1380 CLINICAL CHEMISTRY, Vol. 26, No. 10, 1980

JIEAnalytical Concepts

CLIN. CHEM. 26/10, 1380-1391 (1980)

New Perspectivesin CoagulationTestingJawed Fareed, Harry 1. Messmore, and Edward W. Bermes

The field of coagulation testing has undergone some majortechnological and conceptual developments, which arebriefly reviewed here. The assessment of coagulationparameters is no longer restricted to the study of clotformation and Its dissolution. The understanding of thebiochemical natu-e of coagulation processes, coupled withthe development of new therapeutic agents in the treat-ment of hemostatic disorders, has brought about the de-velopment of fast, reliable, and clearly defined laboratorytest procedures to evaluate the components of this system.The introduction of methods involving synthetic substrateshas been very significant because many of the coagulationparameters can now be measured with a spectropho-tometer or fluorometer, by methods that lend themselvesto the automation found in most large clinical chemistrylaboratories. In our laboratory, we use automated syn-thetic-substrate methods for antlttombln-lll, plasminogen,and prothrombin, and are developing the synthetic-sub-strate assay equivalent of clot-based prothrombin time andpartial thromboplastin. Immunological methods such aslaser/rate nephelometry, enzyme-linked immunoassays,electroimmunodiffusion, and radioimmunoassays havebeen utilized to evaluate coagulation proteins. The relationof functional and Immunological properties of these pro-teins to their physiological function is being studied. Incoming years the testing of coagulation function will un-dergo some major changes and will require input fromclinical chemists and other laboratory scientists to facilitatethe technology transfer and proper standardization of newmethods.

Addftlonal Keyphras.s: hemostasis . prothrombincoagulation proteins . synthetic coagulation substratesenzymlc methods - immunochemistry . plateletsprostagiandins . fibrinogen

Blood coagulation is now understood to be a well-definedmolecular process regulated by a delicate interaction of thefollowing three systems: coagulation and fibrinolytic proteins,platelets, and endothelium.

Hemostasis is the containment of circulating blood. Bloodconsists of a fluid phase (plasma) and a particulate phase(cellular elements); blood vessels are lined with the vascularendothelial cells, which form the blood-contacted surface ofthe normal vessel lumen. The three systems-coagulation andfibrinolytic proteins, blood cells, and vascular endothe-lium-exist in concert and function in hemostasis. The extentto which they participate in thrombotic and hemorrhagicprocesses depends on the severity of the damage, circulatorycomplications, and influences of other physiological systems.

A pathophysiological process is capable of altering the com-position of each of the components of the total hemostaticsystem. Because the molecular nature of each of these pro-cesses is now understood and many alterations are detectableby newer techniques, a new era in the testing of hemostaticfunction has begun (1-10). Major breakthroughs have oc-curred in the understanding of coagulation enzymology, im-munology, and drug modulation of the components of he-mostatic pathways. The understanding of hemostatic pro-cesses and alterations produced by pathophysiologic changesand drug modulation of this system is facilitated by input fromvarious disciplines. The introduction of synthetic peptidesubstrates and immunological methods represent new di-mensions in coagulation testing. We will review some of therecent tiends in this area, emphasizing the role of clinicalchemistry in this developing field.

Concept of Serine Proteases and Their InhibitorsIn the past few years coagulationists have studied intensely

the inhibitors of the coagulation, fibrinolytic, kallikrein, andother serine protease enzymes. The coagulation cascade(Figure 1) of fibrinolytic and prekallikrein/kallikrein pathwaysplays a key role in the regulation of hemostasis. Most of theenzymes participating in these processes mimic trypsin andare known as serine proteases; this group includes the fol-lowing enzymes (and their precursors): Factor XIIa (XII),Factor XIa (XI), Factor IXa (IX), Factor Xa (X), Factor Vila(VII), kallikrein (prekallikrein or Fletcher’s factor), thrombin(prothrombin or Factor II), and plasmin (plasminogen). Boththe intrinsic and extrinsic pathways of coagulation result inthe eventual activation of Factor X, which in turn convertsprothrombin into thrombin. The thrombin formed transformsthe soluble fibrinogen into an insoluble fibrin clot, which iseventually digested by plasmin into the soluble fibrin-splitproducts. It is generally believed that, in the coagulationcascade, fibrinolytic pathways are simultaneously activated;that is, once the coagulation process has been activated, theseclotting enzymes are capable of triggering the fibrinolyticpathways through a feedback mechanism. If the activities ofthese enzymes proceed without any regulatory mechanism,the net result is defibrination. The activities of coagulationand fibrinolytic enzymes are, however, modulated by nu-merous serine protease inhibitors, including C1’ inhibitor;a2-macroglobulin, a,-antitrypsin, a2-antiplasmin, and mostimportant, antithrombin-III (AT-Ill).’ Although the otherinhibitors are also involved in the regulation of hemostaticfunction, AT-Ill plays a central role in the modulation ofcoagulation processes. Some of the properties of the majorinhibitors of clotting and of the fibrinolytie enzymes in humanplasma are given in Table 1. The response to therapeuticanticoagulants, thrombolytic agents, and immunosuppressive

Departments of Pathology, Pharmacology, and Medicine, LoyolaUniversity Medical Center, Maywood, IL 60153.

Received June 9, 1980; accepted July 3, 1980.

‘Nonstandard abbreviations used: AT-Ill, antithrombin-llI; pNA,p-nitroaniline; P/SK, plasminogenlstreptokinase complex; Vifi RAg,Factor Vu-related antigen.

1. Collagen2. Ka1lIkretn-- - - -

(Plasmin)

Factor XII Factor XUa Kininogen

Prekelltkreln Kalflkretn(Fletcher Factor)

Ktntnase C3InactivatorII

IIIIIIII

Fig. 1. Present state of knowledge of coagulation cascade

Table 1. Fibrinolytic Enzymes and MajorInhibitors of Clotting in Human Plasma

Inhlbftor

a1-Antitrypsina1-Antichymotrypsinlnter-a-trypsin-inhibitorAntithrombin-lllC11-lnactivatora2-Macroglobulina2-Antiplasmin

Plasma concn amg/L Mmol/L

2900 ± 450 54.0487±65 7.0500±71 3.1290 ± 29 4.5

M,

54 00069 000

160 00065 000

104 000820 000

65 000

(ni nf (TIT I.,a..ti,..f.,.. a Th.n...hia

235 ± 302600 ± 700

116±19

2.33.3

1.5-1.8

Values from Davie, E. W., Fujikawa, K., Kurachi, K., and Kisiel, W., Adv.Enzymol. 49, 277 (1979); and Baugh, A. F., and Hougle, C., The chemistry ofblood coagulation. C/in. Hematol. 8, 3 (1979).

