Thrombin Generation in Normal Subjects and …circres.ahajournals.org/content/circresaha/14/5/436.full.pdfThrombin Generation in Normal Subjects and Cardiac Patients By Kurt N. von

Thrombin Generation in Normal Subjects

and Cardiac Patients

By Kurt N. von Kaulla, M.D., and Edith von Kaulla

• A reliable and sensitive test for both hypo-and hypercoagulability would be very desira-ble for clinical and research purposes. Rapiddetection of hypercoagulability would be ofvalue in conditions such as spontaneous clot-ting fluctuations which occur during stress inpatients with congenital or acquired valvularheart diseases 1< - and with previous myocar-dial infarction.

It is difficult to find a test with equal sensi-tivity for both hypo- and hypercoagulability.Thrombelastography, the thromboplastin gen-eration test, and earlier thrombin genera-tion tests do not fulfill these requirementscompletely. The thrombelastograph undoubt-edly can detect marked hypercoagulability 8~r'but it is less suited for recording finer devia-tions from the normal; it is also a ratherexpensive device. The thromboplastin genera-tion test has been used in the past to detecthypercoagulability. It is inferior to thrombingeneration B-7 although recently a modifica-tion designed for hypercoagulability has beendescribed.8

The thrombin generation test was originallydevised for studies on hemophilia.9"1' Thetest was subsequently used to study clottingabnormalities in cirrhosis of the liver andother diseases,12'13 iron loading deficiencies,14

and Hageman factor deficiency.15 We haveused it in the method described below tostudy the neutralizing effect of the humanprocoagulant from urine on circulating anti-coagulants.10- 1T It was instrumental in the dis-covery of spontaneous clotting fluctuations incertain patients including those subjected tomoderate treadmill stress.1--

From the Department of Medicine, University ofColorado School of Medicine, Denver, Colorado.

Supported by Crant HE-05538 from the NationalInstitutes of Health, U. S. Public Health Service.

Received for publication October 18, 1963.

436

We have found that thrombin generation,after considerable modification and strictstandardization, is a promising test for detect-ing various degrees of both hypo- and hyper-coagulability. Hypercoagulability is definedhere as an increase of the in vitro-clottingkinetics brought about by endogenous varia-tions of the patient's clotting system. Thesevariations may or may not reflect in vivohypercoagulability predisposing to intravas-cular clotting. In the present investigation,thrombin generation was used to study clot-ting deviations in cardiac patients subjectedto controlled stress.

MethodsPRINCIPLE

The concentration of thrombin, which rises andthen falls during the clotting of recalcified citratedplasma, is measured by transferring serial sam-ples from the recalcification mixture to a fibrino-gen solution and recording the resulting clottingtime of this solution. The speed with which thefibrinogen solution clots depends upon the amountof thrombin present in the aliquot of recalcifica-tion mixture transferred into it.

THROMBIN GENERATION TESTEquipment

1) International centrifuge, clinical model CL.2) All-glass thermostated waterbath without stir-rer set at 37°C and equipped with a rack to hangthe test tubes in the water. 3) Multifit syringeswith straight metal tip, 5 ml and 10 ml (Luer-Lokis unsatisfactory). 4) Yale disposable hypodermicneedles, 20 gauge, VA inches long. 5) Two stopwatches. 6) Wooden applicator sticks. 7) Centri-fuge tubes, 12 ml, heavy wall. 8) Test tubes withrim, 10 X 75 mm, and 13 X 100 mm. 9) 1 ml/100 and 0.2 ml/1000 pipettes, serological. 10)Tissue paper. 11) Surgical sponges, 4" X 4".Syringes and centrifuge tubes but not the needlesare siliconized before use (silicone gas method).18

Reagents

1) Sodium citrate, USP, 3.8%. 2) 0.5 M calciumchloride. 3) Buffered saline, pH 7.42, consistingof four parts 0.85% sodium chloride and one part

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THROMBIN GENERATION 437

barbital acetate buffer, pH 7.42.19 4) Fibrinogen(bovine fraction I, Armour) solution, 1%, in buf-fered saline. For each ml solution, 1 mg BaSO4is added and the solution is stirred several timesat room temperature, centrifuged clear, and thesupernatant stored in a freezer in aliquots of 1 ml.

Performance of the Test

Step One. Blood is drawn by a two-syringetechnique. A 4 X 4 gauze pad is placed on thearm where the hub of the needle will be. Im-mediately after the tourniquet has been applied,a venipuncture is performed, a few ml of bloodwithdrawal, the needle left in the vein, and thesyringe removed. The blood should now flowfreely from the hub of the needle. The test sampleis withdrawn to precisely the 10 ml mark of a10 ml syringe which has previously been filledwith 2 ml of sodium citrate, 3.8%, with exclusionof air. Syringe and needle are removed from thevein, the needle taken off the syringe, the barrelmoved one-quarter inch back, and the contents ofthe syringe mixed by tilting it gently back andforth. All these steps are performed in rapid suc-cession. If difficulty is encountered with thevenipuncture, the tourniquet is released for about30 seconds after the first syringe is in place andthen reapplied before the second syringe is filled.The patient must not "pump" his hand but onlyclose his fist tightly during the procedure.

