dissolution - dm5migu4zj3pb.cloudfront.netgen of 350,000 (15), the degree of iodination varied...

THE MECHANISMOF CLOT DISSOLUTION BY PLASMIN *

By NORMAALKJAERSIG, ANTHONYP. FLETCHERAND SOL SHERRY

(From the Department of Medicine, Washington University School of Medicine, St. Louis, Mo.)

(Submitted for publication October 24, 1958; accepted February 6, 1959)

Normal human and animal sera contain a glob-ulin, plasminogen, which in the presence of acti-vators is rapidly converted to plasmin, a pro-teolytic enzyme active at neutral hydrogen ionconcentrations. Plasmin is an enzyme of widespecificity and will attack such varied substratesas gelatin, casein, certain synthetic esters, accelera-tor globulin, complement, fibrinogen and most im-portantly fibrin. Plasminogen activation occursspontaneously (1), or as a result of contact withactivators of tissue (2), body fluid (3, 4), orbacterial origin (5). The conversion of plas-minogen to plasmin involves the loss of a peptidemoiety and there is evidence to suggest that theplasmin obtained by different modes of activationmay vary in composition (6).

Biochemically, considerable species differencesexist not only between the plasminogen systemof man and animals, but more particularly betweenthe systems of various animals; this variability ismost extreme with regard to the differential effec-tiveness of streptokinase. Rigid kinetic studies(6) reveal that the activation of human plasmino-gen under the influence of streptokinase (an ex-tracellular product of hemolytic streptococcal me-tabolism), trypsin and urokinase (prepared fromhuman urine) results from a first order enzymaticreaction.

Since physiological fibrinolytic phenomena re-sult from activity of the plasminogen system, at-tempts to use plasmin or plasminogen activatorsto effect therapeutic thrombolysis have been nu-merous. Animal experiment, despite the difficul-ties and confusion of species variability, has yieldedstriking findings (7, 8, 9). Though the demon-stration of experimental thrombolytic action hasbeen of an unequivocal nature, the precise mecha-nism of its production has hitherto been obscure.

The present communication describes in vitro* This work was supported by grants from the Na-

tional Heart Institute (H3745), United States PublicHealth Service, Bethesda, Md., and Lederle LaboratoriesDivision, American Cyanamid Co., Pearl River, N. Y.

experiments and in vivo observations bearingupon thrombolytic' mechanisms in man. Theresults indicate that since plasminogen is found bothin plasma and also as a constituent of thrombi,clot lysis occurs by a dual mechanism. The chiefand primary mechanism of thrombolysis involvesthe diffusion or adsorption of plasminogen acti-vator to the thrombus, activation of intrinsic clotplasminogen and thrombolysis. The secondarymechanism involving digestion of the thrombusby extrinsic plasmin action appears to be of littlequantitative importance.

MATERIALS AND METHODS

Streptokinase2 was a highly purified preparation, bio-physically though not immunochemically homogenous; thespecific activity was 600 to 700 streptokinase units perlug. nitrogen.

Human plasminogen (1) contained 100 to 150 caseinunits per mg. tyrosine.

Plasmin was prepared by autocatalytic activation ofplasminogen in glycerol (1) and contained 95 caseinunits per mg. tyrosine (see Casein assay).

Epsilon amino caproic acid2 (6-amino hexanoic acid)had a melting point of 2080 C. and an elemental analysiscorresponding in composition to that of the pure com-pound.

Urokinase 3 contained 5,100 Ploug units per mg. dryweight (10).

Fibrin plate test was modified from Mullertz (11) byusing a highly purified fibrinogen preparation4 in a finalconcentration of 0.2 per cent. Heated fibrin plates (12)

1 The term thrombolytic is used to designate a processby which preformed clots are lysed. This usage corre-sponds to the literal meaning and original derivation ofthe word (Oxford English Dictionary) and its use doesnot imply specific reference to pathological states whereintravascular thrombi may be of variable composition.

2 Kindly supplied by Dr. J. Ruegsegger, Lederle Labor-atories, Pearl River, N. Y. Streptokinase activity ex-pressed in Christensen units (J. clin. Invest. 1949, 28,163).

3 Kindly supplied by Dr. J. Ploug, Leo Pharmaceuti-cals, Copenhagen, Denmark.

4 Bovine fibrinogen, salt free, 92 per cent of the ni-trogen being clottable by thrombin; kindly supplied byDr. Kent Miller, New York State Institute of Health,Albany, N. Y.