(Inhibited)Ari,’it/vomb.1n-flZHeporm CorrpIi Hspsrrn ThromA’, Coa’/s,

CLINICAL CHEMISTRY, Vol. 26, No. 10, 1980 1381

Factor XI Factor XIa

Factor DC Factor DC - - -,

a

Ca, Pt Factor VIII’ Factor VIII

[Factor IXa-Factor VIII’-Ca, I’L]

JlFactorX

Ca, Pt Factor V’ Factor V

[Factor XaFactOr V’-Ca

Prothrombtn Thrombin

Ftbrinogen FibrinPlasminogen I

Actjvators Ca Factor XIII Factor xmPlasmin 4

Fibrin Split Procicts 4 Fibrln (cross linked)

drugs is also dependent on the functional concentrations ofthese inhibitors. Laboratory evaluation of these proteins isan extremely important area, requiring sophisticated meth-odologies and an in-depth understanding of the physiologyof hemostasis. Newer methodologies offer a unique opportu-nity to utilize an enzyme-specific substrate for the study ofthe regulators of hemostasis without significant interferenceby the other proteins.

Antithrombin-III (Heparin CofaCtor)AT-Ill, also known as heparin cofactor and Factor Xa in-

hibitor, is a plasma protein that migrates during electropho-resis in the region of a2-globulin; its relative molecular massis 65 000. It circulates in blood in a nonactivated state, whichis the form responsible for the progressive inhibition of nu-merous serine proteases such as thrombin, Factors XIIa, IXa,Xa, and XIa. Although AT-Ill is a key inhibitor capable ofinhibiting various serine proteases at different sites of thecoagulation cascade, its main action is against thrombin and

Factor Xa. In its natural state AT-Ill is a relatively weak in-hibitor of these enzymes; in the presence of heparmn, however,AT-Ill is activated and becomes a very potent inhibitor ofFactors Xa and thrombin (Figure 2). The therapeutic efficacyof heparin depends almost totally on the presence of AT-Ill,so that individuals lacking this inhibitor fail to respond toheparin therapy. AT-Ill concentrations are decreased in thefollowing conditions: liver diseases, disseminated intravascularcoagulation, carcinoma, acute leukemia, advanced stages ofsickle-cell anemia, major post-surgical trauma, deep venousthrombosis, thrombophlebitis (hereditary deficiency of AT-III), Gram-negative septicemia, endotoxemia, and oral con-traceptive therapy.

ANTlTHROMBIN- ASSA’

(I) Throabin Anftfficombin and Hisin

L$/i!e S/I*

f1r’ J in

Sr/av S/Is IAr*i5,ombrn C/dC St

.-(Non-a(jj

Thrombin / \(/A#g/nrn. S/F.

Th,onub in(Activ.)

Fig. 2. Mechanism of antithrombin-llI activation by heparin andsubsequent inhibition of thrombin by AT-Ill/heparin complex

Table 2. Synthetic Chromogenic Peptlde Substrates and Their Applications

1382 CLINICALCHEMISTRY,Vol. 26. No. 10, 1980

SUBSTRATE

Chroun,zym-TH

S-2160

5-2238

S- 2366

Abbott “?“

CHEMICALSTRUCTURE

Tos or CBz-Gly-Pro-Arg-pNA

Bz-PIte-Val-Arg-pNA

H-D-P$ie-Pip-Arg-pMA

Pyro-Gi u-Pro-Arg-pMA

CH3-Gly-Pro-Arg-pNA

APPLICATIONS

Prothroatin, Antithroetin-III,Antithroutins, Jbnitoring ofOral Anticoagulants, HeparinPlatelet Factor III andPlatelet Factor IV

S-2222

5-2337

Bz-Ile-Glu(’r-OR)-Gly-Arg-pMA

Bz-Ile-Glu-(v-O-Piperidyl )-Gly-Arg-pNA

X, Xa, Anti-Ia and PlateletFactor IV

S-2251

Chrozy.-PL

H-D-Val-Leu-Lys-pNA

Tos-Gly-Pro-Lys-pNA

Plasuiin, Plasainogen, Anti-plasmin, and PlasminogenActivators

S-2322 H-D-Val-Gly-Arg-pliA Tissue Activator of Plasmino-gen

5-2302

Chrozyiu-PK

H-D-Pro-PIte-Arg-pliA

Bz-Pro-P$ie-Arg-p$A

Plasma Kallikrein, Anti-Kallitrein and KallikreinActivators

5-2266 H-D-Val-Leu-Arg-pMA Glandular Kallikrein, Anti-Kalllkrein and KallikreinActivators

Chrouzym-UK

S-2444

Bz-Val -Gly-Arg-pNA

Pyro-Gl u-Gly-Arg-pMAUroki nase

Chrozym-Try H-D-Val -Gly-Arg-pNA

Cbo-Val -Gly-Arg-pNATrypsin and Antitrypsin

5-2422

S-2433

Bz-Ile-Glu-Gly-Arg-pNA

Acet- 11e-Gly-Gly-Arg-pNA

Endotoxin, Liijlus Pro-coagulant enzyma

The substrates of the Chrozym series are manufactured by Pentaphar. (Basel, Switzerland)and the substrates of the 5C series are manufactured by Kabi A B (Stockholm. Sweden).

Synthetic Peptide Substrate Methods forCoagulation Testing

Since the introduction of enzyme-specific synthetic sub-strates for thrombin, the whole field of coagulation enzy-mology has undergone a dramatic change. Although it has onlybeen a few years since the first chromogenic tripeptide, Bz-Phe-Val-Arg-pNA (S-2160), was introduced by Svendsen etal. (11) as a specific substrate for thrombin, more than 30synthetic chromogenic and fluorogenic peptides have beenintroduced for the specific evaluation of thrombin, Factor Xa,plasmin, plasma and glandular kallikrein, plasminogen acti-vator, urokinase, trypsin, limulus-coagulant enzyme, andmany other serine protease enzymes (11-53). Methodologiesfor the active enzymes, their zymogens, and their inhibitorsare continually being developed. In addition, coagulation andrelated pathways have been investigated with these substratesand with specific reagents. Because the peptide substrate-based methods follow the principles of enzymology, a wholenew concept of coagulation analysis has evolved.

