Possibilities and Limitations of Reaction Product Analysis JOHN OWEN Depcrrtment of Meduine The Bowman Gmy School of Meduine Wake l3m-t Unitmity M e d d Cenw Eouhd Winrton-Salem, Nmth Gdit~ 27157 Under normal conditions, in hplasmin action is confined to specific sites, and usually the site is not directly accessible. This generalization holds b r thrombolysis and for tissue remodeling. Direct analysis of the reactions involved is not h i b l e , and we are forced to use indces that reflect the actual events. Plasmin is a proteolyac enzyme, and characteristically degrades its insoluble substrate proteins into small, soluble fragments. The fragments are released into the circulation from whence they are cleared. The clearance mechanism can involve the reticuloendothelial system, notably the liver, or if the fragment is small it can be cleared through the kidney. In the latter situation it may be possible to measure the excretion of peptides in the urine, and use this figure to infer the rate and/or extent of in vim plasmin activity. The greatest amount ofinbrmation comes from analyzingthe concentration of peptides in blood.1 Unfortunately, the increased inbrmation comes at the expense of increased complexity. Plasma is a complex mixture, and acts as a hostile environment in which to measure low concentrations of reaction products. Nonetheless, reaction products can be analyzed quantitatively and qualitatively to give information on the localized fibrinolytic process.2 Electrophoresishas been used to follow fibrinogen proteolysis during thrombolytic therapy.3.4 This technique clearly showed the conversion of virtually all fibrinogen to Fragment X, a modification likely to result in impaired hem~stasis.~ Along similar lines, electrophoresis has been used to characterize the larger fibrinogen derived complexes formed by plasmic lysis of brmed thrombi.G6 From these studies it has become clear that, under pathologc conditions, reaction products appear in the blood. The precise nature of most of these reaction products is yet to be determined, but the promise is evident even now. Semi-quantitative assays b r plasmic +tion products enjoy considerable popularity. No clinical laboratory is complete without offering at least one test aimed at reflecting the presence in the blood of D-dimer. The logic and rationale b r this test is impe~cable.~ Thrombin, fictor XIIIa and plasmin all acting in sequence are required to brm a soluble moiety containing the y cross-link. In practice the theory is often violated, though the test has proved to be u s e l l in the clinical arena.10 This observation leads to the prime question, usually asked after the event. 'What inbrmation do we wish to derive from application of the test?" There is a world of difference be305
306
ANNALS NEW Y O U ACADEMY OF SCIENCES
tween diagnosis and pathophysiology. In diagnosis an empiric test is often as good as one with “sound” scientific underpinnings. Probes of pathophysiology on the other hand can not be empiric. In developinga pathophysiologicprobe for in PiPo plasmin action, a number of important issues need to be addressed. Some issues can be resolved at the design stage, some during development and some will remain as limitations on the system. Specificity is essential: not diagnosticspecificity, but biochemical specificity. Ideally, the probe should be unique to the reaction of interest. Measurement of a product of the reaction gives a great deal of specificity, but only if the product is unique in regard to both enzyme and substrate. In this re@ substrate Specificity is particularly important since plasmin will produce many of the same products either from fibrin or from fibrinogen. The implication in either situation is that plasmin is active, but the interpretation in pathophysiologic terms may be very diflkrent. Sequential enzyme actions are particularly difficult to deal with. Consider the measurement of FpBfl1542 as an index of plasmin action on a thrombus.” Such an interpretation presupposes that thrombin action to release FpB occurred betbre plasmin action. But FpBfl142 is a good substrate tbr thrombin action, and the possibility exists that FpBfl15-42 could be produced by plasmin action preceding thrombin action. This latter situation may well exist in patients undergoing therapy with streptokinase, where FpBflI-rlZlevels exceed 10 000 nM. This concentration exceeds that of the residual fibrinogen, opening the door to the action of thrombin to release FpB and FpBfllsAz from the circulating FpBfl142.3 Is the assay system able to discriminate product from substrate?For most of the coagdation-related systems, this issue has proved to be problematic.2 Measurement of fibrin- or fibrinogen-derived peptides in the presence of native intact fibrinogen