Chromogenic peptide substrate assays and their clinical applications

Chromogenic Peptide Substrate Assays and their Clinical Applications

M. J. Gallimore, P. Friberger S UM M A R Y. Chromogenic peptide substrates were first introduced into research laboratories in the early 190s 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 timeconsuming 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.

Plasma and cellular proteases, their activators, inhibitors and cofactors play vital roles in various defence mechanisms in man and other animals. In healthy individuals enzyme generation and inhibition is in a dynamic balance ensuring that excessive amounts of free enzyme are not produced. In inherited deficiences or deficiences acquired because of some disease process this equilibrium is disturbed, often to such an extent that the life of the organism is threatened. M. J. G-ore, Channel Diagnostics, 26 Sydney Road, Deal, Kent CT14 9JW, UK, P. Friherger, Kabi Diagnostica, Molndal, Sweden. Correspondence to M. J. Gallimore. BIoodReviews (1991) 5, 117-127 0 1991 Longman Group UK Ltd

Walmer, S-43133

Several proteolytic systems have been identified, including coagulation, fibrinolysis, kallikrein-kinin, renin-angiotensin and complement, composed of inactive zymogens (proenzymes) whose activities are regulated by activators and inhibitors as well as by positive and negative feedback mechanisms. They are connected to each other in several ways (Fig. 1), primarily through the activation of FXII and as such, form components of the contact system of blood. A list of inhibitors of enzymes in these systems is shown in Table 1. (For reviews on these systems see’- 5, Historically assays for determining components of these systems involved the use of natural protein

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CHROMOGENIC

PEPTIDE SUBSTRATE ASSAYS COAGUIATIONSYSTEM

nill

KAJLIKREIN-KININ SYSTFMS FREKALLIKREIN

HIGH MO G]

IBI MOLFCULAR PROKALLIKREIN

&Ew

“““i’“‘TdEN

BRADYKININ \

y-

KININASES

INACTIVE PEPTIDES

INIRINSIC PATHWAY

EXTRINSIC PATHWAY FVll-

FX __L

FXa

TISSUE FAC’l0R

I PROTHRCMBIN-T~~R~~BIN-FIBRIN(X;EN

INHIBITION-ACTIVATED tOF IWXEIN C PROTEIN S COAGULATION

--,

FIBRIN-FDP’S

1 PROTEIN C THRCPlBOMODULIN

Fig. 1 Schematic outline of the coagulation, fibrinolytic, kallikrein-kinin protein C and complement systems. Table 1

Plasma proteinase inhibitors and the enzymes which they inhibit Inhibitor

Molecular weight

Enzymes inhibited

a-2-Macroglobulin

725000

Inter-a-trypsin inhibitor Cl-esterase Inhibitor

168000

Trypsin, Plasmin, Plasma kallikrein, Urokinase, Elastases thrombin, a FXII a Trypsin

104000

a-1-Antichymotrypsin

69000

a-2-Antiplasmin Heparin cofactor II Antithrombin III

68000 66000 61000

Protein C inhibitor

57000

a-I-Antitrypsin

51000

Plasminogen activator Inhibitor I Plasminogen activator Inhibitor 2

50000 48000

Cl-esterase, Plasma kallikrein, a and p FXIIa, FXIa Chymotrypsin, Cathepsin G Plasmin Thrombin Thrombin, FXa, a FXIIa Activated protein C, Plasma kallikrein Trypsin, Neutrophil elastase, FXIa Tissue plasminogen activator, Urokinase Tissue plasminogen activator, Urokinase

substrates. Although these give a high degree of specificity the proteins are difficult to isolate and purify in a uniform manner and assays utilising them are often cumbersome and the results difficult to interpret. This led to the introduction of simple amino acid ester substrates, e.g. tosyl-arginine-methyl ester and benzoyl-arginine-ethyl ester, and chromogenic single amide substrates, e.g. benzoyl-arginine-p-nitroanilide. A new generation of substrates was born when, after the elucidation of the amino acid sequence around the thrombin cleavage site of fibrinogen and

