Chromogenic Peptide Substrate Assays Research Articles

Discrepancies between ADAMTS13 activity assays in patients with thrombotic microangiopathies

ADAMTS13 activity assays are sometimes useful in confirming the clinical diagnosis or to distinguish different thrombotic microangiopathies (TMA). We investigated the commonly used clinical assays for ADAMTS13 activity. 159 samples from normal subjects or acquired TMA patients were studied in collagen binding (CBA), Fret and chromogenic peptide substrate assays. Frozen aliquots of pooled normal plasma gave similar values by CBA, Fret-VWF73 peptide, Fret-VWF86 and chromogenic VWF73 ELISA (chr-VWF73). Two lyophilised commercial calibrants gave lower ADAMTS13 activity by CBA than peptide substrate assays. The addition of solid HEPES to normal plasma caused a significant fall in CBA, but not Fret-VWF73 activity and might partly explain the differences, since lyophilised plasmas are often HEPES buffered. Normal plasmas showed good agreement between CBA and Fret assays, although chr-VWF73 gave slightly higher values. In acquired TMA, there was reasonable agreement between assays for samples with <11% ADAMTS13 activity (83% of samples showed agreement between CBA, Fret-VWF73 and chr-VWF73), but samples with moderate deficiency frequently showed lower CBA levels (only 41-52% agreement). However, there were also some discrepancies among the peptide substrate assays, with Fret-VWF86 sometimes giving slightly higher values than the VWF73 substrate assays. An International reference plasma might improve standardisation, but is not the only problem. It is unclear which assay has greatest clinical utility, this may depend on the nature of the sample. If the activity does not match the clinical picture, an alternative method should be performed. Where therapeutic monitoring is required, the same activity assay should be used throughout.

Coagulation, fibrinolysis and cytokine responses to intramedullary nailing of the femur: An experimental study in pigs comparing traditional reaming and reaming with a one-step reamer-irrigator-aspirator system

Introduction Operations in trauma patients represent a second insult and the extent of the surgical procedures influences the magnitude of the inflammatory response. Our hypothesis was that a reamer-irrigator-aspirator (RIA) system would cause a lesser inflammatory response than traditional reaming (TR). Materials and methods Coagulation, fibrinolysis and cytokine responses were studied in Norwegian landrace pigs during and after intramedullary nailing (IMN) with two different reaming systems using ELISA and chromogenic peptide substrate assays. The TR ( n = 8) and the RIA ( n = 7) reaming systems were compared to a control group ( n = 7). The animals were followed for 72 h. Arterial, mixed venous and femoral vein blood were withdrawn simultaneously peroperatively and until 2 h after the nail was inserted for demonstration of local, pulmonary and systemic activation of the cascade systems. At 6 h, 24 h, 48 h and 72 h postoperatively arterial blood samples were withdrawn. Results Significantly procedure-related increased levels were found for thrombin–antithrombin (TAT) and tissue plasminogen activator (t-PA) in the TR group and TAT in the RIA group. The local and the pulmonary activation of coagulation and fibrinolysis were more pronounced in the TR than in the RIA group, the difference reached significance for plasminogen activator inhibitor-1 (PAI-1) (arterial blood). The cytokine response, mainly represented by IL-6 increase, was more pronounced in the TR than the RIA group, and was significant for IL-6 in femoral vein blood. The arterial levels of IL-6 exceeded the mixed venous levels indicating an additional pulmonary activation of IL-6. Two animals in the TR group, who died of pulmonary embolism (PE) prior to planned study end point, had a more pronounced response compared to the rest of the TR group. Conclusion A procedure-related coagulation and fibrinolytic response was demonstrated in both reaming groups, with more pronounced response in the TR than in the RIA group. Elevated levels of cytokines were demonstrated related to reaming and nailing, with significantly higher IL-6 levels in the TR than in the RIA group.

