Summary
1. Choline chloride, 0.1 M (in 0.25 M Tris. HCl buffer, pH 7.4 or 8.0, 37°), doubles
the rate of hydrolysis of TAME by bovine thrombokinase but has no effect on the hydrolysis
of this ester by either human or bovine thrombin. Only when 1.0 M or more choline
chloride is present is the hydrolysis of BAME by thrombokinase or thrombin weakly
inhibited. Evidence is presented that shows that these effects are due to the quaternary
amine group.
2. Tetramethyl ammonium bromide or chloride has about the same effects on the hydrolysis
of esters by these enzymes as does choline chloride but tetra-ethyl, -n.propyl and
-n.butyl ammonium bromides (0.1 M) are stronger accelerators of the thrombokinase-TAME
reaction and they also accelerate, but to a lesser degree, the thrombin-TAME reaction.
In addition, they inhibit the hydrolysis of BAME by both enzymes. Their effects on
these reactions, however, do not follow any regular order. The tetraethyl compound
is the strongest accelerator of the thrombokinase-TAME reaction but the tetra-ethyl
and -butyl compounds are the strongest accelerators of the thrombin-TAME reaction.
The ethyl and propyl compounds are the best (although weak) inhibitors of the thrombokinase-BAME
and the propyl compound of the thrombin-BAME reactions.
3. Tetra-methyl, -ethyl, -n.propyl and -n.butyl ammonium bromides (0.01 M) inhibit
the clotting of fibrinogen by thrombin (bovine and human proteins) at pH 7.4, imidazole
or pH 6.1, phosphate buffers and they also inhibit, but to a lesser degree, a modified
one-stage prothrombin test. In all cases the inhibition increases regularly as the
size of the alkyl group increases from methyl to butyl. Only the ethyl com pound (0.025
M but not 0.01 M), however, significantly inhibits the polymerization of bovine fibrin
monomers. It was concluded that inhibition of the fibrinogen-thrombin and the one-stage
tests by the quaternary amines is not due to any effect of the com pounds on the polymerization
process but probably due to inhibition of thrombin’s action on fibrinogen by the quaternary
amines.
