong the PT 1.8-fold at 120 microM and to prolong the APTT 2.4-fold at 90 microM, whereas 1-naphthoyl-Arg-CF3 only prolonged the APTT 1.7-fold at 100 microM.

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The molecular-weight dependence of the rate-enhancing effect of heparin on the inhibition of thrombin, factor Xa, factor IXa, factor XIa, factor XIIa and kallikrein by antithrombin

Biochemical Journal ◽

10.1042/bj1930395 ◽

1981 ◽

Vol 193 (2) ◽

pp. 395-400 ◽

Cited By ~ 180

Author(s):

E Holmer ◽

K Kurachi ◽

G Söderström

Keyword(s):

Molecular Weight ◽

Factor Xa ◽

Low Molecular Weight ◽

Clotting Factors ◽

Factor Ixa ◽

Molecular Weight Dependence ◽

Factor Xiia ◽

Factor Xia ◽

Heparin Fractions ◽

Inhibition Of Thrombin

Heparin fractions of different molecular weight and with high affinity for antithrombin were studied with respect to their ability to potentiate the inhibition of activated clotting factors by antithrombin. Inhibition of thrombin, Factor IXa and Factor XIa showed similarities in the dependence on the molecular weight of heparin and was found to decrease with decreasing molecular weight. Inactivation of Factor Xa, Factor XIIa and kallikrein was, however, less dependent on the size of the polysaccharide and, to a great extent, was potentiated even by low-molecular-weight heparin fractions that had virtually no effect on the inhibition of thrombin, Factor IXa and Factor XIa.

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Demonstration and mode of action of an inhibitor for activated Hageman factor (factor XIIa) of the intrinsic blood coagulation pathway from Schistosoma mansoni

Blood ◽

10.1182/blood.v49.4.619.bloodjournal494619 ◽

1977 ◽

Vol 49 (4) ◽

pp. 619-633

Author(s):

VC Tsang ◽

RT Damian

Keyword(s):

Schistosoma Mansoni ◽

Anticoagulant Activity ◽

Factor Xii ◽

Contact Activation ◽

Factor Xi ◽

Heat Stable ◽

Normal Plasma ◽

Factor Xiia ◽

Factor Xia ◽

Coagulation Pathway

An anticoagulant activity from adult Schistosoma mansoni whole worm homogenate is described. The inhibitor appears to be specific for the contact activation step of the intrinsic pathway. Experiments with both human and mouse plasmas have defined the specificity of the inhibitor as follows: (1) It lengthens the partial thromboplastin time of normal plasma. (2) It has no effect on the prothombin time and Russell's viper venom time of normal plasma. (3) Preactivation of normal plasma by a contact activator such as Celite eliminates essentially all inhibitory activity. (4) The inhibitor appears to be heat stable and can be precipitated by centrifugation above 27,000 g. (5) The inhibitor has no effect on the activation of factor XII by Celite. (6) The activation of factor XI by factor XIIa, however, is inhibited by the schistosomal inhibitor. The above data are consistent with the view that S. mansoni adults possess an anticoagulant that is capable of specifically inhibiting the conversion of factor XI to factor XIa by factor XIIa.

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