the sequencing of fibrinopeptide A, a chromogenic tripeptide substrate (Bz-Phe-Val-Arg-pNA, S-21 60) with an affinity for thrombin was synthesised. This led to the rapid development of chromogenic peptide substrate assays for components of the coagulation and fibrinolytic systems. ‘-s Subsequently a battery of synthetic tripeptide chromogenic substrates (CS) have been synthesised enabling a large number of proteinases, activators, inhibitors and cofactors to be assayed (Table 2) and has led to the widespread clinical use of CS assays. The development of automated and acute type assays has ensured that all laboratories are able to utilise these assays for the diagnosis and monitoring of treatment of a wide variety of clinical conditions. A list of the clinical situations where these assays are now used is shown in Table 3.

Haemostasis Because chromogenic peptide substrates were first synthesised for enzymes of the coagulation and fibrinolytic systems they rapidly became important in assays designed to detect haemostatic abnormalities. The use of chromogenic substrates in haemostasis has already been excellently reviewed in this journal9 we would however like to add and elsewhere, “J some comments of our own in this area. The fact that defects in the coagulation or fibrinolytic systems can lead to haemorrhagic or thrombotic complications are illustrated by the following well recognised relationships. Deficiencies in coagulation factors or fibrinolytic inhibitors frequently lead to bleeding complications. Excess production of fi-

BLOOD Table 2 Chromogenic peptide substrate activators, cofactors and inhibitors

assays

for proenzymes,

Thrombin, FXa, FXIa, a FXIIa, g FXIIa, plasmin, trypsin plasma kallikrein, glandular kallikrein, urokinase, tissue plasminogen activator, activated protein C, Cls, Clr, chymotrypsin, pancreatic elastase, leucocyte elastase, cathepsin G, acrosin, snake venoms, limulus and crayfish lysates, papain-like enzymes, bacterial and fungal proteases

Proenzymes

Prothrombin, FXII, urinary prokallikrein, urokinase

Activators

FIXa, FVIIa, a FXIIa, S FXIIa, tissue plasminogen activator, urokinase, streptokinase, endotoxins

Inhibitors

Antithrombin III, heparin cofactor II, a-2-anti-plasmin, a-2-macroglobulin, a-1-antitrypsin, tissue plasminogen activator inhibitors, Cl-esterase inhibitor, protein C inhibitors, aprotinin, hirudin, chymotrypsin inhibitors, pancreatic elastase inhibitors, leucocyte elastase inhibitors

Cofactors and moderators

FVIII, FV, platelet dermatan sulphate, weight kininogen

Table 3

Medical

Internal

medicine

disciplines

1 I9

enzymes,

Enzymes

-

REVIEWS

FXI, FX, FVII, plasma prekallikrein, plasminogen, protein C. pro-

factor III, platelet factor 4, heparins, fibrin monomers, high molecular

where chromogenic

peptide

substrate

assays are used

Ischemic heart disease, allergic diseases, acute leukemia, diabetes, hypertension, liver disease, dysfunction of the reticula endothelial system, haemodialysis, cancer

Haematology

Coagulation profile, fibrinolysis profile, disorders, angina, deep vein thrombosis, cancer

Obstetrics/ gynecology

Preeclampsia. hypertension, disseminated coagulation, deep vein thrombosis

Nephrology

Nephrotic

Surgery

Major surgery, tumor surgery, embolism

Orthopedics

Deep vein thrombosis,

Anaesthiology/ intensive care

Severe burns, pancreatitis, disseminated intravascular

Infectious diseases

Sepsis, bacteremia,

Geriatrics

Circulatory

syndrome,

transplant

liver cirrhosis, vascular thromboembolism,

intra-vascular

rejection

cardiopulmonary bypass, abdominal surgery, major trauma, sepsis, vascular surgery,