Some of the commercially available synthetic chromogenicpeptide substrates are listed in Table 2. All of these substratesare coupled with p -nitroaniline (pNA) as a chromophore atthe terminal carboxyl group and contain arginine or lysine.Table 3 lists commercially available fluorogenic substratescurrently available for the determination of serine proteaseenzymes and their inhibitors. The action of serine proteaseson these fluorogenic substrates results in the release of various

fluorophores, which can be measured with a suitable instru-ment.

Currently, reliable methods for the determination of AT-Ill,plasminogen, a2-antiplasmin, and absolute concentrationsof heparin are available. Recently, chromogenic assays forFactors X and II have also become available (46-50). Ami-dolytic assays equivalent to prothrombin time and partialthroinboplastin time are being developed (51-53). Assaymethods based on synthetic substrates are expected to con-tribute significantly to the laboratory testing of coagulationfunction.

Development of Synthetic SubstratesFibrinopeptide A is a known inhibitor of the action of

thrombin on fibrinogen. Svendsen et al. (11) and Blomback(12) have reported on the amino acid sequence of fibrino-peptide A in various animal species. They found that certainamino acids in the carboxyl terminal of fibrinopeptide A wereidentical in all species, which suggested that the affinity tothrombin may be determined by these amino acids. At thepositions 9, 2, and 1 of fibrinopeptide A are phenylalanine,valine, and arginine, respectively. This peptide fragment wasshown to possess an anticoagulant activity and an affinity forthrombin.

Moat synthetic substrates are developed after the peptidesequence of the site of action on the natural substrate has beenestablished. The tripeptide or tetrapeptide sequence is iso-lated and chemically synthesized, and a chromophore or

+ residual thrombin

9

w(-.3z

0U,

Fig. 4. AbsorptIon spectra of S-2160 and pNA in water

WAVELENGTH(nm)

CLINICAL CHEMISTRY, Vol. 26, No. 10, 1980 1383

Table 3. Synthetic Fluorogenic Substrates and Their Applications

#{163}112Y)(

Thrcin

CHEMICALSTRUCTURE

Boc_Val_Pro_Arg_NCA*Cbz, Gly-Pro-Arg-BNAH-D-Phe-Pro-Arg-AIE

APPLICAT1011$

Prothroin, Ant1throin-I1I,Ibnitoring of Oral Anticoagu-lants, Ileparin, Platelet FactorIII and IV

Factor Ia Boc_Ile_Glu_Gly_Arg_MCA** I, Ia, Anti-Ia, Platelet Facto,IV, Heparin and Endotoxin

Urokinase Glut-Gly-Arg-MCA Urokinase, Urokinas Activatorsand Inhibitors

Plasal n H-D-ValBoc-Gly-Leu-Lys-MCA’Boc-Val -Leu-Lys-MCA”

Plasminogen, Antiplasmin,Plasmin, and PlasminogenActivators

Urinary Kallikreln Pro.PheArg_NCAS Kallikrein and Antikallikrein

Pancreatic Kallikrein Pro.Phe.Arg_MCA** Glandular Kallikrein andPrekallikrein

Plasma Kallikrein Z-Phe-Arg-MCA Plasma Kallikrein andPrekal I lkreln

Litlus Clotting Enzyme Boc-Leu-Gly-Arg-MCA Endotoxin, Bacterial Activators

iXa, XIIa Boc-Phe-Ser-Arg-NCA’Boc-Leu-Thr-Arg-MCA

Contact Factors

5Substrate available in the Dade Protopath Kits.Substrate available from Peptide Institute, Protein Research Foundation, Osaka, Japan.

fluorophore is attached at the carboxyl terminal. Because thisattachment is via an amide bond (CONH), the enzyme cancleave this bond and release a chromophore or fluorophorewhose optical properties are significantly different from thoseof the native substrate.

The chemical structure of the first peptide substrate, S.2160, is shown in Figure 3. The amino acid sequence of thissubstrate is identical to the peptide sequence in fibrinogen atwhich the thrombin acts during the clotting process. The pNAgroup attached to the carboxyl end is released during the en-zymic process, the release being proportional to the activityof the enzyme. Most of the other peptide substrates are de-signed on the same concept (17). The representative absorp-tion spectra of equal amounts of S-2160 and pNA are shownin Figure 4. Obviously, these two compounds have quite dis-tinct spectral characteristics. Note that when pNA is boundto the synthetic tripeptide Bz-Phe-Val-Arg, it does not absorbsignificantly in the 400-410 nm range, whereas in the free formit absorbs strongly there. This is the basic principle of theapplication of chromogenic substrates, where both kinetic andendpoint methods are in use.

Application of Synthetic Peptides in ClinicallyImportant Coagulation Parameters

A list of various synthetic peptide substrate methods for thecoagulation parameters is given in Table 4. At present, the

CH -NH-C-NH22

CU3 CU3 CU2 NH

0 CH2O CU 0 H2OI II, II I ii

.NH -CU -C NH -CU -C NH -CU -C .jNH - NO2

I I I

Ri. Phe Val Arg p - NA

Fig. 3. Structure of Bz-Phe-Val-Arg-pNA (S-2160), the firstsynthetic peptide chromogenic substrate

following commerically available methods have been devel-oped (11-43): AT-Ill, plasminogen, heparin, and a2-antip-lasmin.

Monitoring AT-Ill: AT-Ill activity is measured with spe-cific substrates, S-2238 and chromozym-TH. A known amountof thrombin is incubated with a sample of diluted plasma inbuffer containing heparin. The residual amidolytic activityof thrombin is measured and compared with standard AT-Illor a normal human plasma preparation.

Heparin + AT-Ill - activated AT-Ill

Thrombin + activated AT-Ill

thrombin/activated AT-Ill complex

Table 4. Status of Synthetic SubstrateMethodologies for the Evaluation of Coagulation

FunctionAnalyts

AT-Ill

Plasminogen

aAntiplasmin

Heparin

Platelet factor 4

Platelet factor 3

Prothrombin (Factor II)

Prekallikrein

Factor Xa

Factor X

tivity. An exactly identical principle is applicable to the assaysinvolving fluorogenic substrates. Bovine Factor Xa andthrombin can be used instead of human a-thrombin.