has proved particularly troublesome. Nonetheless, unwanted cross-reacting material can be removed by suitable sample pretreatment, and hgrnents of interest determined with high sensitivity and specificity.JJ2J Another approach has been to search fbr neoantigens exposed on the product but not on the substrate. This is the basis for the Dimeflat”. A monoclonal antibody was selected which reacts with an epitope on y cross-linked D domains which is not present on fibrinogen.9 Assays that use this antibody can be perfbrmed directly on plasma samples. A similar approach has given us the assays for reaction products such as the activation peptides of the coagulation hctors.14 This list continues to grow, and in addition to derivatives of fibrinogen,l5J6 includes propeptides of collagen.17J8 These assays have recently been used to demonstrate the breakdown of type I11 collagen during thrombolytic therapy with streptokinase. What is likely to be the magnitude of the signal in the abnormal situation? We are able to set some limits on expectations. We need to take into account the substrate concentration, the rate at which substrate can be formed and the clearance rate of the product from the circulation.19 Using fibrinogen conversion to fibrin as an example, we need the concentration of fibrinogen and the clearance times fbr fibrinogen and fbr FpA fiom the circulation. An average fibrinogen concentration would be 2.5 gn, and the turnover rate for fibrinogen is approximately 25% per day. The clearance of FpA is biphasic, but for this purpose can adequately be described as a single exponential with t%of 2 min. The difference of some three orders of magnitude in these rates of clearance leads to an intractable problem. Assume that a l l fibrinogen disappears from the circulation by being converted to fibrin. Then the amount of fibrinogen converted to FpA must equal the rate of formation of fibrinogen. This can be calculated to be
OWEN: REACTION PRODUCT ANALYSIS
307
0.4 mg/L/min, or 1.2 nmoles/L/min. Assume the liver can increase its output some
fourfold above baseline, then the maximum steady state conversion of fibrinogen would be 4.8 nmoles/L/min. Each molecule of fibrinogen releases two molecules of FpA, so the maximum rate of production of FpA would be 9.6 nmoles/L/min. Using a half dlsappcarance time fbr FpA of 2 rnin, the maximum sustainable steady state plasma concentration would be 27.5 nM. Similar calculations can be made fbr any reaction product; if actual clearance data are not available, reasonable guesstimates can often be made. Not all data are collected under steady state conditions. Take fbr example the infusion of streptokinase. The concentration of the reaction product of the action of plasrnin on fibrinogen, FpBO1-42, was found to exceed 9000 nM.j Steady state assumptions would clearly be unwarranted, but so too would a simple calculation asking if peak FpB6142 concentration exceeded fibrinogen available. If we assume a normal fibrinogen concentration of 2.5 g/L (this is the same as 7350 nM) then complete release of all FpBB1-42 would give a peptide concentration of 14700 nM. But this would be an instantaneous value, which would immediately begm to decrease with a half disappearance time of 20 min. In order to probe deeper we need to integrate the concentration versus time curve fbr the peptide, and use that value to calculate the actual total amount of FpBB142 fbrmed. Such a calculation was used in a study of patients receiving streptokinase, and the amount of FpBB142 fbrmed was fbund to have been 14400 nmoles/L, equivalent to 2.45 g/dl of fibrinogen. This figure was possible, the patients had a mean level of fibrinogen of 3.5 g/d. Yet another level of complexity is added by the circulation of the blood. In a wellstirred vessel reactions are typically ‘’well behaved.” In the blood, most of these reactions take place on suhces, causing the system to manifest nonlinear kinetics. As if this is not enough, recirculation combined with positive and negtive feedback makes fbr an intrinsically unstable system. In this formulation thrombus fbrmation can be thought of as the onset of chaos. A proposition which can not be adequately tested as of this date. Recirculation introduces another problem. One way of increasing the sensitivity of the index is to sample as close as possible to the site of product generation. The basis fbr this is to avoid the dilution effect of sampling at a site remote h m the point of generation. Ultimate sensitivity would be expected when samples are collected “at” the site of generation, but this will hold only if the ultimate effector remains localized. In the coagulation system, thrombin generation is delayed h m the time of initiation. If the later steps occur in the flowing blood, sampling