diseases,

brinolytic activators (or excessive use of these activators during therapy) or a large potentiation of coagulation inhibition (e.g. during heparin therapy) has the same effect. On the other hand deficiencies in coagulation inhibitors (e.g. antithrombin III, heparin cofactor II), insufficient release of plasminogen activators or excessive production of plasminogen activation inhibitor can lead to thrombosis. A powerful activation of coagulation following for example trauma has the same effect. We can therefore illustrate these protease systems and their inhibitors as a dynamic balance model influenced by four forces (Fig. 2). One of these forces can be reduced by hereditary deficiency which usually means a risk for

embolism peritonitis, respiratory coagulation. shock

viremia,

acquired

rheumatoid

immune

distress,

deficiency

arthritis

Inhibitors of coagulation

Inhibitors of fibrinolysis

A t

t

Coagulation

Fibrinolysis

Fig. 2 Coagulation and fibrinolysis can be viewed as antagonising systems regulated by inhibitors. The amplitude and direction of these forces are important for the balance in the normal state. In pathological states the balance is disturbed and hyper-coagulation or hyper-fibrinolysis results. Similar balances occur in other plasma defence systems.

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the individual. Often however no complications appear until some stressful1 situation, e.g. surgery or pregnancy leads to thrombo-embolic complications. This indicates that both poor balance and a trigger are needed to cause bleeding or thrombosis (Fig. 3). In some cases a balance exists because counterbalancing forces counteract one or more abnormalities, in others an inbalance occurs because several slightly abnormal factors add up to create a pathological condition. Similar balances exist in the other plasma defence systems and in all of these systems there is an interplay between cellular and fluid elements. It is evident therefore that all forces acting on the balance should be investigated to ensure a correct diagnosis, often this is impossible however and the parameters which have been proven to yield the best diagnostic results have to be determined. It is in this area where CS assays have proved to be so valuable. As with all of the defence pathways, deficiences in particular haemostatic parameters can be inherited or acquired. In hereditary deficiencies the abnormality can first be identified by the use of CS assays and then therapy introduced and monitored by these assays. For example in antithrombin III deficiency therapy with antithrombin III concentrate has produced excellent clinical resultsi and currently most of the deficiencies can be successfully treated either by concentrates or by the use of heparin or oral anticoagulants. Recent developments in the diagnosis of abnormal haemostasis have, after initial studies with coagulation or fibrinolytic assays, depended heavily on CS assays. Parameters which have been identified as very important in this area include: tissue plasminogen activator (tPA),i3 plasminogen activator inhibitor 1 (PA1 1),r4 protein C,15 protein S,16 heparin cofactor 11,l’ FVIIr* and extrinsic pathway inhibitor.” The utilisation of several of these assays together with CS assays for antithrombin III, plasminogen and alpha2-antiplasmin have led to thrombophilia screening in a large number of haematology and coagulation laboratories. Over the past 5 years CS assays for tissue plasminogen activator and its major inhibitor (PA1 1) have been increasingly used in studies on myocardial infarction, deep vein thrombosis and pulmonary embolism. This topic has been excellently reviewed elsewhere.20 CS assays for FXI121 FXI,22 high molecular weight kininogen,23 and Cl-esterase inhibitor24 have widTRIGGER

Fig. 3 Schematic representation of the pathophysiology of haemostatic diseases.