1086

MECHANISMOF CLOT LYSIS BY PLASMIN

were prepared by subjecting fibrin plates to 800 C. for30 minutes. This procedure resulted in denaturation ofthe contaminating plasminogen, rendered the plates in-susceptible to the action of plasminogen activators, butalso reduced their sensitivity to proteolysis (pure chy-motrypsin5 0.05 mg. per ml. gave a zone of 473 mm.2on unheated plates and 276 mm.' on heated ones, whilethe figures for spontaneously activated plasmin (1.3casein units per ml.) were 350 mm.2 and 172 mm.',respectively).

Fibrinogen was estimated by the method of Ratnoffand Menzie (13) modified only by the addition of 0.1ml. of 1 per cent soy bean trypsin inhibitor 5 to preventfurther fibrinolysis.

I' labeled plasma clot test. Fibrinogen4 was iodi-nated by a method modified from that of Eisen andKeston (14). Assuming a molecular weight for fibrino-gen of 350,000 (15), the degree of iodination varied be-tween 0.6 and 1.3 I.3. atoms for each molecule of fibrino-gen. Free I"i was removed by passage through an Am-berlite IRA-400 column (40 mesh) and integrity of thefibrinogen molecule demonstrated by the fact that afteriodination treatment the nitrogen clottable by thrombinwas 92 per cent (the starting value). Eighty-nine to 93per cent of the radioactivity was clottable by thrombin.

Preformed isotopically labeled human clots were madeby adding 1 to 4 A of the iodinated fibrinogen (1 to 2 percent solution) to 0.5 ml. of bulk plasma containing addedplasminogen (vide infra) in an 8 X 80 mm. serologicaltube. The quantity of added radioactivity was calcu-lated to give approximately 100 cpm per Ag. of containedfibrinogen. For convenience, throughout the test, ob-served counts per minute have been mathematicallytransformed so that a single count per minute corre-sponds to the lysis of 0.8 Ag. of fibrin. A wire, nichrome26 S. W. G., coiled at its lower end, was inserted intoeach tube and 0.1 ml. of thrombin (50 units per ml.) wasadded. The clots were incubated for one to two hoursin a 37° C. water bath and after retraction were with-drawn by means of the wire. Approximately 50 suchclots were washed in 2 L. of normal saline overnight at2° C. This procedure resulted in a low and consistentblank value for release of radioactivity.

IJ" labeled plasminogen deficient, plasminogen normaland plasminogen rich clots

Plasminogen deficient clots. P"31 labeled human plasmaclots vigorously washed in a large excess of phosphatesaline buffer (0.1 M) at pH 7.6 for 24 hours at 20 C.were shown to contain less than 0.15 casein units ofplasminogen per clot (unwashed drained clots contained0.3 to 1 casein unit). These clots were relatively resist-ant to lysis by plasminogen activators, while retaining anormal sensitivity to the action of plasmin. Residualplasminogen could be denatured by heating these clotsin buffer at 80° C. for 30 minutes. Such clots, desig-nated as "heated clots," were completely resistant to ly-sis by plasminogen activators but the heating process had

5 Worthington Biochemical Corp., Freehold, N. J.

reduced their sensitivity to the action of plasmin so thatit was approximately 60 per cent of that shown by un-heated clots.

Clots of normal and high plasminogen content. Sinceclots had to be washed to reduce blank radioactivityvalues and this process reduced clot plasminogen, thepreparation of clots of normal plasminogen content re-quired reinforcement of the original plasma with puri-fied plasminogen. This problem was simplified by thefact that while plasma plasminogen could be washedfrom plasma clots, purified plasminogen, owing to itsdifferent physical state of dispersion at pH 7.6, was com-pletely taken up from the plasma into the clot and wasnot washed out. Plasma clots of normal plasminogencontent were made by fortifying the original plasmawith 0.5 to 1 casein unit plasminogen per clot. Plasmino-gen rich clots were made similarly except that the addedplasminogen was 2 casein units plasminogen per clot.These latter clots were extremely sensitive to the pres-ence of plasminogen activators and exhibited normalsensitivity to lysis by plasmin.