too dose to the site will miss the event of interest.20 In the 1940s E g e Astrup proposed that in p)Pg there is ongoing activity of both coagulation and of fibrinolysis, and that these processes are normally in balance.21 This elegant proposition has been shown confirmed in many ways. In our hands by examining the relationship between plasma levels of the reaction products of both coagulation and of fibrinolysis, and finding a correlation in basal values. We further fbund that correlation is disrupted in pathologic situations.22 This lends further support to the notion that the balance of coagulation and fibrinolysis is central to hemostatic homeostasis. This essential basal activity complicates the detection of the products of the reactions in pathologic states. This problem has been extensively studied in other fields, and appears under the general heading of signal detection in a noisy environment. From such studies we have become h i l i a r with the notion of the signal to noise ratio. Familiarity however does
308
ANNALS NEW YORK ACADEMY OF SCIENCES
not immediately translate into proficiency. Once more the question of whether we are interested in basal levels or in transient activation becomes critical. Changcs in basal activity can only be detected with refrence to an external standard. And the magnitude of the minimum detectable difterence will depend on the precision of measurement. We have control over this to some extent, replication can be used to reduce the random e m r of measurement to any arbitrary value. Selecting a measurement technique with small intrinsic random e m r will help. But, no matter how precise the assay, biologic variability imposes noise on the reference group. The true biologic range fbr a plasma peptide is always going to be tighter than the observed range of normal values. Remember the introductory statistics: variances are additive. A single sample would need to be substantially higher than the upper limit of the normal range befbre we could f e l confident the mult is abnormal, and the less precise the assay, the greater the elevation needs to be. The signal must clearly exceed the noise, and the noise has two components: biology and methodology. Transient elevation can be detected in an individual with relative ease if baseline samples fbr the individual are available. For most of the peptides we are considering here, there is a profound absence of data concerning the tracking of levels. There is an assumption that values do track, but where this has been tested directly it has not been possible to unequivocally confirm tracking. In the short term-over minutesthere is some support fbr this notion, but similar data collected under other conditions has raised the specter of considerable minute-to-minute variability. Detection of transient changes can be achieved by frequent sampling, and meticulous attention to detail.23 On the basis of the above considerationswe can identify the most important issues in selecting a technique fbr a specific purpose. In all cases a simple, rapid, robust technique is desirable. All too frequently we are fbrced to accept complex, time-consuming, brittle assays.This is almost always driven by the need for exquisite sensitivity. In most cases sensitivity fbr the target analyte carries with it unwanted sensitivity to all fbrms of intemrence. Precise timing, exact temperature, details of mixing all have been known to influence results. Batch to batch variability in reagents, reagent instability, and operator effects are likewise common. We,the designers, must shoulder the blame for many of these problems. Attention to these issues during development of the assay obviates many of the potential difficulties down the road. Often a simple change in time of incubation can change a brittle assay into one of great robustness. This same change will often dramatically “improve the skills” of those pertbrming the assay. Despite the multitude problems and manibt weaknesses of these systems, usefd information has been garnered by measuring reaction products. I am ever hopeful that even better assays will be developed soon. I particularly look forward to having available a battery of such assays able to pinpoint the precise step at which a pathway is either interrupted or stimulated. With such systems in place, our understanding of the pathophysiology of pmteolytic processes will be immeasurably increased. REFERENCES 1. OWEN, J., B. A. GROSSMAN,J. SOBEL& B. KUDRYK.1991. Fibrinogen pmteolysis and coagulation system activation during thrombolytic therapy. Adv. Exp. Med. Biol. 281: 401-408. 2. OWEN.T. 1989. The urilitv of plasma fibrinoDeDtide assays. Thromb. Haernostasis 62: 8071810. 3. OWEN, J., K. D. FRIEDMAN, B. GROSSMAN,C. WILKINS,A. D.BERKE& E. R POWERS.
, .
A .