Plasminogen assay: Plasminogen has always been a very

difficult assay for coagulation laboratories, because of potentantiplasmin substances in human plasma. With the intro-duction of S-2251 and chromozym-PL, it has become easierto perform amidolytic assays on samples having true plasminactivity and devoid of antiplasmin substances (which is rarelythe case). Methods for plasminogen determination have notbeen studied as extensively as those for AT-Ill. However, itis possible to measure plasminogen as a plasminogen/strep-tokinase complex (P/SK), whose amidolytic activity is directlyproportional to the plasminogen content of the sample:

Plasminogen (P) + streptokinase (SK) -#{247} P/SK

H-D-Val-Leu-Lys-pNA (S-2251)

PISK-k H-D-Val-Leu-Lys-COOH + pNA

Tos-Gly-Pro-Lys-pNA (chromozym-PL)

P/SK- Tos-Gly-Pro-Lys-COOH + pNA

All plasminogen assays are based on two reactions. First,the plasminogen in plasma reacts with a known amount ofstreptokinase to form a complex (P/SK). Second, the ami-dolytic activity of the complex is determined by using S-2251or chromozym-PL as a substrate; the i.A4o5/min is propor-tional to the amount of plasminogen. In the fluorogenic sub-strates the fluorophore released after the action of P/SK ac-tion is measured to quantitate the plasminogen.

Heparin methods: An assay based on the heparin-acceler-ated inhibition of the activated Factor X was available longbefore the emergence of synthetic chromogenic substrates.In standard procedures for the synthetic-substrate methods,heparmn is analyzed as a heparmn/AT-Ill complex. The pres-ence of AT-Ill in the test plasma is cruicial; either normalplasma or purified AT-Ill can be used to assure proper corn-plexation. These assays can be adapted for both endpoint andkinetic methods. Recently, a kit based on the chromogenicmethodologies has been introduced by Kabi AB and OrthoDiagnostics: it contains the substrate, Factor Xa, AT-Ill,buffer, and normal human plasma in lyophilized form. Theprocedure is as follows:

Heparin + AT-Ill - heparmn/AT-Ill

Heparin/AT-Ill + factor X

- heparin/AT-III/Xa + residual XaResidual Xa + R-pNA

- R-COOH + pNA (S-2222 or S-2337)

Because the purified Xa preparations are difficult to obtain,Larsen et al. developed a heparin assay involving S-2238 andthrombin (39):

Heparin + AT-Ill - heparmn/AT-Ill

Heparmn/AT-Ill + thrombin - heparmn/AT-Ill/thrombin

1384 CLINICAL CHEMISTRY, Vol. 26, No. 10, 1980

Partial thromboplastin time(amidolytic equivalent)

Prothrombin time (amidolyticequivalent)

Factor VIIIFactor VIIFactor XIITissue activator of

plasminogenTotal serine protease activity

Status

Satisfactory, all reagents usedare readily available. Manycommercially available kits.Standardization needed.Diagnostic valueestablished.

Satisfactory. Reagentsareavailable. Standardization?

Reagents are not availablereadily. Availability ofplasmin is limited. One kitavailable.

Only measures absoluteconcentrations of heparin.Of questionable benefit forthe monitoring oftherapeutic heparinization.

Methodologies arestandardized. Measuresantiheparin activity and onlyreliable in samples notcontaining heparin.

Complex method; ultra-pure

non-activated prothromblncomplex is needed.

Still in developmental stages.A reliable assay is possible.

Satisfactory. All reagentsavailable. Diagnosticsignificance is to beestablished.

Satisfactory. Helpful in thediagnosis ofhypercoagulable state.

Reliable method available.Useful In the managementof oral anticoagulants.

Numerous methods available,clinical efficacy not proven.

Numerous methods available.

Under development.Under development.Under development.Under development.

Under development.

+ residual thrombinResidual thrombin + H-D-Phe-Pip-Arg-pNA (S-2238)

-* H-D-Bz-Phe-Pip-ArgCOOH + pNA

Residual thrombinor

+ Tos-Gly-Pro-Arg-pNA(chromozym-TH)

- Tos-Gly-Pro-ArgCOOH + pNA

In the presence of heparin, suppression of the amidolyticactivity of thrombin is inversely proportional to AT-Ill ac-

Residual thrombin + R-pNA -*R.COOH+ pNA (S-2238 or chromozym-TH)

Fluorogenic substrates for the assay of thrombin and factorXa can also be used for the quantitation of absolute amountsof heparin.

Antiplasmin methods: a2-Antiplasmin is now known to bethe primary antiplasmin in blood because of its rapid andrelatively specific inhibition of plasmin activity. Numerous

CLINICAL CHEMISTRY, Vol. 26, No. 10, 1980 1385

studies have shown that the amount of a2-antiplasrnin fluc-tuates in certain disorders (54,55). Various chromogenic andfluorogenic methods have been proposed for the quantitationof this inhibitor. A known amount of plasmin is incubated withthe test material for 30 to 60 s. The amount of residual plasminis quantitated with plasmin-specific substrates, such as S-2251or chromozym-PL:

Plasmin + plasma (a2-antiplasmin)

-. Plasmin/a2-antiplasmin + residual plasmin

Residual plasmin + R-pNA (S-2251 or chromozym-PL)-. R-COOH + pNA

Automation of Synthetic-Substrate MethodsBecause synthetic-substrate methods are based on the re-

lease or the suppression of release of a chromophore or fluo-rophore, most of the enzyme analyzers used in clinicalchemistry laboratories can be used for these analyses. Scullyand Kakkar have used an LKB analyzer (LKB Instruments,Rockville, MD 20852) to measure thrombin, AT-Ill, and an-tithrombin, with S-2238 as substrate (21). Using the LKBanalyzer, Bergstrom and coworkers automated a method fordetermining plasma prothrombin (56, 57). The applicationof various centrifugal analyzers for the determination ofAT-Ill have been reported (58, 59). Recently, automateddetermination of antiplasmin and AT-Ill with the ABA-100system (Abbott Diagnostics, N. Chicago, IL 60064) has beendescribed (60). Yamada and Meguro (61) have recently de-scribed a novel method of activated partial thromboplastintime, using the chromogenic substrate for thrombin and anAutoAnalyzer (Technicon Instruments, Tarrytown, NY10591). A fully automated chromogenic-substrate method formeasuring plasma Factor Xa to monitor patients receivingcoumarin therapy has also been reported (62).

Standardization of Synthetic-SubstrateMethodologies

Because most of the synthetic substrates are used in acti-vator, zymogen, enzyme, and inhibitor assays and follow theprinciples of enzymology, eventually their use will have tofollow the recommendations of the International Commissionof Enzymology.

