Summary
Shorter clotting times were found in the presence of 50 mM Hepes (N-2-hydroxyethylpiper-azine-N1-2-ethanesulfonic acid) buffer than of 50 mM Imidazole buffer in one-stage assays
of factors V and VIII, in modified APTT and PT tests and in tests of the clotting
of human plasma by purified human thrombin. All tests were performed at ionic strength
0.155 in the presence of either Hepes. NaOH or Imidazole. HC1 buffer, pH 7.4 at 37°.
The faster clotting in the presence of Hepes buffer, therefore, is probably due, at
least in part, to acceleration by Hepes of thrombin’s enzymatic action on fibrinogen
and/or of the polymerization of the fibrin monomers.
Hepes may also have effects on other blood clotting reactions. Rates of hydrolysis
of TAME or BAME (p-toluenesulfonyl-or benzoyl-L-arginine methyl ester) at pH 7.4,
37° by purified human or bovine thrombin were essentially the same in 200 mM Hepes
as in 250 mM Tris. HQ buffer (rates in Hepes. NaOH or Hepes. KOH buffers were compared
with those in Tris. HQ plus NaCl for KC1). However, with purified bovine thrombokinase,
rates of TAME hydrolysis in Hepes buffer were accelerated and rates of BAME hydrolysis
slightly inhibited. Hepes, therefore, reacts with thrombokinase but whether this accelerates
(or inhibits) the rate of converting prothrombin to thrombin remains to be determined.
In addition, Hepes has an inhibitory effect on clotting since increasing the concentration
of Hepes from 50 mM to 200 mM inhibits clotting in the PT, APTT and bovine thrombin-human
plasma tests.
Hepes buffer is being added to some plasmas and to some reagents used in clotting
tests. It is, therefore, important to realize that its concentration must be monitored
closely or erroneous results may be obtained in clotting tests and assays of clotting
factors.
The clotting times were the same in the presence of 50 mM Tris. HC1 as in Imidazole.
HC1 buffers in APTT tests at three ionic strengths but they differed slightly in plasma-thrombin
tests. Depending upon the ionic strength, 17 mM Barbital Sodium. HC1 buffer inhibited
APTT tests but accelerated plasma-thrombin tests. All the buffers tested, therefore,
have individual effects on the clotting tests.