Inhibition of Coagulation Activation and Inflammation by a Novel Factor Xa Inhibitor Synthesized from the Earthworm Eisenia andrei

We have cloned an earthworm-derived Factor Xa (FXa) inhibitor, with an excellent inhibitory specificity from the midgut of the Eisenia andrei. We designate this inhibitor eisenstasin. An eisenstasin-derived small peptide (ESP) was synthesized and we examined whether ESP played an essential role in FXa inhibition. Compared to antistasin-derived small peptides (ASP) originating from leech, ESP primarily exhibited a high level of FXa inhibition in chromogenic peptide substrate assays and revealed an approximately 2-fold greater inhibition of FXa cleavage of a target protein than ASP. This suggests that ESP could be an effective anti-coagulant that targets FXa during the propagation step of coagulation. ESP also inhibited proteinase-activated receptor 2-mediated FXa activation, which may trigger endothelial inflammation. Endothelial nitric oxide (NO) was significantly reduced by ESP (p<0.0001), indicating that protease-activated receptor-2 (PAR-2) was effectively inactivated. We also found that ESP reduced the expressions of pro-inflammatory cytokines (IL-1alpha, IL-1beta, IL-8, IL-16, MCP-1, MIP-1alpha and MIP-1beta) by cultured cells treated with both ESP and FXa. Our results provide the first evidence that ESP might interrupt coagulation cascades by inhibiting FXa, and thereby may effectively control the bidirectional alternation between coagulation and inflammation.

Changes in high molecular weight kininogen levels during and after cardiopulmonary bypass surgery measured using a chromogenic peptide substrate assay.

High molecular weight kininogen (HK) is a co-factor in the blood-contact activation system. A chromogenic peptide substrate assay for HK (HKcs) has been developed in which test plasmas are mixed with diluted HK-deficient plasma and incubated with a soluble contact system activator that activates prekallikrein and factor XII. Calcium chloride, a synthetic thrombin inhibitor and a chromogenic peptide substrate for activated factor X (FXa) are then added. The FXa generated cleaves the FXa substrate releasing p-nitroanaline, which is measured photometrically. Test plasma HK values were calculated from a standard curve generated using a pooled normal plasma. Acceptable intra-assay and inter-assay precision values were obtained and levels of HK up to 200% were measurable. The assay measured HK in plasmas deficient in factor XII, prekallikrein and factor XI, was not affected by antiphospholipid antibodies and gave an acceptable correlation (r = 0.95) when normal plasmas and mixtures of HK-deficient and normal pooled plasma, calculated to give HK levels of 25 and 50%, were compared using HKcs and a HK one-stage clotting assay. The HKcs was used to measure HK levels in seven patients undergoing cardiopulmonary bypass (CPB). HK levels fell significantly during CPB (P = 0.0014) and were significantly higher (P = 0.016) 6 days after CPB, suggesting that HK may be a positive acute-phase reacting protein.

Veno-venous bypass in liver transplantation: heparin-coated perfusion circuits reduce the activation of humoral defense systems in an in vitro model.