Standardization of reagents available for the synthetic-substrate methodologies is a growing concern. Although thewhole field of synthetic-substrate methodologies has grownvery rapidly, the availability of purified reagents is stills se-rious limitation. Commercially available reagents such asthrombin, Factor Xa, plasmin, AT-Ill, and kallikrein varygreatly from source to source and from batch to batch. Aclassic example is the available human thrombin preparation:despite the potency values assigned to a known thrombin lot,wide variations have been detected. Moreover, most of thecoagulation enzymes are standardized in terms of their relativecoagulant or lytic properties, which are not the same as theiramidolytic activity. Availability of a complete kit for AT-Ill,antiplasmin, prekallikrein, heparin, and their coagulationparameters undoubtedly puts the responsibility on themanufacturer’s shoulders, and we have seen good qualitycontrol measures taken by these manufacturers.

Future Trends in Measurement of AT-Ill andHeparin

The introduction of synthetic-substrate assays will play akey role in the development of diagnostic methodologies totest hemostatic function. AT-Ill determination has alreadybecome a routine diagnostic test in major medical centersthroughout Europe and the United States. The amounts ofheparmn/AT-Ill complex may possibly be used in the study ofkinetics of heparin and the evaluation of heparin products by

clinical pharmacologists. One area where the application ofsynthetic-substrate methodologies for heparmn may have avalue is the study of the release of endogenous heparin orheparin-like substances in certain pathophysiologic condi-tions. The clinical significance of endogenous heparin has yetto be established. The ratio of the functional AT-Ill andheparmn concentrations may also have some diagnostic value.Although methods based on the clotting principle for themeasurement of heparmn are available, these tests are timeconsuming, expensive, and difficult to perform. In addition,reproducibility is often a problem, and the biologic reagents,once reconstituted, cannot be used repeatedly.

Mass screening of AT-Ill is possible where automatedmethodologies are available; at the present, automated andsemiautomated methods are used with the LKB analyzer, theAbbott Bichromatic analyzer, the Beckman Model 35 kineticanalyzer (Beckman Instruments, Fullerton, CA 92634), theGilford Model 3500 analyzer (Gilford Instrument Labs., Ob-erlin, OH 44074), and Technicon analyzers. The applicationof centrifugal analysis is quite attractive, as is the use of adiscrete analyzer for the determination of antithrombin andmost of the other coagulation parameters.

Testing of hemostatic function is at present considered tobe a highly specialized test battery, performed only in thecoagulation laboratories. With synthetic substrates the de-termination of AT-Ill may also be within the scope of theroutine clinical chemistry laboratory. In the near future

coagulation panels based on clinical chemistry principles willbe available. The role of industry in the development of suchpanels is a crucial one. Preliminary studies in many labora-tories indicate that AT-Ill, antiplasmins, plasminogen,anti-Xa, antikallikrein, and progressive antithrombin activitycan be run as one panel.

Methods based on synthetic peptide substrates are cur-rently more expensive than the existing methods, with re-agents and sub8trates being the main contributors to the cost.Improving the methods for synthesizing these substrates mayalso lower costs. There is no serious problem with the speci-ficity with the existing substrates; however, it may be profit-able to provide more sensitive substrates. The developmentof agents such as the tetrazolium in dyes is desirable. Couplingsubstrates with a fluorophore may also help reduce costs andincrease the sensitivity of the assay system.

Education of laboratory personnel is important. At this timethe biochemical concepts involved in the coagulation processesare generally poorly understood by laboratory personnel.Extensive educational material and notes on applicationshould be provided for review of the biochemical concepts andtheir pertinence to the pathology of hemostatic defects.

The role of various government agencies in the developmentof chromogenic substrates can be summarized as follows:

1. Promotion of basic research in the developmentalareas.

2. Standardization of methodologies by the National Bu-reau of Standards and the Bureau of Biologic Standards.

3. Coordination of clinical trials in conjunction with pro-fessional organizations such as the American Society ofClinical Pathologists and the American Association for Clin-ical Chemistry.

4. Implementation of well-defined regulations regardinguse of these new concepts.

Because they involve a new concept, the synthetic-substratemethodologies need a great deal of work to be accepted in theroutine laboratory. An important consideration is the avail-ability of reliable reagents, especially the Factor Xa andthrombin preparations. These enzymes must be consistent,and a known standard should always be available for com-parison.

Previously stated problems with the chromogenic-substrate

Method

Kabi

Table 5. AT-Ill Determination by Commercially Available Methods and by a Method Developed at OurLaboratory

Normal

Pacific Hemostasis

Table 6. Comparison of AT-Ill, Plasminogen, andHeparin, As Determined with Chromogenic and

Fluorogenic SubstratesConan. _______

AT-Ill (n = 100)Plasminogen (n = 100)Heparin (n = 50)

Chromogunlc Fluorogenicmethods methods

78.1 ± 11.881.9 ± 12.60.48 ± 0.08

82.0 ± 13.684.6 ± 12.90.51 ± 0.01

‘Protopath” System was used for all three analyses. Kinetic

methods developed at Loyola were used for the AT-Ill and plasminogen deter-minatIons, whereas a commercially available kit (Ortho Diagnostic) was usedto quantitate heparin.

1386 CLINICALCHEMISTRY,Vol. 26. No. 10, 1980

StagoBoehringer-MannheimAbbott

Loyola UniversityRadial Immunodiffusion

(Calbiochem)

Hum. n plasma AT-ill concn.Coagulopathy Plasma St andard AT-Ill

107±16 67±31 110±11 108±9

113±16 17±26 113±15 103±6

118± 13 79±24 116±10 103± 13

107±21 64±30 103±15 104±16

94± 13 58± 11 101±7 103± 12

109±31 89± 11 104±9 105± 10

Kabi, Stockholm. Sweden; Stago, Asnieres, France; Boetwinger-Mantheim. Garmlsch. F.R.G.; Abbott, North Chicago, IL 60064; PacifIc Hemostasis, Bakersfield,CA 93301; Calblochem, San Diego, CA 92112.

methodologies such as nonspecificity, formation of fibrin inthe reactionmixture,possible inhibition of thrombin bysubstrateorproducts,appearanceofturbidity, instability ofthrombin,substrateinstability,and precipitationofthepNAhave alreadybeen worked out,and thepresentassaysdo notsufferfrom these problems. Any diagnostic laboratory testthatcorrelateswellwiththe clinical status of the patient andthatwillaccuratelyand reproduciblydetectbiologiceventsshould be available to the physician. The synthetic substrateslend themselves extremely well to the assay of the enzymesof coagulation and related systems, and are equally useful intheassayoftheinhibitorsinthesesystems.