Thrombolytic 6 assays were made by inserting a pre-pared clot into 0.5 ml. of plasma or other specimen in a10 X 100 mm. tube and incubating at 370 C. for 30 min-utes. The clot was then withdrawn, removed from thewire and centrifuged in 2 ml. of normal saline; thesupernatant was added to the residual plasma and theradioactivity determined in a well-type scintillation de-tector (efficiency 45 per cent). Comparison of residualclot nitrogen content with residual radioactivity dem-onstrated that clot lysis and radioactivity release werehighly correlated.

Assay of isotopically labeled clots for plasminogen.Single clots were ground with powdered glass in a tubecontaining 0.5 ml. 1/6 N hydrochloric acid. After 15minutes at room temperature, the supernatant solutionwas neutralized and assayed for plasminogen by thecasein method. The validity of this assay procedure wastested on a series of 15 plasmas, to some of which hadbeen added plasminogen and to others fibrinogen. Themean recovery of plasminogen from clot plus supernatantserum was 94 ± 9 per cent (range 79 to 109 per cent) ofthe original plasma aliquot.

Assay for plasminogen and plasmin in plasma

Casein assay. The casein assay (16) was modifiedfrom that of Remmert and Cohen (17), our unit being

6 The presence of plasma inhibitors requires that aclear distinction be drawn between total demonstrableactivator or enzyme activity and that portion that is physi-ologically active (uncombined with inhibitor). Test sys-tems requiring dilution or other changes in biophysicalconditions are unacceptable for the assay of physiologi-cally active components in the complex milieu of plasma,since these changes alter the ratios of combined to un-combined activities. Assay of these individual activitiesin undiluted plasma by means of isotopically labeledplasma clots of varying plasminogen concentration ful-fills these stringent requirements.

1087

NORMAALKJAERSIG, ANTHONYP. FLETCHERAND SOL SHERRY

0/

ACTIVATOR ACTIVITY

NO PLASMiNOGEN

1.1 1.7 2.3 2.9 3.5 4.1

.

PROTEOLYTIC ACTIVITY

1.1 1.7 2.3 2.9 3.5 4.1

LOG SK CONC.

FIG. 1. ACTIVATOR AND PROTEOLYTIC ACTIVITIES OF

HUMAN PLASMINOGEN AT VARIOUS STREPTOKINASE

(SK) CONCENTRATIONSPlasminogen concentration, 5.6 casein units per ml.

Activator activity was approximately related to the

logarithm of the streptokinase concentration (indicatedon the figure as streptokinase log units per ml.) and

was independent of the proteolytic activity.

aproximately 50 per cent that of the latter authors.

Assay of untreated plasma for plasminogen yielded low

values and recovery of added plasminogen was incompleteowing to the presence of antiplasmin activity in untreated

plasma. However, direct assay of plasma antiplasmincontent revealed that this activity was largely or com-

pletely destroyed by adding 0.5 ml. of 1/6 N hydro-chloric acid to 0.5 ml. of plasma, leaving at room tem-

perature for 15 minutes and then neutralizing with 0.5ml. of 1/6 N sodium hydroxide and 1 ml. of 0.1 Mphos-phate buffer at pH 7.6. With this modification recovery

of added plasminogen or plasmin became quantitative andassay values for plasma plasminogen were substantiallyincreased. All plasma used for casein assay was treatedin this manner.7

7 The use of this method for the assay of plasmin inplasma may, under certain conditions, yield spuriouslyhigh values. There is evidence that, if the plasma con-

tains both activator and plasminogen, the plasma manipu-lation (dilution, and so forth) consequent upon the as-

say procedure may facilitate plasminogen activation.

Esterase activity was determined by the hydrolysis ofbenzoyl arginine methyl ester (BAMe) (18). The de-gree of hydrolysis was measured by the Hestrin colori-metric method (19). In contrast to the findings withcasein plasminogen assay, prior acidification of theplasma was unnecessary if BAMe were used as a sub-strate. Though the reason for this discrepancy is un-certain,8 the experimental difference has been adequatelyvalidated.

Plasminogen "activator" assay. The activation ofplasminogen by streptokinase occurs as a two stagereaction (20, 21). Streptokinase first reacts with aplasma protein, termed proactivator,9 to form activatorand the activator converts plasminogen to plasmin byenzymatic transformation (20, 21). Since proactivatoris absent from bovine plasma, bovine plasminogen maybe used to assay "streptokinase activator" present inthe plasma of other species.