OWEN: REACTION PRODUCT ANALYSIS
4. 5. 6. 7. 8.
309
1987.Quantitation of fragment X formation during thrombolytic therapy with streptokinase and with tissue plasminogen activator. J. Clin. Invest. 79: 1642-1649. GRON,B., A. BENNICK, W. NIEUWENHUIZEN, S. BJABNSEN & F. BROSSTAD. 1988.Immunovisualization of fibrinogen A alpha-chain hetemgcneity in normal plasma and plasma frompatients with DIC or on streptokinase therapy. Thromb. RCS. 52: 413-424. MENTZER,R L.,A. Z. BUDZYNSKJ & S. SHERRY.1986. H e d o s e , briefduration intravenous &ion of streptokinase in acute myocardial inhaion: Description of efEcts in the circulation. Am. J. Cardiol. 57: 1220-1226. FRANCIS,C. W., V. J. MARDER&S. E. MARTIN.1980.Plasmic degradation ofcrosslinked fibrin. I. Structural analysis of the particulate clot and identification of new macromolecular-soluble complexes. Blood 56: 456-464. GAFPNEY, P. J., F. JOE& M. MAHMOUD.1980.Giant fibrin fragments derived from crosslinked fibrin: Structure and clinical implications. Thromb. RCS. 20: 647662. FUNCIS,C. W.,D. G. C~NNAGHAN & V. J. MARDER.1986.Assessment of fibrin degradation products during fibrinolytic therapy for acute myocardial i n h a i o n . C i t i o n
7 4 1027-1036. 9. ELMS,M. J., I. H.BUNCE,P. G. BUNDESEN,D. B. RYLAIT,A. J. WEBBER, P. P. MASCI & A. N. WHITAKER.1983.Measurement ofcrosslinked fibrin degradation products-an immunoassay using monoclonal antibodies. Thromb. Haemostasis (Stuttgart) 50:
591-594. C. S.,D. V. DEVINE& K. M. MCCRAE.1987.Measurement of plasma fibrin 10. GREENBERG, Ddimer levels with the ux of a monoclonal antibody coupled to latex beads. Am. J. Clin. Pathol. 87: 94-100. 11. KUDRYK, B., D. ROBINSON, C. NETRE,B. HESSEL,M. BLOMBACK & B. BLOMBACK. 1982. Measurement in human blood of fibrinogen/fibrin fiagrnents containing the B&l542 sequence. Thromb. Res. 25: 277-291. 12. KOCKUM, C. & S.FRBBELNS.1980.Rapid radioimmunoassay of human fibrinopeptide Aremoval of cmss-mcting fibrinogen with bentonite. Thromb. RCS. 19: 589-598. E. A. STOEPMANVAN DALEN,C. J. GINKEL, 13. LEEKSMA,0.C., F. MEIJERHUIZINGA, W. G. VAN AKEN& J. A. VAN MOURIK.1986. Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated i n t r a d coagulation. Blood 67: 1460-1467. 14. BAUER,K. A. & R D. ROSENBERG. 1987.The pathophysiology of the prethrombotic state in humans: Insights gained from studies using markers of hemostatic system activation. Blood 7 0 343-350. 1985. A monoclonal anti15. KOPPERT,P. W., C. M. HUIJSMANS& W. NIEUWBNHUIZEN.
16. 17. 18. 19. 20. 21. 22. 23.