The demonstration of the clinical usefulness of these testsis the first step in their adoption as routine laboratory meth-ods. More clinical studies comparing the standard methodswith these newer methods are needed, but we feel that all in-dications at present are that they are well suited for routineuse.

Commercially Available KitsWe have examined commercially available AT-Ill methods

and have found them reliable. The assay methods for pre-kallikrein and antiplasmin need further clinical work to beutilized in the diagnosis of a given coagulopathy. A comparisonof results obtained with various commercially available kitsfor AT-Ill is given in Table 5; obviously, most of these meth-ods give comparable results for AT-Ill. The results for theradialimmunodiffusionmethods ina normal populationandwith a standard preparation of AT-Ill are comparable. Inabnormal plasma samples higher results were obtained withradial immunodiffusion; however, both the clotting and thechromogenic methods gave comparable results (unpublisheddata).

Fluorogenic Substrate MethodsRecently, Dade Division (American Hospital Supply Corp.,

Miami, FL 33152) introduced a complete system, includinga dedicated instrument (“Protopath”), for the fluorometricquantitation of AT-Ill, plasminogen, and heparmn in plasma(63-65). The principles of the fluorometric method areidentical to the ones used in the chromogenic substratemethod except that instead of pNA a fluorogenic material,5-amidoisophthalic acid dimethyl ester, is released andkinetically measured with the Protopath fluorometer.

In addition, the heparmn methodologies in this system makeuse of thrombin whenever Factor Xa was used in the chro-mogenic methods. The assay methods for the quantitation ofAT-Ill and plasminogen determination are exactly the sameas those applied in the chromogenic analysis, except that thefluorophore released is designed to be measured only with theProtopath enzyme analyzer. These kits cannot be conve-

niently used with other spectrophotofluorometers, whichlimits the user to only one instrument. Table 6 compares theconcentrations of AT-Ill, plasminogen, and heparin as de-termined by the chromogenic and fluorogenic methods; bothmethods gave comparable results.

Although we believe that AT-Ill and plasminogen analysisbased on the synthetic peptide methods provide reliable re-sults in our hands, we still feel that the therapeutic antico-agulant action of heparmn is best monitored with the activatedpartial thromboplastin time and other coagulant assays. Thedetermination of absolute amount of heparin in a patient’splasma in meaningless, in that many endogenous factors suchas AT-Ill, platelet factor 4 (antiheparin factor), and some oftheir proteins affect the anticoagulant action of heparmn. Thesestudies are limited, and we need to gather more data.

Immunological Method in Coagulation TestingThe specificity and sensitivity of the antigen/antibody re-

action has provided a unique tool to determine minutequantities of antigenic proteins in serum and other biologicfluids. Immunologic methods have been widely utilized for theabsolute quantitation of coagulation proteins (2-9). Newermethods such as radioimmunoassays, laser and rate nephe-lometry, enzyme-linked and other fluoroimmunoassays havebeen introduced and are being used in most clinical and re-search laboratories. For a general account of immunologicalprinciples and techniques, see the reviews by Clausen (66) andby Rose et al. (67).

Radial ImmunodiffusionSince the introduction of radial immunodiffusion methods

(68-70), many commercially prepared radial immimodiffusionplates for coagulation proteins have become available. For acritical review on this methodology see Ritzman (71). Al-

CLINICAL CHEMISTRY, Vol. 26, No. 10, 1980 1387

though these procedures are reliable, their turnaround timeoften exceeds 48 h, making them somewhat undesirable in aclinical situation where results are required immediately.

Rocket lmmunoelectrophoresisRocket immunoelectrophoresis is extensively used in im-

munology and clinical chemistry (72, 73). The technology hasbeen used extensively in the study of Factor-VIlI-relatedantigen, AT-Ill, and a2-antiplasmin.

The rocket method offers several advantages over con-ventional immunodiffusion methods. Quantitation is achievedin only a few hours instead of days, and antigenically relatedcoagulation proteins can be identified and quantitated. Re-cently, an automated electro-immuno quantitation system(“EIQ”; ICL Scientific, Fountain Valley, CA) has becomeavailable for quantitating a1-antitrypsin and other serum andplasma proteins.

RadioimmunoassayEver since radioimmunoassay was introduced by Yalow and

Berson (74) to measure endogenous human plasma insulin,efforts have been made to apply this technique in practicallyall areas of laboratory medicine. An extensive review on thistechnology as it pertains to clinical chemistry is available (75).Fibrinopeptide A, platelet-release-specific proteins, AT-Ill,and many of the other components of coagulation system havebeen measured by radioimmunoassay (1).

NephelometrySeveral laboratories are now utilizing commercially avail-

able laser and rate nephelometers to quantitate coagulationproteins. In 1977 Sternberg (76) introduced a rate nephe-lometer to monitor the rate of light-scatter change. Thisparticular feature eliminated the necessity of blank correc-tions, which is necessary in equilibrium nephelometry (i.e.,light scatter is measured at the equilibrium of antigen/anti-body reactions). At present, laser and rate nephelometers aresatisfactorilyutilizedforestimatingthequantitiesofseveralproteins. Coagulation proteins that can be quantitated bynephelometric methods include: fibrinogen, AT-Ill, plas-minogen, a2-macroglobulin, a2-antiplasmin, fibrinopeptides,fibrin-split products, and prothrombin. The methods are re-portedly reproducible and highly sensitive, and correlate wellwith radial immunodiffusion methods (77).

FluoroimmunoassayA solid-phase fluoroimmunoassay has been developed re-

cently to quantitate Factor VIlI-related antigen (VIII R:Ag)(78). In this assay a fluorescein-labeled anti-Vill R:Ag is al-lowed to react with the test plasma, and the unbound antibodyis then removed by using a specially designed sampler (“Stiq”)precoated with a purified VIII R:Ag. The fluorescence on thepolymeric surface of the sampler is measured with a specialfluorometer system (FlAX; International Diagnostics Tech-nology, Santa Clara, CA 95050). The precision of the assayreportedly compares favorablywithelectro-immunoassay. Wehave adopted this system in our laboratory to quantitate VIIIR:Ag, and the results so far indicate a correlation coefficient(r) of 0.96 between the FlAX and the electro-immuno detec-tion methods. Almost all of the coagulation protein/serineprotease-inhibitor complex can be detected with the FlAXand electro-immuno diffusion methods.