We studied the effects of bypass circuit surface heparinization on kallikrein-kinin, coagulation, fibrinolytic and complement activation in a closed model system for simulating veno-venous bypass (WBP) in orthotopic liver transplantation (OLT). The circuits were identical to those in routine use during clinical OLT in our institution. Fresh whole human blood diluted 1:2 with Ringer's acetate was circulated at a non-pulsatile flow (2 l/min) and at a constant temperature (37.5 degrees C) for 12 h. In 10 experiments, the entire inner surface of the circuits was coated with end-point attached heparin (HC). In the remaining 10, non-treated PVC tubing was used (NC). Components of the plasma kallikrein-kinin, coagulation, fibrinolytic and complement systems were analyzed using functional techniques (chromogenic peptide substrate assays) and enzyme immunoassays at baseline, 3 and 12 h. Significant activation of the initial (C3bc) and terminal (TCC) components of the complement system were found in both the NC and HC groups after 3 and 12 h: C3bc: NC: baseline = 4 (3.5-7.7), 3 h = 17.3* (12.5-27), 12h = 31* (17.7-63.6), HC: baseline = 4.9 (3.2-6.8), 3h = 9* (6-14.4), 12h = 13.7* (7.4-18.1). TCC: NC: baseline = 0.4 (0.2-0.6), 3h = 5*(0.8-11.9), 12 h: 13.1* (4.2-25.7). HC: baseline = 0.5 (0.1-0.6), 3 h = 0.6* (0.1-0.8), 12 h = 1.2* (0.3-2) AU/ml; median and range (*: p < 0.05). The C3bc and TCC concentrations were significantly higher in the NC group at 3 and 12 h, compared to the HC group: C3bc (NC vs. HC group): 3 h, p < 0.001; 12 h, p < 0.001. TCC (NC vs. HC group): 3h, p < 0.001; 12 h, p < 0.001. Significant increases in the values of thrombin-antithrombin complexes (p = 0.003), prothrombin fragment 1 + 2 (p = 0.006) and plasmin-alpha2-antiplasmin complexes (p = 0.016) were found in the non-coated group, but not in the heparin-coated group during the observation period, showing that the coagulation and fibrinolytic systems were activated in the non-coated circuits. We conclude that heparin-coating of the internal surface of the extracorporeal perfusion circuit used for WBP reduces activation of the plasma cascade systems in a closed venous system in vitro.

FXII levels, FXIIa-like activities and kallikrein activities in normal subjects and patients undergoing cardiac surgery

A new chromogenic peptide substrate assay kit was used to measure factor XII (FXII) levels in plasma samples from 115 male patients with heart disease awaiting cardiac surgery, 40 age-matched normal healthy male blood donors and 20 patients before, during and after cardiopulmonary bypass surgery (CPB). Kallikrein-like and FXIIa-like activities were also determined in the CPB patient group. FXII levels were significantly lower (p=0.0049) in the heart disease patients awaiting surgery when compared with values for the healthy donors and 13 patients (11.3%) had FXII levels below 50% compared with 1 normal donor (2,5%). During CPB significant decreases in FXII levels and significant increases in FXIIa-like and kallikrein-like activities were found indicating activation of the FXII-plasma kallikrein pathway during CPB.

A Chromogenic Substrate Assay Kit for Factor XII: Evaluation and Use for the Measurement of Factor XII Levels in Cardiopulmonary Bypass Patients

Factor XII levels were determined in plasma samples from 75 patients before undergoing aortocoronary bypass grafting and from 40 healthy age-matched donors by using a microtitre plate adaptation of a new chromogenic peptide substrate assay kit for factor XII. The chromogenic peptide substrate assay values for factor XII correlated well with those obtained in clotting r=0.90; y= 15.811+0.8236 x and immunochemical r=0.88; y=17.90+0.817 x assays in the normal donor samples. Factor XII levels in the patients were significantly lower than those in the normal donors 83.3±23.2% versus 103.4±23.1; p=0.004 , and nine patients (12%) had factor XII values below 50% compared with only one of the normal donors (2.5%). Factor XII levels and kallikrein-like activities (a measure of contact system activation) were followed before, during, and one day after cardiopulmonary bypass in 20 patients. Factor XII levels were significantly reduced, and kallikrein-like activities significantly elevated after 5 and 30 minutes cardiopulmonary bypass. One day after cardiopulmonary bypass both factor XII levels and kallikrein-like activities were significantly lower than preoperation values.

An automated chromogenic peptide substrate assay for coagulation factor XII.

