The enzyme activity expressed as katal is related to the type of substrate, to the nature of the enzyme and to the experimental conditions. In the following paragraphs, a tentative correlation between the different types of units and the enzyme activity with Chromogenix substrates is reported. The purpose is to help the researchers in developing chromogenic methods, by using such information as a starting point.
The conversion factors, have been taken from journal articles or from laboratory results within the Chromogenix R&D department, thus the consultation of such references or of the assay conditions reported in the kinetic tables is recommended.
Thrombin
The current International Standard for thrombin is the Human a-thrombin 89/588 available from NIBSC. This is a high purity preparation of a-thrombin prepared from Cohn fraction III and assayed by a clotting time method against the first International Standard for thrombin, 75/157 (1).
The National Institute of Health standard (Lot J) is also commonly used for calibration and a study conducted by Gaffney PJ et al. (2) was focused on the relationship between the two standards, and between the International Units and the NIH Units. As a result of this study, based both on a clotting and a chromogenic assay (with the substrate S-2238), 1 NIH-U corresponds to 1.15 IU.
In an article (3) it was shown that bovine thrombin has a higher amidolytic activity than human thrombin when the same NIH-U are compared. It was also underlined that the influence of b and g-forms, that were probably contaminating the bovine enzyme, might be the reason for this discrepancy. In the same article it was concluded that 1 NIH-U bovine thrombin was equivalent to 3.4 nkatS-2238, and that 1 NIH-U of human thrombin was equivalent to 2.7 nkatS-2238.
From an earlier publication (4) 1 NIH-U of human thrombin corresponded to 2.5 nkatS-2238 . The correspondence between NIH-U or IU of thrombin and the enzyme activity expressed in nkat, depends on the substrate, the enzyme preparation (content of a-, b- and g-thrombin) and the assay conditions. From the article of Friberger (4), 1 micrograms thrombin corresponds to 2.2 NIH-U or 5.5 nkatS-2238 or to 0.02 plasma equivalent units. In another study (5), 1 micrograms thrombin corresponds to 3.1 NIH-U.
In the experiments done in Chromogenix (see table 3 of the catalogue) 1 micrograms thrombin was equivalent to 3 nkatS-2238 (human) or 4.4 nkatS-2238 (bovine). It might also be added that if all prothrombin is activated in 1 ml of human plasma, about 1.5 nanomoles or 17.5 NIH-U of thrombin are formed (5).
Urokinase
Urinary type plasminogen activator (u-PA) is present either as a single chain proenzyme form (scu-PA) with a very low plasminogen activating activity or as an activated two-chain form (tcu-PA). tcu-PA occurs as both high molecular weight (HMW tcu-PA) or low molecular weight (LMW tcu-PA) enzyme species. HMW tcu-PA is 2-3 times more potent than LMW tcu-PA in biological assays such as the clot lysis method but their amidolytic activity on the chromogenic substrate S-2444 is equivalent (1). The International Reference Preparation (IRP) of urokinase (66/46) was established by the Expert Committee on Biological Standardisation of the WHO in 1968 (2). Until then urokinase was expressed in casein Ploug (Leo standard) or CTA units (CTA standard). These units were related to the amount of urokinase capable to hydrolyse a certain amount of casein in a defined time. 1 CTA-U was assumed to be identical to 1 International Unit (IU). Although there was no official numerical relationship between Ploug units and IU, 1.5 IU has been accepted to be equivalent to 1 Ploug unit. The IRP 66/46 is a mixture of 66% LMW and 34% HMW forms of tcu-PA (3).
Following the need of a HMW International Standard, a new international standard (87/594) was established in 1989 (4). Due to the complexity of u-PA it is difficult to determine a direct relationship between IU, CTA-U, Plough-U and nkat S-2444. In 1983 Friberger (5) reported that 0.34 nkatS-2444 correspond to 110 Plough-U, 160 CTA-U or about 160 IU. In an earlier work by Paar D and Marhulm D, on urokinase purified from urine (6) , 100 CTA-U corresponded to 0.27 nkatS-2444.
Tissue-plasminogen activator (t-PA)
Tissue plasminogen activator (t-PA) exists in two forms: as single-chain (sct-PA) and as two-chain t-PA (tct-PA), the latter form being generated from sct-PA through proteolytic cleavage by plasmin. They both activate plasminogen to plasmin (plasminogenolytic activity; refer to the Coaset t-PA kit). Also they both hydrolyse chromogenic substrates (amidolytic activity; refer to the Research Method for t-PA in purified preparations).
A quantitative analysis for the composition of a mixture of one-chain and two-chain t-PA by amidolytic assay has been proposed (1). The first International Standard for t-PA, 83/517, was established in 1985 (2) by using a melanoma extract. The potency assignment was done with a fibrin clot lysis method.
In 1987, the second International Standard for t-PA, 86/670, was established (3). It was again a purified preparation from a cultured melanoma cell supernatant containing about 98% of single-chain t-PA. Chromogenix t-PA reagent (Art. No. 821157), is composed mainly of single-chain t-PA (>95%) with a specific fibrinolytic activity of about 500,000 IU/mg of enzyme (batch-specific reagent) assessed against the second international standard. The enzyme activity in terms of nkat with the substrate S-2288 is 0.087 nkat with 400 IU (see kinetic tables).