Five-tenths ml. of bovine Fraction III2 (Armour) (20mg. per ml. or 5.6 casein units per ml.) was incubatedfor 15 minutes at room temperature with 0.1 ml. of thetest solution. The activation reaction was then stoppedby the addition of epsilon amino caproic acid to a finalconcentration of 0.08 M (16) and the solution assayedfor plasmin activity by the casein assay. The resultswere expressed as casein units bovine plasminogen ac-tivated per 15 minutes.

RESULTS

The action of streptokinase on plasminogen prepa-rations

Figure 1 illustrates activator and proteolytic ac-tivity assays made after the addition, 15 minutespreviously, of increasing quantities of streptokinaseto a purified human plasminogen preparation. Itis evident that the two distinct activities of plas-minogen preparations developed under the in-fluence of streptokinase, the proteolytic and theactivator activities, are differently related to thestreptokinase concentrations employed. Whereas,proteolytic activity reaches a plateau at 100 strep-tokinase units per ml., which is maintained with

8 Presumably the affinity of plasmin for BAMe isgreater than for antiplasmin.

9 There is still controversy as to whether proactivatoris an entirely separate substance or is derived from plas-minogen itself (22, 23, 24), but the term is used to desig-nate a plasma factor capable of reacting with streptokinaseto form plasminogen activator. Though divergent em-phasis has been placed on the significance of proactivatorin the human as apart from the bovine system (25, 26,27) for descriptive purposes we acknowledge its sepa-rate existence and assay for its biochemical activity eventhough its individual identity apart from plasminogenor its derivatives has not been clearly established.

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- NO PLASMINOGEN*-* LOWPLASMINOGEN

0-0 NORMALPLASMINOGEN--K HIGH PLASMINOGEN

SK- U/ML UK-MICROGRAM/ML PLASMIN-CAS U/ML

FIG. 2. THE Lysis RATE OF I" TAGGEDPLASMACLOTS AS A FUNCTION OF THE PLASMINOGENCONTENTOF THE CLOTANDOF THE ACTIVATORAND PLASMIN CONCENTRATIONS

Lysis rates for streptokinase and urokinase varied as the logarithm of plasminogen concen-

tration being zero in clots devoid of plasminogen. Lysis rates with plasmin were uninfluencedby the clot plasminogen concentration except that where clots were devoid of plasminogen, a

lesser rate of lysis was found, due to denaturation caused by the heating process.

increasing streptokinase concentrations until itultimately commences to decline around 3,000streptokinase units per ml., activator activity be-haves differently. Activator activity continues toincrease with increasing streptokinase concentra-tions and is still rising at the highest streptokinaseconcentration used (25,000 streptokinase units per

ml.). Since thrombi normally contain significantquantities of plasminogen, it is apparent that un-

der the influence of streptokinase, plasminogenpreparations develop two types of activities, pro-

teolytic and activator, either of which is potentiallycapable of mediating the lysis of a thrombus.

The effect of streptokinase, urokinase and plasminupon plasma clots

Figure 2 shows the rate of supernatant radio-activity release determined when isotopically la-beled plasma clots of differing plasminogen con-

centration (four concentrations displayed in eachpanel) were incubated at 370 C. for 30 minuteswith several concentrations of streptokinase, uro-

kinase and spontaneously activated human plasmin(in 0.1 Mphosphate buffer at pH 7.6). The as-

say technique was that described under the head-ing of thrombolytic assay in the Methods section.

The results for streptokinase and urokinase(Panels 1 and 2) show that not only is the rateof clot lysis linearly related to the surrounding

activator concentration,'0 but that it is also, at any

given activator concentration, related, though notdirectly proportional, to the clot plasminogen con-

centration. In fact, in this and other experiments,over a "physiological" range of clot plasminogenconcentrations, clot lysis rates at a given activatorconcentration have been approximately propor-tional to the logarithm of the clot plasminogenconcentration. It is evident that clots lysing in a

streptokinase solution do so because there are

ample amounts of proactivator as well as plasmino-gen in the clot.

Panel 3 illustrates the effect of spontaneouslyactivated human plasmin. The lysis rate riseslinearly with enzyme concentration but the releaseof radioactivity was independent of clot plasmino-gen concentration. Though clots of high, normaland low plasminogen concentration were lysed atidentical rates, the lysis rates of those clots marked"no plasminogen" were about half those of theothers. This occurred as a consequence of theirmethod of preparation, which involved heating,and the diminished sensitivity to plasmin actionwas similar to that recorded with the heated fibrinplates (see Methods section). The sensitivity ofthe unheated clots to the action of plasmin in

10 This relationship should not be interpreted as

favoring either of the two alternative views concern-ing passive diffusion of or active absorption of strepto-kinase or urokinase to a clot.