We have developed an automated chromogenic peptide substrate assay for factor XII (FXIIcs) on a Cobas Mira S Plus clinical chemistry analyser using a new commercially available kit. This was used to determine factor XII (FXII) levels in plasma samples from 320 blood donors, 206 patients with a history of venous thrombosis and 74 lupus anticoagulant positive (LA+) patients. Results were compared with those obtained in a clotting assay for FXII (FXIIct) and an immunochemical assay (FXIIag). A satisfactory correlation coefficient of 0.92 and a regression line equation of y = 7.898 + 0.871x was obtained between FXIIcs and FXIIct in the 320 blood donors. Levels of FXII below the calculated normal range were found in nine blood donors (2.8%) and 16 venous thrombosis patients (7.8%). The blood donors and patients with venous thrombosis with low FXIIcs values had FXII levels below our lower limits of normal for both FXIIct and FXIIag; all were lupus anticoagulant negative. When FXII levels were determined in the 74 LA+ patients, 27 (36.5%) gave markedly lower FXII values in the FXIIct when compared with the FXIIcs. FXIIag levels corresponded with FXIIcs. The automated FXIIcs assay is therefore lupus anticoagulant insensitive and allows us to measure FXII levels accurately and routinely in large numbers of patient samples.

The effect of antithrombin III substitution therapy on components of the plasma protease systems in surgical patients

Objective: To evaluate the possible effects of antithrombin III (AT III) concentrate therapy on components of the plasma protease systems in surgical intensive care patients. Design: Open randomised, prospective study. Setting: General intensive care units in secondary and tertiary care hospitals. Subjects: Eighty-three surgical patients with functional plasma activity of AT III below 80% of normal plasma pool value the day after multiple trauma or major abdominal surgery. Interventions: Intravenous infusion of AT III concentrate. Endpoints: To maintain plasma AT III activity above 90% of normal plasma pool value in the therapy group. Measurements and main results: AT III, prothrombin, prekallikrein, kallikrein inhibition, kallikrein-like activity, plasminogen, antiplasmin activity and plasmin-like activity were measured with chromogenic peptide substrate assays. AT III, prothrombin, prekallikrein and plasminogen were reduced on the inclusion day in all patients. The other variables were in the normal ran...

Release of tissue plasminogen activator during reperfusion after different times of ischaemia in isolated, perfused rat hearts

Tissue plasminogen activator (t-PA) is a potential marker of endothelial cell activation or injury. The relationship between duration of ischaemia and release of t-PA during reperfusion was investigated in isolated rat hearts exposed to either 5, 10, 20, 30, 40, or 60 min of global, normothermic ischaemia followed by 30 min of reperfusion (n = 8 in each group). t-PA activity was measured (chromogenic peptide substrate assay) in the effluent before ischaemia, and after 2.5, 5, 7.5, 10, 20, and 30 min of reperfusion. Release of lactate dehydrogenase (LD), a marker of myocyte injury, was measured before ischaemia and after 5 min reperfusion. Left ventricular pressures were measured by a balloon in the left ventricle. Ischaemia for 20 min or less had only minor effects on cardiac function. Thirty min or more of ischaemia induced ventricular fibrillation during reperfusion in most hearts. After ischaemia t-PA outflow increased, but without any significant difference between groups. Peak release occurred after either 2.5 or 5 min of reperfusion. After 10 min reperfusion the release was not different from the basal value. In contrast, postischaemic release of LD correlated to the length of ischaemia. To conclude, t-PA release from the ischaemic-reperfused rat heart is independent of the length of ischaemia. Thus the potential of t-PA to quantify endothelial injury appears to be limited.