body, specific for human fibrinogen, fibrinopeptide A-containing fragments and not reacting with tire fibrinopeptide A. Blood 66: 503-507. SCHEEPERS BORCHBL, U.,G. MULLERBERGHAUS, P. FUGHE, R EBERLE& N. HEIMBERGBR. 1985. D h i n a t i o n between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide. Pmc. Natl. Acad. Sci. USA 82: 7091-7095. RISTELI, J., S. NIEMI,P.TRIVEDI, A. P. MOWAT& L. RISTBLI. 1988.Rapid equilibrium radioimmunoassay for the aminoterminal propeptide of human type I11 proco@n. Cli.Chem. 34:7l5-718. MELKKO,J., S. NIEMI,L. RISTELI & J. RISTELI. 1990.Radioimmunoassay of the carboxy terminal propeptide of human type I procollagen. Clin. Chem. 36: 1328-1332. OWEN, J. 1987. On the quantitative interpretation of plasma fibrinopeptide levels. :237-240.Elsevier Science Publishers BV. OWEN,J. & K. L. KAPLAN. 1987. Blood tests for the detection of thrombosis. Efkts of flow and location of the sampling site. Ann. NY. Acad. Sci. 516 621-630. ASTRIJP,T. & P. M. PERMIN.1947.Fibrinolysis in the animal o@m. Nature 159: 681. OWEN,J., D. KVAM,H. L. NOSSEL,K. L. KAPLAN & P. B. KERNOFF. 1983.Thrombin and plasmin activity and platelet activation in the development of venous thrombosis. Blood 61: 476-482. OWEN, J., M. HUNTHR-LASZLO, J. K. WILLIAMS & M. ADAMS.1990.Thrombin activity induced by balloon angioplasty of the coronary artery in M n m c a f k c c i n r ~(cynomolgus monkey). Blood Coap. Fibrin. 1: 505-507.
306
ANNALS NEW Y O U ACADEMY OF SCIENCES
tween diagnosis and pathophysiology. In diagnosis an empiric test is often as good as one with “sound” scientific underpinnings. Probes of pathophysiology on the other hand can not be empiric. In developinga pathophysiologicprobe for in PiPo plasmin action, a number of important issues need to be addressed. Some issues can be resolved at the design stage, some during development and some will remain as limitations on the system. Specificity is essential: not diagnosticspecificity, but biochemical specificity. Ideally, the probe should be unique to the reaction of interest. Measurement of a product of the reaction gives a great deal of specificity, but only if the product is unique in regard to both enzyme and substrate. In this re@ substrate Specificity is particularly important since plasmin will produce many of the same products either from fibrin or from fibrinogen. The implication in either situation is that plasmin is active, but the interpretation in pathophysiologic terms may be very diflkrent. Sequential enzyme actions are particularly difficult to deal with. Consider the measurement of FpBfl1542 as an index of plasmin action on a thrombus.” Such an interpretation presupposes that thrombin action to release FpB occurred betbre plasmin action. But FpBfl142 is a good substrate tbr thrombin action, and the possibility exists that FpBfl15-42 could be produced by plasmin action preceding thrombin action. This latter situation may well exist in patients undergoing therapy with streptokinase, where FpBflI-rlZlevels exceed 10 000 nM. This concentration exceeds that of the residual fibrinogen, opening the door to the action of thrombin to release FpB and FpBfllsAz from the circulating FpBfl142.3 Is the assay system able to discriminate product from substrate?For most of the coagdation-related systems, this issue has proved to be problematic.2 Measurement of fibrin- or fibrinogen-derived peptides in the presence of native intact fibrinogen has proved particularly troublesome. Nonetheless, unwanted cross-reacting material can be removed by suitable sample pretreatment, and hgrnents of interest determined with high sensitivity and specificity.JJ2J Another approach has been to search fbr neoantigens exposed on the product but not on the substrate. This is the basis for the Dimeflat”. A monoclonal antibody was selected which reacts with an epitope on y cross-linked D domains which is not present on fibrinogen.9 Assays that use this antibody can be perfbrmed directly on plasma samples. A similar approach has given us the assays for reaction products such as the activation peptides of the coagulation hctors.14 This list continues to grow, and in addition to derivatives of fibrinogen,l5J6 includes propeptides of collagen.17J8 These assays have recently