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terms of fibrin lysis rates was one-fifth to one-tenththat shown by purified fibrinogen or purified fibrinin a diffuse state. Whether this diminished sen-sitivity was due to the relative impurity of thefibrin in the washed clot or due to the mechanicalbarrier to enzyme penetration posed by the struc-ture of the clot is uncertain, but the findings dosuggest that digestion of thrombi, in vivo, by ex-trinsic plasmin action may be somewhat impeded.

Thrombolytic activity in plasma

The results of adding an excess of streptokinaseto plasma are illustrated in Figure 3. Seventystreptokinase units were added per ml. of plasma,but since the antibody content of the plasma wasequivalent to 30 streptokinase units per ml., theresults illustrated refer to the presence of 40 freestreptokinase units per ml. plasma. The upperportion of the figure shows that rapid activation

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FIG. 4. THE DEPENDENCEOF STREPTOKINASE EFFECTUPONCLOT PLASMINOGENCONCENTRATION

Clots devoid of plasminogen were unaffected by strep-tokinase treated plasma. Clots containing plasminogenwere lysed by streptokinase treated plasma at rates de-pendent upon their plasminogen concentration.

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FIG. 3. BIOCHEMICAL CHANGESPRODUCEDBY STREPTO-KINASE IN PLASMA

Seventy streptokinase units were added per ml. plasmaat zero time. The plasma activity developed was pri-marily thrombolytic in nature and fibrinogenolysis was

minimal. This comparison is most strikingly seen in thebottom section of the figure.

of plasminogen occurred, so that at the end of 10minutes only 10 per cent of the original quantityremained and after 20 minutes a zero assay read-ing was obtained. Concomitantly with the fall ofplasminogen concentration, plasmin activity of lowdegree was detected, but it is noteworthy thatthis activity was only detectable after plasma anti-plasmin activity had been destroyed by acidifica-tion (see Methods section). Assay of unacidifiedplasma for plasmin activity (not illustrated inFigure 2) revealed the presence of little or noproteolytic activity.

Fibrinogen levels declined to 75 per cent oftheir original value and then became stable; theseassay values have been calculated as rates offibrinogen lysis (pug. lysed per minute) and areplotted as such in the lower portion of Figure 3.However, in contrast to the low degree and shortduration of plasma fibrinogenolytic activity, as-say for plasma thrombolytic activity by means ofthe isotopically labeled clot technique (using clotsof normal plasminogen content) disclosed an in-tense and sustained state of clot lysing activity.This activity, calculated as rates of fibrin lysis(fig. clot fibrin lysed per minute), has beenplotted in the lower portion of Figure 3, whereit may be compared with the rate of fibrinogenlysis calculated similarly. Comparison of thesebiochemical activities shows extreme divergence

1090


between their magnitudes and durations, plasmaclot lysing power being predominant in bothrespects. The fibrin plate assay results, displayedin the middle portion of Figure 3, confirm thisconclusion. Assay on unheated fibrin platesshowed a high and sustained degree of plasmathrombolytic activity that paralleled that revealedby the isotopic clot dissolution test. However,all readings made on heated fibrin plates werezero, indicating a plasmin activity of less than 0.15casein unit per ml. (sensitivity limit of themethod).

The biochemical findings illustrated in Figure3 are typical of those found on addition of strep-tokinase to plasma. Variation of the streptokinaseconcentration, at least within the range of fiveto 50 free streptokinase units per ml. plasma,while altering the speed and degree of the reactiondoes not influence its general qualitative nature.

The relationship of thrombolysis, plasma throm-bolytic activity and clot plasminogen concen-trationFigure 4 shows the degree of lysis produced in

clots of differing plasminogen content immersedin plasma containing varying concentrations ofstreptokinase. The four clot plasminogen con-centrations tested were those used in the experi-ment shown in Figure 2 and the plasma antibodycontent was equivalent to 15 streptokinase unitsper ml. Lysis failed to occur in any of the clotsimmersed in plasma containing 10 streptokinaseunits per ml. and it has been an invariable findingthat significant thrombolysis has always been ab-sent unless the quantity of added streptokinase hasbeen sufficient to exceed the antibody bindingpower of the plasma. Though under special cir-cumstances using different techniques a limiteddissociation of the streptokinase antibody com-plex can be demonstrated, provided that the sys-tem is not diluted, dissociation of the streptokinaseantibody complex exerts no practical effect.