ened the scope for studies on the intrinsic coagulation pathway. Intensive Care In intensive care CS assays are increasingly being used to monitor the status of the patient, control therapy and aiding in the introduction of new therapies. Trauma Trauma may lead to major changes in components of the plasma defence systems and CS assays have played a major role in identifying these changes and monitoring treatment. 25 In particular the plasma levels of prekallikrein, antithrombin III and FXII appear to be reasonably good indicators of recovery following trauma. Septic Shock In septic shock CS assays are increasingly being used to measure components of the plasma defence systems.26 Special attention has been paid to the FXII related defence pathways as FXII is known to be activated by endotoxins2’ and endotoxin has been found in the peripheral blood of patients with septic shock.28 In a canine endotoxin shock model, CS assays were used to monitor components of the FXIIrelated pathways. 29,30 The course of lethal canine endotoxinemia was characterised by progressive changes in components of these pathways. Activation of the plasma kallikrein-kinin system was a major feature of the terminal vascular collapse. Falls in levels of several plasma protease inhibitors were also observed and granulocyte elastase release was demonstrated. It was suggested that defective inhibitory regulatory mechanisms are associated with the pathophysiological changes observed in the canine shock model.3’ In another study in a canine model endotoxin infusions caused significant falls in components of the coagulation, fibrinolytic and complement systems.32 By using aprotinin or a specific granulocyte proteinase inhibitor (Bowman-Birk inhibitor from soybeans) the changes induced by endotoxin were reduced. Subsequently studies using CS and other assays in plasma samples from humans who either died during, or recovered following, septic shock, revealed that extensive pathological plasma proteolysis occurs during sepsis and that significant differences between values for some parameters between the two groups of patients in the study were seen.33 This study gave valuable information about the pathophysiology and prognosis in septic shock and showed that persistent sepsis caused by an unrecognised septic focus was associated with continuous low values for prekallikrein, kallikrein inhibition, plasminogen and antithrombin III. In the patients where successful therapy was effected, values for these par-

BLOOD REVIEWS

ameters returned to normal. In this and other studies the role of protease inhibitors in septic shock was apparent and in particular the functional activity of Cl-esterase inhibitor (the major inhibitor of contact system enzymes)34 and antithrombin III35 were highlighted. These studies led to the use of the so-called proenzyme functional inhibition index (PFI index) as a diagnostic aid in critically ill patients. The PFI index is defined as the sum of deviations from the normal plasma pool values for the proenzyme and functional inhibition values of the coagulation, fibrinolytic and kallikrein-kinin systems.36 The paramprothrombin measured eters include, and antithrombin III, plasminogen and antiplasmin, and prekallikrein and kallikrein inhibition. The PFI index gives reliable information on the severity of the illness and prognosis from the first days of therapy in severe septicemia. Using the PFI index new therapies in septic shock are undergoing evaluation including infusions of antithrombin III, Cl-esterase inhibitor and aprotinin either singly or in combinations. Recently high levels of plasminogen activator inhibitor 1 (PAIl) have been reported in septic shock3’ and an association between high PA1 1 levels and mortality has been reported. 2o Also high urokinase-type plasminogen activator (U-PA) levels have been found in sepsis patients suggesting that this parameter may also be a useful diagnostic indicator in septic shock. Disseminated

Intravascular

Coagulation

As a result of severe trauma, sepsis, obstetric complications, pancreatitis, leukemia, etc. the clinical condition known as disseminated intravascular coagulation (DIC) can occur. In this syndrome an increased intravascular coagulation occurs often followed by bleeding and is the result of hyperactivation of the coagulation, fibrinolytic and often the plasma kallikrein and complement systems. As DIC can rapidly lead to multiple organ failure (MOF) and/or adult respiratory distress syndrome (ARDS) it is of vital importance to diagnose DIC as soon as possible and this is where CS assays are becoming increasingly important. In particular analyses of antithrombin III, prekallikrein, kallikrein inhibition, antiplasmin, Clesterase inhibitor and alpha-2-macroglobulin are being used as diagnostic indicators.36,38-40 Also prothrombin, plasminogen, plasminogen activation inhibitor (PAI-I), protein C, FVIII, PMN elastase inhibition and endotoxin levels are increasingly being utilised. All of these parameters are analysed by CS assays. The use of the PFI index has been reported to be an excellent indicator for predicting MOF.41 The recently introduced CS assay for fibrin monomers42 is increasingly being utilised in DIC and other life threatening conditions. In DIC fibrin monomer levels above 50 nmol/l is a specific finding. After surgery fibrin monomer levels rise to between 50175 nmol/l. Levels above 200 nmol/l indicate a life threatening hypercoagulation. This assay has been