been used to demonstrate the breakdown of type I11 collagen during thrombolytic therapy with streptokinase. What is likely to be the magnitude of the signal in the abnormal situation? We are able to set some limits on expectations. We need to take into account the substrate concentration, the rate at which substrate can be formed and the clearance rate of the product from the circulation.19 Using fibrinogen conversion to fibrin as an example, we need the concentration of fibrinogen and the clearance times fbr fibrinogen and fbr FpA fiom the circulation. An average fibrinogen concentration would be 2.5 gn, and the turnover rate for fibrinogen is approximately 25% per day. The clearance of FpA is biphasic, but for this purpose can adequately be described as a single exponential with t%of 2 min. The difference of some three orders of magnitude in these rates of clearance leads to an intractable problem. Assume that a l l fibrinogen disappears from the circulation by being converted to fibrin. Then the amount of fibrinogen converted to FpA must equal the rate of formation of fibrinogen. This can be calculated to be
OWEN: REACTION PRODUCT ANALYSIS
307
0.4 mg/L/min, or 1.2 nmoles/L/min. Assume the liver can increase its output some
fourfold above baseline, then the maximum steady state conversion of fibrinogen would be 4.8 nmoles/L/min. Each molecule of fibrinogen releases two molecules of FpA, so the maximum rate of production of FpA would be 9.6 nmoles/L/min. Using a half dlsappcarance time fbr FpA of 2 rnin, the maximum sustainable steady state plasma concentration would be 27.5 nM. Similar calculations can be made fbr any reaction product; if actual clearance data are not available, reasonable guesstimates can often be made. Not all data are collected under steady state conditions. Take fbr example the infusion of streptokinase. The concentration of the reaction product of the action of plasrnin on fibrinogen, FpBO1-42, was found to exceed 9000 nM.j Steady state assumptions would clearly be unwarranted, but so too would a simple calculation asking if peak FpB6142 concentration exceeded fibrinogen available. If we assume a normal fibrinogen concentration of 2.5 g/L (this is the same as 7350 nM) then complete release of all FpBB1-42 would give a peptide concentration of 14700 nM. But this would be an instantaneous value, which would immediately begm to decrease with a half disappearance time of 20 min. In order to probe deeper we need to integrate the concentration versus time curve fbr the peptide, and use that value to calculate the actual total amount of FpBB142 fbrmed. Such a calculation was used in a study of patients receiving streptokinase, and the amount of FpBB142 fbrmed was fbund to have been 14400 nmoles/L, equivalent to 2.45 g/dl of fibrinogen. This figure was possible, the patients had a mean level of fibrinogen of 3.5 g/d. Yet another level of complexity is added by the circulation of the blood. In a wellstirred vessel reactions are typically ‘’well behaved.” In the blood, most of these reactions take place on suhces, causing the system to manifest nonlinear kinetics. As if this is not enough, recirculation combined with positive and negtive feedback makes fbr an intrinsically unstable system. In this formulation thrombus fbrmation can be thought of as the onset of chaos. A proposition which can not be adequately tested as of this date. Recirculation introduces another problem. One way of increasing the sensitivity of the index is to sample as close as possible to the site of product generation. The basis fbr this is to avoid the dilution effect of sampling at a site remote h m the point of generation. Ultimate sensitivity would be expected when samples are collected “at” the site of generation, but this will hold only if the ultimate effector remains localized. In the coagulation system, thrombin generation is delayed h m the time of initiation. If the later steps occur in the flowing blood, sampling too dose to the site will miss the event of interest.20 In the 1940s E g e Astrup proposed that in p)Pg there is ongoing activity of both coagulation and of fibrinolysis, and that these processes are normally in balance.21 This elegant proposition has been shown confirmed in many