The assays conducted at streptokinase concen-trations of 20, 30, 40 and 50 units per ml. plasmashowed an increasing divergence between the lysisrates of clots containing different plasminogenconcentrations. Where clot plasminogen was lowor absent, thrombolysis was negligible or absent.In contrast, clots containing a normal plasminogenconcentration were rapidly lysed and this action

was greatly enhanced if plasminogen rich clotswere used. Thrombolysis rates with clots of agiven plasminogen concentration were approxi-mately linear with increasing concentrations offree streptokinase and the somewhat concavecurves shown in Figure 4 probably resulted as aconsequence of the varying time relations of re-actions in the complex matrix of plasma. As inthe earlier experiment shown in Figure 2, therates of clot lysis, at any given activator concen-tration, were approximately related to the loga-rithm of the clot plasminogen concentration,though the data fitted this relationship far less ex-actly than in the former case.

These experimental findings, which closelymimic those found in vivo, demonstrate that clotlysis, at a given activator concentration, is de-pendent upon the plasminogen content of theclot. A corollary to these findings indicates thatthrombolysis, at least under physiological condi-tions, must be dependent upon activation of in-trinsic clot plasminogen. Moreover, the illustra-tion that plasminogen deficient clots are lysedpoorly, if at all, by streptokinase activated plasmaspeaks strongly against the concept that thrombo-lysis is related to extrinsic plasmin action underphysiological circumstances.

The influence of an inhibitor of plasminogen ac-tivation upon thrombolytic activity displayed bystreptokinase activated plasma

Plasminogen activation, in the presence of plas-minogen activators, is inhibited competitively bylow concentrations of epsilon amino caproic acid,themselves insufficient to inhibit plasmin action(16). Figure 5 (lower portion) displays suchevidence, obtained by the fibrin plate method andpertaining to the experimental conditions used inthe upper portion of Figure 5. The lower partof the figure shows that the high assay readingproduced by plasma containing 50 streptokinaseunits per ml. was completely abolished by the ad-dition to the mixture, prior to assay, of 0.02 Mepsilon amino caproic acid. The action of plas-min on the plate, adjusted in amount so as to yielda zone of lysis comparable with that of the strep-tokinase activated plasma, was virtually unaffectedby the addition of 0.02 M epsilon amino caproicacid.

1091


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Plasma + 50 SK U/ML. 325mm2Plasma + 50 SK U/M +.02 Mf-Am. Cap. A. 0

Plasmin 390 mm2Plasmin +.02 ME-Amino Caproic Acid 360mm2

FIG. 5. EFFECT OF C-AMINO CAPROIC ACID ON PLAS-MINOGENACTIVATOR (MEASUREDON PLASMINOGENRICHPLASMACLOTS)

The bottom portion of the figure demonstrates that£-amino caproic acid added to streptokinase treatedplasma in a concentration of 0.02 M acts solely to in-hibit plasminogen activation. The top portion of thefigure demonstrates the abolition of thrombolytic ac-tivity in streptokinase treated plasma to which e-aminocaproic acid has been added.

The upper portion of Figure 5 shows that thepowerful thrombolytic action of plasma containingvarying concentrations of streptokinase on plas-minogen enriched clots was totally suppressed bythe addition of 0.02 Mepsilon amino caproic acid.Though plasminogen enriched clots were used forthis experiment precisely similar results have beenobtained when clots containing different plasmino-gen concentrations were used. These findingsoffer powerful support to the concept that plasmathrombolytic activity is related to its content ofplasminogen activator and that free plasmin ac-tivity plays little or no part in thrombolysis.

Evidence from in vivo experiment

In vitro experiments, such as have been de-scribed here, may not be directly applicable tothe in vivo state, since changes in plasma com-position may alter the experimental conditions.The biochemical changes occurring in man as aresult of massive and prolonged streptokinase in-fusion have been described elsewhere (24), but

two problems, relevant to the application of thepresent findings to in vivo circumstances, are con-sidered in this communication.