Enzyme-Linked ImmunoassayVarious competitive and noncompetitive enzyme-linked

immunoassays have been developed to measure severalcoagulation proteins (79, 80). The principle of the assay issimilar to fluoroimmunoassay, except that an enzyme-con-jugate is utilized instead of the fluorescein-labeled antibody.

Substrate hydrolysis by the enzyme-conjugate is inverselyproportional to the amount of test protein in plasma. Ness andPerkins (81) have quantitated VIII R:Ag by this method andclaim to have increased sensitivity over the Laurell method.However, enzyme immunoassays do not seem to offer serious

advantages over the Laurell method or fluoroimmunoassays.The technique requires additional steps of: (a) purifying VIIIR:Ag on a polyacrylamide agarose column, (b) isolatinggoatanti-rabbit IgG by immunoadsorption, (c) conjugating theenzyme to the antibody, (d) preparing substrate solution andbuffers, and (e) performing several incubation steps. Asidefrom the increased sensitivity with this method, no particularadvantage is seen in the diagnosis of Factor Vill-related dis-orders.

Immunologic Studies of Serine Protease InhibitorComplexes

The role of various serine protease inhibitors in regulatingthe function of coagulation and fibrinolytic enzymes such asthrombin, Factor Xa, and plasmin is quite clear. The existenceof an AT-Ill/serine protease complex, which has been reportedpreviously (9, 10,88), indicates that the process of activationhas taken place and that the active enzyme has formed acomplex with a given inhibitor. The detection of an enzyme/inhibitor complex will provide a useful mean to evaluate theactivationofa givensystem.

Clinical ConsiderationsDuring the last two decades, rapid advances have been

made in immunological concepts and immunologic methods.Clinical investigators must incorporate these newer conceptsand methods to interpret results of laboratory tests performedto aid in diagnosis, therapeutic monitoring, and prognosis ofa hemostatic and thrombotic disorder; therefore, specificityand sensitivity of a given method are important consider-ations. Although immunodiagnosis depends to a large extenton antibody specificity, there are no widely available inter-national standards for most coagulation proteins. Commer-cially available antisera to most coagulation proteins and re-lated proteins show inappropriate specificities when assayedwith a panel of antigens (82). To overcome this problem,several recommendations on the characterization and clinicaluseof immunologic reagents have been made (83). At present,each immunologic method should be critically evaluated forthe significance of its results and its suitability in a coagulationlaboratory. Immunologic methods provide important infor-mation on the qualitative nature of a coagulation protein. Inrocket immunoelectrophoresis the genetically variant andcomplex forms of coagulation protein are distinguishable.Immunologic methods, however, quantitate coagulationprotein in terms of absolute amounts, which does not trulyreflect the function of a given coagulation protein. It is,therefore, important to consider the limitations of thesemethods before making any firm diagnosis.

Study of Fibrinogen and Its DerivativesMolecular elucidation of fibrinogen has played a key role

in the understanding of coagulation process and has provideduseful diagnositc information in certain coagulation disorders.The study of fibrinogen can be divided into the followingcategories: the study of the native fibrinogen and its variants,cleaved fibrinopeptide A and B, proteolytic degradationproducts of fibrinogen and fibrin, soluble fibrin monomer/polymer complexes, and fibrinolytic enzymes and inhibi-tors.

Fibrinopeptide A and B can be analyzed by thin-layerchromatography and thin-layer high-voltage electrophoresis.Reliable radioimmunoassays are also available for the quan-titation of fibrinopeptide A (84). The quantities of fibrino-

1388 CLINICALCHEMISTRY, Vol. 26, No. 10, 1980

peptide A are increased in hypercoagulable states and relateddisorders (85). Gel electrophoresis has been extensively usedto study fibrinogen and its derivatives (86,87). From a diag-nostic standpoint increased amounts of fibrinopeptide A inplasma are indicative of the production of fibrin. Nossel et al.have developed a radioimmunoassay for fibrinopeptide A thatis applicable in clinical situations (88). A commercial kit(IMCO Corp., Stockholm, Sweden) is also available. As dis-cussed previously, the serine protease inhibitor complex canbe quantitated by radioimmunoassay methods. Pre-existingplasmin/antiplasmin complexes that circulate in blood forsome time after the induction of pathologic or therapeuticfibrinolysis may reflect the degree of activation (10). Collenand de Cock (10) have used the appearance of a neoantigenin such complexes to produce an agglutination test readilyapplicable in clinical situations. They reported a high titre ofsuch complexes in plasma samples from patients with suchfibrinolytic conditions as disseminated intravascular coagu-lation.

Platelet Functions and ActivitiesOur understanding of the platelet physiology and bio-

chemistry has recently been enhanced by the availability ofnew methodologies; only some aspects, related to clinicalchemistry, are considered here. Several recent reviews covernewer tests for evaluating platelet function (89-97), for which,from a diagnostic standpoint, the following areas are of in-terest: platelet-release-specific proteins, prostaglandins andtheir derivatives, and biochemical aspects of platelet aggre-gation and adhesion.

Most existing platelet-function tests such as bleeding time,platelet retention, aggregation, release reaction, platelet factor3 availability, circulating aggregates, and survival studiessuffer from certain limitations. The problem arises from at-tempting to evaluate cellular function in an in vitro situation,where an essential constituent (vascular endothelium) is ab-sent and where coagulation is controlled by use of exogenouslyadded anticoagulants. Except for imaging and other sophis-ticated tests, a reliable test to evaluated the endothelialfunction is not available. However, the study of prostaglandinderivatives in circulating plasma indirectly gives an indicationof the role endothelium plays in the regulation of prosta-glandins and theirderivatives,thereby modulating platelet.function (98-100). Obviously, in vivo tests are highly desir-able, but except for bleeding time tests, which can be in-fluenced by factors other than platelets, no such easily per-formed test is available.

Release of Platelet-Specific Proteins In ViVOThe biochemical and ultrastructural considerations of

platelet-release reactions have been elegantly reviewed byMaclntyre (101) and White (102). Isolation and character-ization of platelet-specific proteins have made it possible tomonitor the release reaction in vivo by radioimmunoassaymethods. ADP and serotonin (5-hydroxytryptamine) can bemeasured during a release reaction, but because of their rel-atively short half-lives and insensitive methods for their de-termination, the quantities of these two release substancesprovide only limited information.