Triabin, a Highly Potent Exosite Inhibitor of Thrombin

Triabin, a new thrombin inhibitor, has been purified from the saliva of Triatoma pallidipennis, a blood-sucking triatomine bug. It forms a noncovalent complex with thrombin at a molar ratio of 1:1, inhibits thrombin-induced platelet aggregation, and prolongs thrombin clotting time and activated partial thromboplastin time. However, it only minimally suppresses the amidolytic activity of thrombin, as measured by a chromogenic peptide substrate assay. It completely blocks trypsin-catalyzed cleavage of thrombin, probably via protection of the anion-binding exosite and inhibits the effect of thrombomodulin on thrombin in a dose-dependent fashion. These results indicate that the inhibitor is directed toward the anion-binding exosite of thrombin. The protein was partially sequenced and the information used to isolate cDNA clones from a T. pallidipennis salivary gland library. Four slightly polymorphic variants coding for mature proteins of 142 amino acids preceded by a putative leader sequence were obtained. The recombinant protein expressed in the periplasmic space of Escherichia coli has a biological activity similar to that of salivary triabin, as tested in a thrombin-induced platelet aggregation assay. In addition, recombinant triabin inhibits thrombin-catalyzed hydrolysis of fibrinogen with a Ki of about 3 pM.

Activation of the plasma contact system and hemodynamic changes after graft revascularization in liver transplantation.

In this study, the relation between activation of the plasma contact system and hemodynamic changes during orthotopic liver transplantation was evaluated. Nineteen consecutive courses of OLT in 17 adult patients were investigated. Veno-venous bypass was used in all patients. Blood samples were drawn through all phases of the procedure, and analyzed for the following parameters using functional techniques (chromogenic peptide substrate assays): plasma kallikrein (KK), prekallikrein, functional plasma kallikrein inhibition, C1 inhibitor, and alpha 2-macroglobulin. Plasma high molecular weight kininogen (HK) degradation was evaluated using the immunoblotting technique. An abrupt rise in KK activities occurred within 1 min after portal reperfusion of the liver graft (7-16 U/L, P < 0.05). Simultaneously, proteolytic breakdown of HK was seen. The elevated KK activities were maintained the next 1 1/2 hr. Ten min after graft reperfusion, a significant increase in cardiac output compared with the anhepatic phase (7.2-12.4 L/min, P < 0.05) was found. At the same time, systemic vascular resistance fell significantly (817-408 dynes x sec/cm-5, P < 0.05). The increase in plasma KK activities accompanied by simultaneous degradation of HK seen immediately after reperfusion of the liver graft may be due to contact activation as recipient blood contacts with the underlying basement membrane of injured sinusoidal endothelium in the transplanted liver. We suggest that hemodynamic changes associated with the postreperfusion syndrome seen after revascularization of the liver in OLT could at least be caused in part by bradykinin release due to contact activation.

The effects of aprotinin on chromogenic peptide substrate and other assays for components of the coagulation, fibrinolysis and plasma kallikrein systems

Plasma samples containing a range of aprotinin levels (0–400 KIU/ml) were tested in chromogenic peptide substrate assays for antithrombin III, protein C, plasma kallikrein inhibition, plasminogen, antiplasmin, tissue plasminogen activator and plasminogen activator inhibitor. No effect on the antithrombin III assay was seen. Aprotinin produced significantly reduced values in the assays for protein C, plasminogen and tissue plasminogen activator, and significantly increased values for kallikrein inhibition, antiplasmin and plasminogen activator inhibitor. Immunochemical assays for plasminogen activator including a new assay (BIA-t-PA) and plasminogen activator inhibitor were not affected by aprotinin. When plasma samples from patients undergoing cardiopulmonary bypass, with and without high dose aprotinin therapy, were tested in the chromogenic substrate assay for plasminogen activator inhibitor, markedly elevated apparent inhibitor levels were found in the aprotinin group.