ways. In our hands by examining the relationship between plasma levels of the reaction products of both coagulation and of fibrinolysis, and finding a correlation in basal values. We further fbund that correlation is disrupted in pathologic situations.22 This lends further support to the notion that the balance of coagulation and fibrinolysis is central to hemostatic homeostasis. This essential basal activity complicates the detection of the products of the reactions in pathologic states. This problem has been extensively studied in other fields, and appears under the general heading of signal detection in a noisy environment. From such studies we have become h i l i a r with the notion of the signal to noise ratio. Familiarity however does
308
ANNALS NEW YORK ACADEMY OF SCIENCES
not immediately translate into proficiency. Once more the question of whether we are interested in basal levels or in transient activation becomes critical. Changcs in basal activity can only be detected with refrence to an external standard. And the magnitude of the minimum detectable difterence will depend on the precision of measurement. We have control over this to some extent, replication can be used to reduce the random e m r of measurement to any arbitrary value. Selecting a measurement technique with small intrinsic random e m r will help. But, no matter how precise the assay, biologic variability imposes noise on the reference group. The true biologic range fbr a plasma peptide is always going to be tighter than the observed range of normal values. Remember the introductory statistics: variances are additive. A single sample would need to be substantially higher than the upper limit of the normal range befbre we could f e l confident the mult is abnormal, and the less precise the assay, the greater the elevation needs to be. The signal must clearly exceed the noise, and the noise has two components: biology and methodology. Transient elevation can be detected in an individual with relative ease if baseline samples fbr the individual are available. For most of the peptides we are considering here, there is a profound absence of data concerning the tracking of levels. There is an assumption that values do track, but where this has been tested directly it has not been possible to unequivocally confirm tracking. In the short term-over minutesthere is some support fbr this notion, but similar data collected under other conditions has raised the specter of considerable minute-to-minute variability. Detection of transient changes can be achieved by frequent sampling, and meticulous attention to detail.23 On the basis of the above considerationswe can identify the most important issues in selecting a technique fbr a specific purpose. In all cases a simple, rapid, robust technique is desirable. All too frequently we are fbrced to accept complex, time-consuming, brittle assays.This is almost always driven by the need for exquisite sensitivity. In most cases sensitivity fbr the target analyte carries with it unwanted sensitivity to all fbrms of intemrence. Precise timing, exact temperature, details of mixing all have been known to influence results. Batch to batch variability in reagents, reagent instability, and operator effects are likewise common. We,the designers, must shoulder the blame for many of these problems. Attention to these issues during development of the assay obviates many of the potential difficulties down the road. Often a simple change in time of incubation can change a brittle assay into one of great robustness. This same change will often dramatically “improve the skills” of those pertbrming the assay. Despite the multitude problems and manibt weaknesses of these systems, usefd information has been garnered by measuring reaction products. I am ever hopeful that even better assays will be developed soon. I particularly look forward to having available a battery of such assays able to pinpoint the precise step at which a pathway is either interrupted or stimulated. With such systems in place, our understanding of the pathophysiology of pmteolytic processes will be immeasurably increased. REFERENCES 1. OWEN, J., B. A. GROSSMAN,J. SOBEL& B. KUDRYK.1991. Fibrinogen pmteolysis and coagulation system activation during thrombolytic therapy. Adv. Exp. Med. Biol. 281: 401-408. 2. OWEN.T. 1989. The urilitv of plasma fibrinoDeDtide assays. Thromb. Haernostasis 62: 8071810. 3. OWEN, J., K. D. FRIEDMAN, B. GROSSMAN,C. WILKINS,A. D.BERKE& E. R POWERS.