The Scottish group isolated two platelet-specific antigens,platelet factor 4 and /3-thromboglobulin, and radioimmuno-assays for both have been developed (103-106). Preliminaryapplications of these tests to clinical situations indicated thatthe tests were a reliable measurement of the platelet-releasereaction in vivo. Ellis et al. have reported that platelet factor4 is increased in patients with coronary diseases (107). Al-though numerous publications on these two proteins haveappeared, our current knowledge of the basic physiology ofplatelet-release reactions in normal and disease states is still

incomplete; hence, the diagnostic efficacy of these two pro-teins needs additional clinical support data.

Localization of various substances in platelet granules hasbeen investigatedindirectly by their susceptibility to differentconcentrationsofaggregatingagents. This technique indicatesthat there are apparently three types of granules (101): a-,orlight, granules with platelet-specific proteins; /3-, or dense,granules with amines and nucleotides; and other a-granuleswith lysosomal hydrolases. Another interesting release proteinis the cell-growth-stimulating (mitogenic) factor, which hasbeen implicated in the proliferation of smooth muscle cellswithin the vascular intima; it is located in the same a-granulesas /3-thromboglobulin and platelet factor 4.The diagnosticroleof this protein is yet to be evaluated.

Prostaglandins and PlateletsMany of the substances formed by platelets during the re-

lease reaction arise through the liberation of arachidonic acidfrom platelet or arterial membrane phospholipids, eitherthrough the action of phospholipase A2 (108, 109) or throughthe sequential actions of phospholipase C and diglyceride li-pase (110). When arachidonic acid is acted upon by cycloox-ygenase, the short-lived aggregating agents PGG2, PGH2, andthromboxane A2 are formed (111-115). Although all investi-gators agree that thromboxane A2 is a potent aggregatingagent, there is some controversy concerning whether PGG2and PGH2, which give rise to thromboxane A2, are themselvesaggregating agents (98, 116). Thromboxane A2 also causesvasoconstriction (117). The stable end-product, thromboxaneB2, is chemotactic (able to stimulate mobility) for leukocytes.A small proportion of the PGH2 may give rise to PGE2 andPGD2 instead of thromboxane A2 (118). PGE2 affects vesselpermeability and causes vessel contraction (119, 120). In some

animal species and in humans PGD2 inhibits platelet aggre-gation (118). Arachidonate molecules that are acted upon byplatelet lipooxygenase are converted to several products, in-cluding L-12-hydroxy-5,8,10,14-eicosatetraenoic acid, whichis also chemotactic (121). Other substances that appear in themedium when platelets interact with release-inducing agentsare collagenase elastase and a factor that interacts with thefifth component of complement (C5) to form a chemotacticsubstance (122-124).

As far as is known, all the agents that cause the release ofplatelet granule contents also activate phospholipase A2. Thelink between the release reaction and activation of this enzymehas not been established, although both probably involve themovement of internal platelet calcium (98, 116, 117).

Malondialdehyde, a metabolite of labile endoperoxidePGG2, is formed during platelet aggregation (125). Becauseit can easily be measured by incubation with thiobarbituricacidto form a pink pigment, itisa usefulindicatorofplateletprostaglandin synthesis. Stuart et al. first estimated plateletsurvival by measuring the rate of return of such synthesis afterits inactivation by aspirin (126).

Serial determinations of malondialdehyde are performedon platelet samples aggregated with thrombin, epinephrine,or N-ethylmaleimide after the ingestion of one 600-mg doseof aspirin. Platelet lipid peroxidation (nanomoles of malon-dialdehyde per iO platelets) is markedly inhibited, with alinear return to normal production as the acytylated plateletsdisappear. Half-life values in both normal persons andthrombocytopenic patients agree closely with those obtainedby using 51Cr, and the technique is considerably less cum-bersome than injectionof radioisotope-labeledautologousplatelets. Uncontrolled aspirin-taking must be excluded for10 days before and during the study period. Roncucci et al.have automated the method, enabling multiple replicate de-terminations of malondialdehyde to be made when necessary(127).

CLINICALCHEMISTRY,Vol. 26, No. 10, 1980 1389

A recent report has compared the plasma concentration ofthromboxane B2 in Prinzmetal’s angina (variant angina) andclinical angina pectoris (128, 129). These investigators con-cluded that spontaneous thromboxane generations in variantangina is associated with coronary artery spasm and may playa key roleinangina and myocardial infarction. ThromboxaneB2 isa stablemetabolite ofthromboxane A2, and has a muchlongerhalf-life.A reliableradioimmunoassay forthromboxanehas been previouslydescribed (130). Undoubtedly, the mea-

surement of prostaglandins and their metabolities by variousconventional methods will aid in the diagnosisof coagulationand vessel defects. Some of the newer immunological methodswill be extensively applied in this area. The antiplatelet actionof various drugs can also be assessed by using techniques tomeasure prostaglandins and their metabolites.

Assessment of coagulation function is no longer restrictedto the measurement of clot formation by such methods as thebleeding, clotting, prothrombin, and partial thromboplastintimes. Newer tests based on biochemical, pharmacological,immunological, chemical, physiological, and radiochemicalprinciples have emerged to identify the locale and nature ofthe hemostatic defect in a given disorder. With the intro-duction of synthetic chromogenic and fluorogenic tripeptidesubstrates, it has become possible to assay for specific coag-ulation enzymes, their activators, and inhibitors. The conceptof coagulation profiling has emerged, and many panels arebeing developed. Newer therapeutic agents have necessitatedthe development of fast and reliable diagnostic tests to mea-sure therapeutic efficacy and effect on the overall coagulationstatus of a patient. The continued development of newermethods to evaluate the hemostatic function and to under-stand the mechanisms involved is made possible by an over-whelming interest among basic and clinical scientists in thestudy of hemostasis. Because of the increased understandingof the biochemistry and pathophysiology of thrombotic andhemostatic disorders, the introduction of synthetic peptidesand immunochemical methods, and the application of auto-mated analyzers, we project a growing role for clinical chemistsin this developing area of laboratory medicine. In the comingyears the testing of hemostatic functions will be affected bythese dramatic developments; clinical chemists, immuno-chemists, and pharmacologists will play an important role inimplementing many valuable tests for diagnosing disordersrelated to the hemostatic system.

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56. Bergstrom,K.,and Egberg,N.,Determinationof vitamin Ksensitivecoagulationfactors inplasma.Studieson threemonths usingchromogenicsubstrates.Thromb. Res. 12,531 (1978).

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