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used to identify thromboembolic complications in pre-eclampsia, septic shock, DVT, stroke and acute myocardial infarction. Cardiopulmonary

Bypass

As F plays a pivotal role in the plasma defence systems, other clinical situations were FXII becomes activated present patho-physiological changes as a result of this activation. One such situation occurs during cardiopulmonary bypass (CPB) where blood comes into prolonged contact with an activating surface. CS assays have been and are still being extensively used to study CPB both in vivo and in vitro. By using CS assays it was shown that the plasma kallikrein system becomes activated early in CPB and this was exemplified by raised kallikrein like activities and falls in FXII, prekallikrein and kallikrein inhibition in the blood. 43 As plasma kallikrein liberates bradykinin from high molecular weight kininogen and both bradykinin and plasma kallikrein are powerful liberators of tissue plasminogen activator& the resultant activation of prekallikrein leads to increased fibrinolysis and this contributes to the blood loss in CPB. Further studies with CS assays revealed that the high doses of heparin used during CPB markedly reduced the inhibition of beta FXIIa by Cl esterase inhibitor.45 Recently high dose aprotinin therapy during CPB has been recommended to reduce blood 10~s.~~ Aprotinin potentiates inhibition of beta FXIIa in heparinised blood both in vitro and in vivo45 and it has been suggested that this effect of aprotinin by blocking the FXII-plasma kallikrein fibrinolytic pathway reduces hyperfibrinoiysis and this contributes to the reduction in blood loss.’ CS assays for aprotinin are available to monitor this protease inhibitor. 47*48It has also been reported that activated components of the FXII-kallikrein system are chemotactic for neutrophils and release of PMN elastase during CPB has been shown.4g During simulated CPB in vitro, Cl-esterase inhibitor was shown to block the release of PMN elastase produced by added beta FXIIa and plasma kallikreinsO and it was suggested that infusions of Cl-esterase inhibitor or PMN elastase inhibitors5,32 could have therapeutic benefits in CPB. Also because of the negative effects of heparin it has been suggested that other anticoagulants such as hirudin or synthetic protease inhibitors might have clinical benefits in CPB.’ Pancreatitis

In pancreatitis CS assays have been used to compare changes in components of the plasma defence systems and to follow the clinical course of the disease. In a study on 26 patients with acute pancreatitis at least 5 patients had marked evidence of activation of the plasma kallikrein system. Two patients had evidence of activation of the coagulation and fibrinolytic systems without involvement of the kallikrein system.51

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PEPTIDE SUBSTRATE ASSAYS

In another study 14 patients with acute pancreatitis were investigated using CS and other assays,52 reduced prekallikrein, antithrombin III and kallikrein inhibition values were observed. In the fatal cases these values were significantly lower than in the patients who recovered. In severe pancreatitis evidence for activation of both the complement and kallikrein systems was found in blood and peritoneal fluid.53 The extent of activation being closely correlated with the severity of the disease. Functional alpha-2-macroglobulin levels and kallikrein inhibition values were significantly lower and activated trypsin levels higher in severe attacks than in other attacks. Changes in proteases and protease inhibitors were most pronounced in the peritoneal fluid. Because of the obvious reduced proteinase inhibitory capacity in severe attacks it was suggested that high dose aprotinin therapy could be of benefit in the disease. Liver Disease

