Assay of prekallikrein in human plasma: comparison of amidolytic, esterolytic, coagulation, and immunochemical assays

Abstract

Using the substrate H-D-Pro-Phe-Arg-p-nitroanilide-HCl, an amidolytic assay was designed to measure prekallikrein in plasma. At a substrate concentration of 1 mM (Km = 0.2 mM), the amidolysis of purified kallikrein at 1 coagulant unit/ml was observed to be 2.47 mumole/min/ml. Conditions for plasma prekallikrein activation were optimized to approach complete activation when compared to the amidolytic activity of the purified plasma kallikrein. Plasma treated with chloroform to destroy inhibitors of kallikrein was activated with dilute kaolin (final concentration 1 mg/ml) for 1 min at 25 degrees C. Activated plasma prekallikrein had 78% (1.92 mumole/min/ml) of activity of purified kallikrein at plasma concentration. Comparison of this amidolytic assay with immunochemical, esterolytic, and coagulant assays of three subject populations (normals, women on birth control pills, and patients with hepatocellular disease) showed good correlation both in normals and in the patient groups between the amidolytic and esterolytic assays (r = 0.89). Each enzymatic assay correlated with the immunochemical assay (r = 0.72, r = 0.68, respectively). However, comparison of each of these assays with the coagulant assay showed no significant correlation due to the large inherent error of the latter assay. This standardized plasma prekallikrein amidolytic assay should facilitate studies of plasma prekallikrein concentration in physiologic and pathologic conditions and help identify activation of the contact phase of coagulation in disease states.

Contact Factor Deficiency in a German Shorthaired Pointer without Clinical Evidence of Coagulopathy

“…14,15 Affected patients invariably have low in vitro PK activity in functional tests, but some affected families have plasma PK concentration within normal limits. 6 There is variation in sensitivity of different aPTT assay systems for detecting PK deficiency. In particular, the use of ellagic acid contact activator in an aPTT reagent has been associated with a decreased and variable ability to detect PK deficiency in test plasmas.…”

“…In particular, the use of ellagic acid contact activator in an aPTT reagent has been associated with a decreased and variable ability to detect PK deficiency in test plasmas. 5,6,9 The findings in this report considered consistent with PK deficiency included absence of a clinical bleeding tendency, variable prolongation of aPTT using an ellagic acid reagent, marked prolongation of aPTT using a silica contact activator reagent, and the inability of specific PK-deficient plasma to correct the long aPTT in a mixing study. Inconsistent with PK deficiency was the finding of only slight shortening of aPTT in the PK screening tests using silica contact activator reagent.…”

“…For more specific confirmation of PK or HK deficiency, an aPTT using each reagent was performed on a 1:1 mixture of patient plasma preincubated for 20 minutes with human PK k or HK l deficient substrate plasma. 6 Clotting times for the dam's plasma in both sets of aPTT assays were within reference range ( Table 1). The aPTT assays for the GSHP using reagent 1 (ellagic acid activator) were within the reference range or only slightly prolonged (2 to 3 seconds) for all test reactions.…”

“…Prekallikrein-deficient plasmas are expected to show a shortening or correction of long clotting time in this modified aPTT. 5,6 The aPTT of GSHP plasma in the screening test corrected to 21.0 seconds (reference range, 10 to 17 seconds), suggestive of PK deficiency.…”

“…These findings were compatible with a contact factor deficiency, either PK or HK, as the cause of long aPTT. [4][5][6] Although differentiation between these 2 factor deficiencies could not be accomplished from these studies, the pattern of abnormalities was most consistent with PK deficiency. 5,9 Specific deficiency of PK has been reported in several species, including dog, horse, rat, and human being.…”