Lipopolysaccharide-induced monocyte procoagulant activity, fibrinopeptide A, and PAI-I levels in persons with high or low levels of HDL-lipoprotein

We have examined basal and stimulated monocyte procoagulant activity (PCA) in 10 persons with high HDL (1.62–2.47 mmol/L) and 10 persons with low HDL (0.43–1.29 mmol/L). Heparinized whole blood was incubated at 37°C for 2 hours with 100 ng/ml of E. Coli lipopolysaccharide (LPS). Monocytes were isolated by density gradient centrifugation (purity >;70%), and PCA measured with a chromogenic peptide substrate assay. LPS-induced PCA was significantly higher in the high HDL group (p=0.02), whereas basal levels were similar. Furthermore, the levels of total HDL, HDL2, as well as apo-A1 were all significantly correlated with LPS-induced monocyte PCA (p<0.01, p<0.01, and p=0.03), whereas VLDL was inversely correlated (p=0.02). PAI-I activity was significantly lower in the high HDL group (p<0.01). LPS recovery in plasma after incubation, by LAL test, was significantly higher in the high HDL group (p=0.02) and correlated to HDL2 (p=0.04), and inversely correlated with triglycerides (p=0.04). There was no significant difference between the two groups in plasma fibrinopeptide A (FPA) levels after LPS incubation.

Sulfur mustard-increased proteolysis following in vitro and in vivo exposures.

The pathologic mechanisms underlying sulfur mustard (HD)-induced skin vesication are as yet undefined. Papirmeister et al. (1985) postulate enhanced proteolytic activity as a proximate cause of HD-induced cutaneous injury. Using a chromogenic peptide substrate assay, we previously reported that in vitro exposure of cell cultures to HD enhances proteolytic activity. We have continued our investigation of HD-increased proteolytic activity in vitro and have expanded our studies to include an in vivo animal model for HD exposure. In vitro exposure of human peripheral blood lymphocytes (PBL) to HD demonstrated that the increase in proteolytic activity is both time- and temperature-dependent. Using a panel of 10 protease substrates, we established that the HD-increased proteolysis was markedly different from that generated by plasminogen activator. The hairless guinea pig is an animal model used for the study of HD-induced dermal pathology. When control and HD-exposed PBL and hairless guinea pig skin where examined, similarities in their protease substrate reactivities were observed. HD-exposed hairless guinea pig skin biopsies demonstrated increased proteolytic activity that was time-dependent. The HD-increased proteolytic response was similar in both in vitro and in vivo studies and may be useful for elucidating both the mechanism of HD-induced vesication and potential treatment compounds.

Plasminogen activation in lesional skin of Pemphigus vulgaris type Neumann

Zymographic and immunological studies revealed that primarily tissue-type plasminogen activator and to a lesser extent urokinase-type plasminogen activator were present in fluids of pemphigus vulgaris (type Neumann) skin blisters. Furthermore, plasmin activity was detected in pemphigus blister fluids using chromogenic peptide substrate assays. In pemphigus, but not in control, suction blister fluids plasmin/alpha 2-antiplasmin and plasmin/alpha 2-macroglobulin complexes were found by immunoprecipitation or by testing in immunoassays after fractionation by molecular-sieve chromatography. Plasmin activity, detected by a low molecular weight chromogenic peptide assay, was ascribed to plasmin/alpha 2-macroglobulin complexes. Since formation of plasmin/inhibitor complexes requires active plasmin, the finding indicates previous activation of plasminogen in pemphigus lesions.

Hemodynamic and proteolytic effects of intravenous injection of purified human plasma kallikrein.

To investigate the precise hemodynamic effects of purified human plasma kallikrein, small doses (0.3 U/kg) of this proteolytic enzyme were infused over 10 s in five pigs. Hemodynamic performance was evaluated by means of a Swan-Ganz catheter, and parameters of the plasma kallikrein-kinin, coagulation, and fibrinolytic systems were determined by means of chromogenic peptide substrate assays. The infusions of plasma kallikrein were followed by rapid decreases in mean arterial pressure (to 43 +/- 2% of baseline values) and increases in cardiac output (to 145 +/- 6% of baseline values), accounting for marked decreases in systemic vascular resistance, to 28 +/- 1% of initial values. Heart rate, mean pulmonary arterial, central venous, and pulmonary capillary wedge pressures did not change after the injection. Pulmonary vascular resistance decreased to 69 +/- 3% of baseline values. The hemodynamic changes gradually normalized during the following 10 min. Functional levels of prekallikrein, kallikrein, kallikrein inhibition, prothrombin, antithrombin III, plasmin, and antiplasmin were not affected by the injection or the resulting hemodynamic changes; the hematocrit and the number of circulating leukocytes or platelets were also unaffected.