Plasmin assays made in 17 patients (total of 88plasma samples), receiving streptokinase therapy,revealed no evidence of greater plasmin releasein vivo than would have been anticipated from invitro plasma assay. Sufficient streptokinase wasinfused to activate all the plasma plasminogenwithin a one to two hour period and then maintainthe plasminogen concentration at zero for the re-mainder of the treatment period. Plasmin activitywas only detectable for the first two to three hoursof the treatment period and peak values in theindividual patient were of short duration. Assayvalues found were always less than 10 per cent andusually less than 5 per cent of the potential ac-tivity (total conversion of plasminogen to plasminwould equal 100 per cent). Indeed it is possiblethat these values may have been even lower, asthe plasma manipulations, required for assay pur-poses, may have resulted in a spurious increase infree plasmin assay values.

Figure 6 illustrates the effect of the variousfibrinolytic moieties found in plasma during strep-tokinase therapy upon an assay system of clots

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FIG. 6. EFFECT OF FIBRINOLYTIC MOIETIES PRESENTIN STREPTOKINASE (SK) ACTIVATED PLASMA UPON

RADIOACTIVE CLOTSThe right hand column shows the thrombolytic ac-

tivity of plasma drawn from a patient receiving strepto-kinase therapy upon clots of different plasminogen con-tent. The general similarity of this pattern of activityto that displayed in the center column (the action ofstreptokinase upon identical clots) and its dissimilarityto the pattern of activity found with plasmin (shownon the left) is obvious. Furthermore the quantity ofplasmin used was much greater than can ever be demon-strated in plasma.

1092


containing four different plasminogen concentra-tions. On the left is shown the effect of the maxi-mal plasmin activities found in vivo (after acidifica-tion of the plasma); in the center, the effect ofstreptokinase (10 units per ml. buffer) on thesame assay system; and on the right, the effect ofa plasma sample withdrawn from a patient threehours after the start of treatment. This patientreceived tracer doses of isotopically labeled strep-tokinase for the purpose of determining plasmastreptokinase concentrations and at the time thatthe sample was drawn the value for free strepto-kinase units per ml. plasma was 6.4. The centerand right hand columns show identical ratios ofactivity on clots of different plasminogen contentand it is apparent that in vivo and in vitro clotlysis mechanisms are similar in nature.

DISCUSSION

Humanplasminogen preparations, when treatedwith streptokinase, develop both activator andproteolytic activity. However a marked dissocia-tion is apparent between the streptokinase/plas-minogen ratios required for the maximal develop-ment of these two activities. With a fixed quan-tity of plasminogen, the addition of a relativelylimited amount of streptokinase will induce fullproteolytic activity but a restricted degree of ac-tivator activity. Increasing concentrations ofstreptokinase cause an increase of activator ac-tivity approximately related to the log concentra-tions of the added streptokinase, but proteolyticactivity does not increase and later, when activatoractivity is still rising, commences to decline.

In a like manner, the addition of an excess ofstreptokinase to plasma produces both a proteo-lytic and also an activator effect. The proteolyticactivity is almost completely inhibited by plasmaantiplasmin, but the activator effect (assayed onthe bovine fibrin plate, which is not sensitive to theaction of streptokinase alone) persists, even whenplasminogen is no longer demonstrable as a con-sequence of its activation. These findings areidentical with those obtained by study in patients,in whomactivator formation remained unimpairedafter plasma plasminogen concentrations had beenmaintained at zero or virtually zero levels for 30hours by streptokinase infusion (24).

Though plasminogen activation occurs as atwo stage process (20, 21) and requires proacti-

vator as an essential component for its completion,clots containing plasminogen also contain a suffi-cient amount of proactivator to allow of their lysisby streptokinase alone. Experiment showed thatclots contained in streptokinase buffer solutionlysed in a manner comparable with clots suspendedin streptokinase treated plasma. Thus the lysisof clots in streptokinase treated plasma, by ac-tivation of their contained plasminogen, couldoccur either through the direct action of preformedactivator or be caused indirectly by streptokinasealone. Insufficient evidence is available to evalu-ate the relative importance of these mechanisms,but conceivably, under in vivo conditions, one maybe more important than another.