Because the liver is the major organ for production of the majority of components of the contact system CS assays have been used to study some of these components in liver disease. Plasma levels of antithrombin III, prekallikrein, plasminogen and alpha-2-antiplasmin have all been studied as prognostic indicators in liver disease and compared with tests which measure the vitamin K dependent coagulation factors and fibrinogen levels. Early studies showed that prekallikrein, antithrombin III, plasminogen and alpha-2-antiplasmin levels are low in chronic liver disease54*55 and all of these parameters have been shown to be superior to fibrinogen and other parameters in diagnosing liver damage. It has been suggested that prekallikrein54 or prekallikrein together with FVI156 are particularly good liver function parameters. Plasma levels of protein C and heparin cofactor II are low and alpha-2-macroglobulin levels are significantly elevated in chronic liver disease. As the liver is the principal organ responsible for clearance of tPA and hepatocytes produce PA1 1, in liver disease both tPA and PA11 levels increase.” Because of reduced synthesis by the liver antithrombin III levels are low in a majority of intensive care patients. It is apparent therefore that infusions of this protease inhibitor can help in protecting the patient from the effects of these reduced levels. Kidney Disease and Transplantation CS assays have also been used in attempts to obtain diagnostic indicators of rejection in liver and kidney transplant patients.57*58 In an attempt to find early indications of kidney transplant rejection before clinical symptoms were noticed CS and other assays were used to follow some of the components of the coagulation, fibrinolytic and kallikrein-kinin system in 19 kidney transplant patients. From all of the parameters studied kallikrein inhibition, beta FXIIa inhibition,

plasminogen and antithrombin III were early indicators of transplant rejection. Significantly elevated plasma levels of these parameters were found 2-3 days before clinical signs of rejection were noticed. No significant changes in these parameters were seen in samples from patients without rejection.58 Because antithrombin III levels were elevated during rejection it was suggested that this might be a response induced to reduce intravascular coagulation in the transplanted kidney. A study was therefore instigated to see whether antithrombin III infusions reduced transplant rejection. In a group of patients who had infusions of antithrombin III and who received donor kidneys which had also been transfused with antithrombin III a significant reduction in the number of rejections (3/15) were observed compared with the control group (12/22) who had no antithrombin IIL5’ In renal disease CS assays have been used to study components of the contact system of blood. Prekallikrein and antithrombin III levels are low in chronic glomerulonephritis, and nephrotic syndrome.60T61 In patients undergoing renal dialysis low levels of prekallikrein, high molecular weight kininogen and FXII were found.60 In the intensive care situation one problem with all assays has been the inability to have bedside assays and the subsequent delayed results on patients samples has restricted their value. The recent introduction of single cuvette CS assays (Coacute antithrombin and Coacute heparin)62 allow rapid monitoring of antithrombin and heparin to be effected in the intensive care department. These assays will play vital roles in monitoring both the status and treatment of the patient in intensive care in the future.

General Medical Disorders Hypertension CS assays have been used to study components

of both blood and urine in hypertension. This is because not only are there blood and urinary kallikreins which liberate kinins from kininogens and whose effects are the opposite to the renin-angiotensin system, (lower blood pressure), but also disturbances in, for example, the coagulation system can lead to microthrombi formation in the kidneys with subsequent hypertensive side-effects. Most studies have been performed using CS assays to measure urinary kallikrein levels following on from the observations that reduced amounts of this enzyme are excreted in essential hypertension. 63 A CS assay was used to monitor urinary kallikrein levels in a study whereby elevated blood pressure in patients with essential hypertension was normalised following oral therapy with pig pancreatic kallikrein.64 Recently it has been reported that in pregnancy-induced hypertension urinary kallikrein levels are reduced with the most pronounced effects being on inactive urinary kallikrein.65 By calculating the inactive urinary kallikrein-creatinine

BLOOD REVIEWS

ratio a predictive indicator of pregnancy-induced hypertension was obtained. The role of plasma prekallikrein in hypertension has not been adequately investigated, although it is well known that infusions of plasma kallikrein in experimental animals produce immediate falls in blood pressure.& In one published study 66 it was suggested that determinations of prekallikrein differentiated between renal hypertension (low prekallikrein values) and essential hypertension (high prekallikrein values). However as it has been reported that activation of the FXII-prekallikrein pathway leads to activation of the plasma reninangiotensin system67 more work is required in this area.