Inhibition of sulfur mustard-increased protease activity by niacinamide, N-acetyl-L-cysteine or dexamethasone.

The pathologic mechanisms underlying sulfur mustard-induced skin vesication remain undefined. Papirmeister et al. (1985) have postulated a biochemical mechanism for sulfur mustard-induced cutaneous injury involving DNA alkylation, metabolic disruption, and enhanced proteolytic activity. We have previously utilized a chromogenic peptide substrate assay to establish that human peripheral blood lymphocytes exposed to sulfur mustard exhibited enhanced proteolytic activity. In this study, compounds known to alter the biochemical events associated with sulfur mustard exposure or to reduce protease activity were tested for their ability to block the sulfur mustard-increased proteolysis. Treatment of cells with niacinamide, N-acetyl-L-cysteine, or dexamethasone resulted in a decrease of sulfur mustard-increased protease activity. Complete inhibition of sulfur mustard-increased proteolysis was achieved by using protease inhibitors (antipain, leupeptin, and 4-(2-aminoethyl)-benzenesulfonylfluoride). These data suggest that therapeutic intervention in the biochemical pathways that culminate in protease activation or direct inhibition of proteolysis might serve as an approach to the treatment of sulfur mustard-induced pathology.

Effect of sulfur exposure on protease activity in human peripheral blood lymphocytes

Sulfur mustard is a chemical warfare blistering agent for which neither the mechanism of action nor an antidote is known. Papirmeister et al. (1985) have postulated a biochemical hypothesis for mustard-induced cutaneous injury involving a sequelae of DNA alkylation, metabolic disruption and activation of protease. Human peripheral blood lymphocytes in cell cultures were employed as an in vitro model for alkylating agent toxicity. A chromogenic peptide substrate assay was used for detection of protease in lymphocytes treated with sulfur mustard or chloroethyl ethyl sulfide. Exposure of human peripheral blood lymphocytes from normal donors to these alkylating agents resulted in an increase in cell associated protease activity. This increase in protease activity may contribute to the pathology or act as an indicator to predict methods of therapeutic intervention for sulfur mustard toxicity.

Chromogenic peptide substrate assays and their clinical applications

Chromogenic peptide substrates were first introduced into research laboratories in the early 1970s and were quickly utilised to develop assays for the determination of enzymes, proenzymes and inhibitors of the coagulation system. These assays were gradually introduced into coagulation and clinical chemistry laboratories as laboratory tools in the diagnosis and treatment of coagulation disorders. From the knowledge of the structures of the natural substrates attacked by enzymes other than those of the coagulation system or by synthesis and random screening, substrates for enzymes of the fibrinolytic, plasma and glandular kallikrein and complement systems were produced. These allowed various research groups to develop assays for components of these systems and subsequently led to the use of these assays in studies on various clinical conditions. Substrates for activated protein C ensured that assays for this enzyme and its inhibitors could be developed and introduced into the haematological routine. With the introduction of substrates for limulus lysate not only were assays for endotoxins in clinical samples produced but the control of all disposable products and injectables for endotoxin contamination can now be effected. Initially high costs and time-consuming manual assays were a hinderence to the general acceptance of the use of chromogenic peptide substrate assays and they were only used routinely in a few specialised laboratories. With the introduction of automated and microtitre plate methods however, these assays are now available in most hospital laboratories. Since the first chromogenic peptide substrate was described thousands of articles have been published on the use of chromogenic substrate assays to measure proenzymes, enzyme activators, enzyme cofactors and inhibitors in blood and other body fluids in normal subjects and clinical material. We have endeavoured to cover as many of these as possible in this review.