Factor Xa and X
Factor Xa, which has a molecular weight of 44 KDa, is the activated form of Factor X (MW: 59 KDa). The International Units of Factor X correspond to the amount of Factor X contained in 1 ml of normal plasma. This is about 8 mg/l or 0.13 micromol/l. Since there is no WHO standard for FXa, one would assume that if all the Factor X in normal plasma was converted to the activated form, the Factor Xa concentration would be approximately 5.7 mg/l. The activity of human Factor Xa as calculated from the kinetic tables is 1.5 nkat/micrograms with the substrate S-2222, and 4.4 microkat/micrograms with the substrate S-2765. The activity of 1 micrograms of Factor Xa as determined by Frieberger (1) is 1.9 nkatS-2222. Thus, 1 plasma equivalent unit of Factor X would correspond to 15.2 nkatS-2222.
Plasminogen and Plasmin
Plasminogen (MW: 92 Kda) is the zymogen form of plasmin (83 KDa). The activation of plasminogen is accomplished by t-PA, urokinase or streptokinase. It is not the purpose of this text to describe the mechanisms of activation of plasminogen and its inhibition, but it is important to underline that they can affect the reaction of the active enzyme with chromogenic substrates, casein or fibrin. For more information refer to Gaffney (1). 1 ml of normal plasma contains about 180 micrograms or 2 nmol of plasminogen. The activity of plasminogen can be expressed in casein units (CU) e.g. for example to Sgouris et al. (2). Friberger (3) found that the amount of plasminogen in 1 ml of normal plasma corresponds to 3.8 CU, as determined by the chromogenic method described (streptokinase activated plasminogen). The Chromogenix Plasminogen Reagent (Art. No. 81 06 63), when activated with streptokinase shows an activity of 7.3 nkatS-2251 per CU.
In 1983 Friberger (4) reported that 1 micrograms of plasmin corresponds to 0.20 nkat S-2251 , or to 0.024 CU or to 0.028 CTA-U. In 1975, the first standard for plasmin was established (5) and the international unit was defined equivalent to CTA-U (5-6). This contained a purified human plasmin in 50% glycerol. When tested by Friberger et al. (7), it showed about the same activity as the Kabi preparation in terms of micromol of substrate hydrolysed per minute per unit of enzyme. Since then the different aspects of caseinolytic, amidolytic and fibrinolytic activity have been explored in details and, the discrepancy between amidolytic and fibrinolytic activity in reference preparations was underlined as in the case of the 2nd International Reference Preparation of plasmin in 1983 (8). The current 3rd International Standard for plasmin, 97/536 was established in 1998, and the results presented at the Fibrinolysis Subcommittee of the Scientific and Standardisation Committee of the International Society of Thrombosis and Haemostasis (Ljubljana, Slovenia, June 1998). The 2nd International Reference Preparation was used as reference material and a chromogenic assay was the method used. There is no established WHO standard for plasminogen, but the National Institute for Biological Standards and Control, established the Ist British Reference Preparation, 78/646 (9). It is a preparation of Glu-plasminogen, assayed by fibrinolytic and chromogenic methods against the 2nd International Standard for plasmin. Complete activation of plasminogen was achieved by both urokinase or streptokinase and the activity was comparable to that of the reference preparation of plasmin.
Protein C
Protein C, a 62,000-dalton glycoprotein with approximately 28% carbohydrates, is synthetised in the liver. Activated protein C (APC) is a key anticoagulant enzyme in the down-regulation of coagulation. Protein C consists of two polypeptide chains with a heavy chain linked by a disulfide bond to a light chain. The heavy chain contains the serine active site and the activation peptide and it also contains proposed binding sites for factors Va and VIIIa. The light chain contains a region of g-carboxyglutamic acid residues (calcium ion and phospholipid binding region) and the epidermal growth factor region (proposed protein S-binding region). The average concentration of protein C in human plasma is 4 mg/mL and its half-life in plasma is 7 to 9 hours. One Unit of Protein C corresponds to the amount of Protein C contained in one mL of freshly pooled normal plasma. In order to facilitate comparison of results from analysis of protein C in plasma, an international standard has been prepared and with the assigned potency expressed in international units (IU). Thus 1 IU corresponds to about 4 mg of functional protein C. A freeze-dried human plasma (denoted 86/622) has been established by the WHO Expert Committee on Biological Standardization as the 1st International Standard for protein C in plasma, which is available from NIBSC.1 The assigned functional activity of this plasma is 0.82 IU/ampoule. Protein C in plasma may be activated by the thrombin/thrombomodulin complex or, more conveniently, by a specific venom enzyme from the snake Agkistrodon controtrix contortrix (Southern Copperhead Snake).2 The most suitable chromogenic substrate for the assay of APC described so far is S-2366.3-5 The Chromogenix Protein C Reagent (Art. No 82 20 98), which contains a purified preparation of the snake venom enzyme allows activation of protein C without interference from other coagulation factors. The amount of activated protein C is determined by the rate of hydrolysis of the chromogenic substrate S-2366. The activity of human APC, derived from the rate of hydrolysis of S-2366 in a purified system under properly standardized conditions (0.1 mol/L Tris-HCl pH 8.3, 0.26 mol/L CsCl, 4 mmol/L CaCl2 and with 0.2% BSA as bulking agent) is about 37 nkat / mg of active enzyme. The corresponding activity obtained in a plasma containing system after snake venom activation, using Coamatic Protein C, is about 25 nkat / mg of active enzyme.