123

Hereditary Angioneurotic Edema

In hereditary angioneurotic edema (HANE) C 1-esterase inhibitor is either absent or functionally inactive.77-79 FXII levels are significantly higher and high molecular weight kininogen levels lower than normal.*’ The disease is characterised by recurrent attacks of mucosal and subcutaneous swelling and the patient can die because of laryngeal edema. During an attack there is a simultaneous activation of FXII and the plasma kallikrein and classical complement pathways. The acute attacks can be controlled by infusions of C 1-esterase inhibitor.

Cold Urticaria Diabetes

Abnormalities of the plasma defence systems have been studied in diabetes and it is thought that in this disease there is an increased risk of thrombosis with increased plasma FVIII levels and reduced levels of tissue plasminogen activator and antithrombin III. Because glandular kallikreins have been shown to convert proinsulin to insulin6* and kinins have insulin-like activity, components of the plasma and urinary kallikrein systems have been studied in diabetes. Plasma prekallikrein levels and kallikrein like activities have been shown to be elevated in diabetes,@j whilst urinary kallikrein levels are 10w.~~

In cold urticaria histamine is released upon cold challenge of the skin and it has been suggested that exposure to cold could generate kallikrein-like activity and liberate bradykinin. *i CS assays have been used to study subjects with cold urticaria and a functional defect in Cl-esterase inhibition with normal antigen levels were found.82 Nine out of 20 patients plasmas exhibited cold activation of FVII and after cold activation a significant fall in functional Cl-esterase inhibition was observed in samples from these subjects. Addition of purified Cl-esterase inhibitor to the plasmas before exposure to cold blocked the activation of FVII.

Emphysema Cancer

Coagulation and fibrinolytic disorders are commonly observed in cancer in particular in acute leukemia and are thought to be caused by the release of thromboplastin-like materials or tissue plasminogen activators from cancer cells.70*71CS assays have been used to look for proteolytic activities in cancer cell lines7’ and to determine components of the plasma defence systems in patients with a variety of cancers.73 It has been reported that carcinoma cells are coated with Cl-esterase inhibitor and that this might protect the carcinoma cells against the humoral immune system. 74 Cl-esterase inhibitor is the major inhibitor of the contact activation pathways of coagulation, fibrinolysis, and kinin generation and of the classical complement pathway. 34 Levels of this proteinase inhibitor and functional kallikrein inhibition were significantly elevated and prekallikrein reduced in plasma samples from patients with breast, gastric and lung cancer.73 The most pronounced changes were seen in patients with lung cancer. It is of interest that following cytostatic treatment of breast cancer Clesterase inhibitor levels fe1175 and in other cancer patients prekallikrein levels were normalised following chemotherapy.66 Increased plasma levels of PA11 have been reported in patients with a variety of tumors.76

Subjects with inherited alpha-1-antitrypsin deficiency (alpha-1-proteinase inhibitor deficiency) have a high incedence of pulmonary emphysema83 in which lung elastin degradation occurs as a result of uncontrolled elastase activity. Neutrophil elastase is thought to be the major enzyme involved in the development of emphysema. Some smokers who acquire emphysema however have normal concentrations of alpha-l-proteinase inhibitor.84 It has been suggested that in these subjects oxididation of the reactive site methionyl residue produces a molecule with markedly reduced capacity to inhibit neutrophil elastase. CS assays for alpha-1-antitrypsin using either trypsin or PMN elastase have been developed and it has been reported that by using these two enzymes it is possible to differentiate between oxidised and normal alpha-lantitrypsin. 84 In cardiopulmonary bypass inhibition of PMN elastase is more markedly reduced than inhibition of trypsin. Other clinical conditions where contact system activation occurs and where CS assays are beginning to be utilised include: acquired immune deficiency syndrome, allergic diseases, rheumatoid arthrytis, dermatitis, cystic fibrosis and peridontitis. Changes in components of the contact systems following intravenous phlebography and prosthesis inplantation are also being studied. These studies are still at an early