These results offer no evidence to support theview that digestion of clots or thrombi by thedirect action of circulating plasmin is of practicalimportance. First, the appearance of plasmin,during and after the process of complete activa-tion of plasminogen in vitro, is transient and itsconcentration is very low. Second, assay ofplasma from patients receiving streptokinasetherapy has shown that no greater quantity ofplasmin is formed in vivo than would have beenexpected from predictions based on in vitro plasmaassay. Third, in vitro experiment showed thatplasmin digested clots, of differing plasminogencontent, at a uniform, relatively slow rate and thatif used in concentrations similar to those found inplasma its effects were negligible. Fourth, plasmadrawn from patients, receiving streptokinase ther-apy, lyses clots of varying plasminogen content ina manner identical to that of plasma to which strep-tokinase has been added in vitro and differentlyfrom that seen with plasmin. Lastly, the additionof a specific inhibitor of activator, epsilon aminocaproic acid (16), in a concentration insufficientto inhibit plasmin, abolishes plasma thrombolyticactivity.

On the other hand, there is strong evidence thatthrombolysis results from the direct or indirectactivation of thrombus plasminogen. First, di-rect assay for plasminogen, within the thrombus,disclosed the presence of sufficient plasminogento account for the observed results. Second, witha fixed activator concentration contained in eitherbuffer or plasma, thrombolysis rates were linearlyrelated to the logarithm of the clot plasminogenconcentration. Third, with clots of fixed plas-

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minogen concentration, thrombolysis rates werelinearly related to the logarithm of the activatorconcentration. Fourth, assays for activator werehighly correlated with thrombolysis rates of clotscontaining normal amounts of plasminogen.Lastly, identical patterns of thrombolysis wereproduced by comparable plasma specimens whetherthese were prepared in vitro or drawn from strep-tokinase treated patients and in both cases lowconcentrations of epsilon amino caproic acid com-pletely inhibited thrombolytic activity.

These experiments demonstrate that the chiefand primary mechanism of plasma thrombolysisinvolves the activation of intrinsic clot plasmino-gen with resulting autodigestion of the thrombusand that the secondary mechanism of thrombolysisby exogenous plasmin action appears to be rela-tively unimportant. Therefore we propose thatthe word thrombolysis be used to designate theentire phenomenon by which preformed thrombiare lysed and which for practical purposes de-scribes a complex series of events inclusive of afinal step involving the local action of plasmin con-fined within the substance of the thrombus. Theword fibrinolysis will then be restricted to itsoriginal meaning and will designate the action ofa proteolytic enzyme acting primarily upon a fibrinsubstrate, but also acting secondarily upon suchother substrates as are susceptible to its action.

The necessity for such a clarification of nomen-clature is emphasized by the finding that the addi-tion of urokinase to plasma produces similarresults to those reported with streptokinase; a find-ing that also serves to establish the general im-portance of these mechanisms to the body econ-omy. These observations exemplify a new bio-logical principle of perhaps considerable signifi-cance as they constitute the first demonstration ofhow an enzyme of wide proteolytic activity (plas-min) may in vivo retain its full spectrum of ac-tivity, but through local mechanisms have its speci-ficity virtually limited to one substrate (fibrin).Indeed, unless such a mechanism existed, phe-nomena causing plasminogen activation might wellbe attended by catastrophic consequences to theorganism.

SUMMARY

1. The respective roles of plasminogen activa-tors and plasmin in human thrombolytic mecha-

nisms have been investigated. Quantitation ofthrombolysis was accomplished by the use of J131labeled human plasma clots. Test systems usinglabeled clots containing varying concentrations ofplasminogen permitted differentiation between theactions of plasminogen activators and plasminitself.

2. Studies on streptokinase treated patients con-firmed that the conclusions drawn from in vitrotest systems were applicable to the in vivo state.

3. It was demonstrated that the chief and pri-mary mechanism of plasma thrombolysis involvedthe activation of intrinsic clot plasminogen withresulting autodigestion of the thrombus. Thesecondary mechanism of thrombolysis by exoge-nous plasmin action appeared to be relativelyunimportant.

ACKNOWLEDGMENTS

It is a pleasure to record our indebtedness to MissGeraldine Nelson and Mr. Charles Chisenhall for tech-nical assistance. Wealso wish to thank those physiciansat the Barnes and Jewish Hospitals who referred pa-tients for treatment.

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19. Hestrin, S. The reaction of acetylcholine and othercarboxylic acid derivatives with hydroxylamineand its analytical application. J. biol. Chem. 1949,180, 249.

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21. Troll, W., and Sherry, S. The activation of humanplasminogen by streptokinase. J. biol. Chem. 1955,213, 881.

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27. Fletcher, A. P., Alkjaersig, N., and Sherry, S. Themaintenance of a sustained thrombolytic state inman. I. Induction and effects. J. clin. Invest.1959, 38, 1096.

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