, .
A .
OWEN: REACTION PRODUCT ANALYSIS
4. 5. 6. 7. 8.
309
1987.Quantitation of fragment X formation during thrombolytic therapy with streptokinase and with tissue plasminogen activator. J. Clin. Invest. 79: 1642-1649. GRON,B., A. BENNICK, W. NIEUWENHUIZEN, S. BJABNSEN & F. BROSSTAD. 1988.Immunovisualization of fibrinogen A alpha-chain hetemgcneity in normal plasma and plasma frompatients with DIC or on streptokinase therapy. Thromb. RCS. 52: 413-424. MENTZER,R L.,A. Z. BUDZYNSKJ & S. SHERRY.1986. H e d o s e , briefduration intravenous &ion of streptokinase in acute myocardial inhaion: Description of efEcts in the circulation. Am. J. Cardiol. 57: 1220-1226. FRANCIS,C. W., V. J. MARDER&S. E. MARTIN.1980.Plasmic degradation ofcrosslinked fibrin. I. Structural analysis of the particulate clot and identification of new macromolecular-soluble complexes. Blood 56: 456-464. GAFPNEY, P. J., F. JOE& M. MAHMOUD.1980.Giant fibrin fragments derived from crosslinked fibrin: Structure and clinical implications. Thromb. RCS. 20: 647662. FUNCIS,C. W.,D. G. C~NNAGHAN & V. J. MARDER.1986.Assessment of fibrin degradation products during fibrinolytic therapy for acute myocardial i n h a i o n . C i t i o n
7 4 1027-1036. 9. ELMS,M. J., I. H.BUNCE,P. G. BUNDESEN,D. B. RYLAIT,A. J. WEBBER, P. P. MASCI & A. N. WHITAKER.1983.Measurement ofcrosslinked fibrin degradation products-an immunoassay using monoclonal antibodies. Thromb. Haemostasis (Stuttgart) 50:
591-594. C. S.,D. V. DEVINE& K. M. MCCRAE.1987.Measurement of plasma fibrin 10. GREENBERG, Ddimer levels with the ux of a monoclonal antibody coupled to latex beads. Am. J. Clin. Pathol. 87: 94-100. 11. KUDRYK, B., D. ROBINSON, C. NETRE,B. HESSEL,M. BLOMBACK & B. BLOMBACK. 1982. Measurement in human blood of fibrinogen/fibrin fiagrnents containing the B&l542 sequence. Thromb. Res. 25: 277-291. 12. KOCKUM, C. & S.FRBBELNS.1980.Rapid radioimmunoassay of human fibrinopeptide Aremoval of cmss-mcting fibrinogen with bentonite. Thromb. RCS. 19: 589-598. E. A. STOEPMANVAN DALEN,C. J. GINKEL, 13. LEEKSMA,0.C., F. MEIJERHUIZINGA, W. G. VAN AKEN& J. A. VAN MOURIK.1986. Fibrinopeptide A and the phosphate content of fibrinogen in venous thromboembolism and disseminated i n t r a d coagulation. Blood 67: 1460-1467. 14. BAUER,K. A. & R D. ROSENBERG. 1987.The pathophysiology of the prethrombotic state in humans: Insights gained from studies using markers of hemostatic system activation. Blood 7 0 343-350. 1985. A monoclonal anti15. KOPPERT,P. W., C. M. HUIJSMANS& W. NIEUWBNHUIZEN.
16. 17. 18. 19. 20. 21. 22. 23.
body, specific for human fibrinogen, fibrinopeptide A-containing fragments and not reacting with tire fibrinopeptide A. Blood 66: 503-507. SCHEEPERS BORCHBL, U.,G. MULLERBERGHAUS, P. FUGHE, R EBERLE& N. HEIMBERGBR. 1985. D h i n a t i o n between fibrin and fibrinogen by a monoclonal antibody against a synthetic peptide. Pmc. Natl. Acad. Sci. USA 82: 7091-7095. RISTELI, J., S. NIEMI,P.TRIVEDI, A. P. MOWAT& L. RISTBLI. 1988.Rapid equilibrium radioimmunoassay for the aminoterminal propeptide of human type I11 proco@n. Cli.Chem. 34:7l5-718. MELKKO,J., S. NIEMI,L. RISTELI & J. RISTELI. 1990.Radioimmunoassay of the carboxy terminal propeptide of human type I procollagen. Clin. Chem. 36: 1328-1332. OWEN, J. 1987. On the quantitative interpretation of plasma fibrinopeptide levels. :237-240.Elsevier Science Publishers BV. OWEN,J. & K. L. KAPLAN. 1987. Blood tests for the detection of thrombosis. Efkts of flow and location of the sampling site. Ann. NY. Acad. Sci. 516 621-630. ASTRIJP,T. & P. M. PERMIN.1947.Fibrinolysis in the animal o@m. Nature 159: 681. OWEN,J., D. KVAM,H. L. NOSSEL,K. L. KAPLAN & P. B. KERNOFF. 1983.Thrombin and plasmin activity and platelet activation in the development of venous thrombosis. Blood 61: 476-482. OWEN, J., M. HUNTHR-LASZLO, J. K. WILLIAMS & M. ADAMS.1990.Thrombin activity induced by balloon angioplasty of the coronary artery in M n m c a f k c c i n r ~(cynomolgus monkey). Blood Coap. Fibrin. 1: 505-507.