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CHROMOGENIC

PEPTIDE SUBSTRATE ASSAYS

stage but CS assays will undoubtedly become important in these areas in the future. Other Clinical Applications Endotoxin Determinations The introduction of highly sensitive CS assays for endotoxins has revealed that not only are endotoxins present in patients with septic shocks5 but that a lot of the parenterals (heparins etc.) and devices (canulae, plastic lines, syringes etc.) were contaminated with endotoxins.86 The areas where CS endotoxin assays have been used are outlined in Table 4. These findings have led to numerous studies on endotoxins in intensive care situations where the identification of a persistant septic focus is vital for the successful treatment of the patient and on all clinical devises etc. where contaminating endotoxins would have a deleterious effect on the clinical condition of the patient. These topics have been extensively reviewed elsewhere. 86 Blood Products Not only can blood products now be analysed for contaminating endotoxins by CS assays but other contaminants such as enzymes can be determined. For example it has been reported that infusions of either albumin or IgG preparations have led to falls in blood pressure. “vE8 Studies with CS assays revealed that Hageman factor fragments or beta FXIIa8’ in albumin and plasma kallikreinE8 in IgG preparations are hazardous contaminants which must be removed to make the preparations safe. Also enzyme activities have been found in FVIII and FIX Table 4 Clinical and other areas where chromogenic peptide substrate assays for endotoxins are being used

concentrates the side effects of which could be harmful to the patient especially because of the prolonged use of these concentrates. CS assays are now widely being used in the pharmaceutical industry to ensure that deleterious contaminants are either removed from blood products by purification procedures or destroyed by heating or solvent treatment.

Monitoring of Therapy CS assays are now widely used to monitor heparin therapyE9 and the introduction and ever increasing use of fractionated heparins which can only be assayed with CS assays” together with the availability of automated and single cuvette acute bedside assays means that CS assays will be vital for controlling heparin therapy in the future. CS assays are also being used to monitor oral anticoagulant therapy’*” but have not been widely utilised and certainly have not replaced clotting assays in routine oral anticoagulant control in the majority of laboratories. One problem has been to decide on which of the vitamin K-dependent factors should be assayed and currently FX seems to be the parameter which is recommended. One earlier problem with CS assays, i.e. cost per assay has now been overcome with the introduction of microtitre plate and automated assays, and it is to be expected that these will be used more frequently in the future. Most of the blood products used to correct inherited or acquired protein deficiences can be assayed by CS assays. These assays are not only used in the purification and production of these products but are also vital for controlling and monitoring therapy. The production of new products especially proenzymes enzymes and inhibitors will certainly be controlled by CS assays.

Determinations of endotoxins in body fluids, including: blood urine, saliva, cerebrospinal fluid, peritoneal fluid, synovial fluid, lymph fluid, amniotic fluid Analysis of drugs for parental administration Control of production of products for substitution therapy, including blood products Analysis of solutions for dialysis and haemodilution Contamination control of medical devices, surgical gloves and bandages (consumables) Control of products made by gene technology Analysis of culture media Control of water, air and air borne particles in pharmaceutical production Hygenic control of foodstuffs (meat, poultry and dairy products) Control of plastics and glassware and biochemicals used in experimental work Control of the removal of endotoxins from products

Conclusion It is almost 20 years since S-2160 was synthesised

and since then chromogenic peptide substrates have been widely used in research laboratories to increase our understanding of the various defence systems in health and disease. Subsequently CS assays have contributed greatly to the improved diagnosis and treatment of inherited and acquired protein deficiencies, to the production of improved blood products and other infusable products and to the identification of septic foci and the production of sterile consumables. CS assays are increasingly being used in intensive care medicine and the recent introduction of acute assays will ensure that these will be more frequently utilised. We can also expect that these assays will be of great value in the diagnosis and treatment of liver disease, hypertension, cancer and organ transplant rejection in the future.

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