Thrombin generation must be carried out with-in 30 minutes after shedding of blood. The fresh,noncentrifuged specimens are kept at room tem-perature until they are tested.

Step Two. Centrifugation. The content of thesecond syringe is carefully emptied into a sili-conized 12 ml centrifuged tube and centrifugedfor exactly five minutes at full speed (1470R.C.F.). Since the test is also sensitive to changesin platelet concentration, correct centrifugation isa critical procedure.

Step Three. Preparation for thrombin genera-tion. In the center of the test tube rack suspendedin the prewarmed thermostated waterbath areplaced seven 10 X 75 mm tubes. One-tenth mlof fibrinogen solution is pipetted into each tube.Then a new nonsiliconized, 13 X 100 mm tube,which has been rinsed previously with distilledwater and dried, is placed in one corner of therack. One ml of plasma from the upper third ofthe plasma phase of the centrifuged specimen iscarefully pipetted into this tube with the 1 mlnonsiliconized pipette without allowing the plas-ma to run down the side of the tube. The plasmais incubated for one minute (not longer).

Step Four. Thrombin generation. After this min-ute, using a 0.2 ml pipette, 0.1 ml 0.5 M CaCl2 iscarefully blown into the citrated plasma, and astop watch is started. After exactly four minutes,


0.1 ml of the recalcified plasma is transferred witha 0.2 ml pipette into one of the small test tubespreviously filled with fibrinogen solution. To pre-vent technical error it is essential to wipe off theend of the pipette with tissue paper after thealiquot has been drawn up and to avoid excessiveblowing when expressing the sample from thepipette.

The pipette should reach the bottom of thetube and at this very instance a second stop watchis started, always leaving the first watch running.The time required by the fibrinogen solution toclot is determined by slowly and rhythmicallytilting the tube which is dipped again into waterat 37°C during each back and forth motion. Theendpoint, when the first fibrin strands are visible,is quite sharp. A globular mass forms rapidly whenthe time interval is short. The procedure is re-peated at the 8th, 12th, 16th, 20th, 24th, and28th minutes of incubation.

When a visible fibrin clot is formed in the re-calcified plasma, a wooden applicator stick ispierced into the clot and the clot wrapped aroundthe stick by rotating it with some pressure alongthe wall of the tube. Clot and stick must be leftin the tube. At the beginning and the end of theincubation period, very small amounts or nothrombin at all might be present in the recalcifiedplasma, hence, transfer of this material to thefibrinogen solution results in a very long or in-definite clotting time. For practical reasons, theclotting time of the fibrinogen solution is not fol-lowed beyond 120 seconds. The peak thrombingeneration in normal plasma occurs around the12th or 16th minute, producing a clotting timeof the fibrinogen solution of 20 seconds or less.

In plasma with poor thrombin generation, aswith hemophilia or with inhibitors interferingwith intrinsic thromboplastin (formation), a clotmay not appear in the recalcified plasma; how-ever, some more or less retarded thrombin gen-eration will result.

Precautions

Strict adherence to the details outlined above isessential to avoid faulty results. The main sourcesof error are storage, uncontrolled contact withglass, alterations of the platelets, and silicone-contaminated recalcification test tubes. It is notnecessary to perform the test in the fasting patientbut fatty meals or exercise are to be avoided.

Method of Graphing. Normal Thrombin Generation Curve

The thrombin generation curve representingthe average values from 25 normal individuals isshown in figure 1. The typical pattern is that ofan increasing yield of thrombin, indicated byshortening of the clotting times of the fibrinogensolution to which an aliquot of the recalcification


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FIGURE 1

Average values of 25 normal thrombin generationdeterminations: black line. Spread of the highest andlowest values encountered: shaded area. Abscissa:incubation time after which an aliquot of the recal-cified plasma is transferred to the fibrinogen solution.Ordinate: clotting times of fibrinogen solution broughtabout hy an aliquot transferred from the recalcifiedcitrated plasma at the incubation times indicated onthe abscissa.

mixture was transferred, at the incubation inter-vals indicated on the abscissa, followed by agradual disappearance of the generated throm-bin, as incubation continues. This normal graph,together with the ranges of the normal values, is,however, slightly misleading because the peakof normal thrombin formation (shortest clottingtimes of the fibrinogen solution) is generallyreached at the 12th or 16th minute but not atboth intervals. By calculating the average valuesa thrombin generation curve resulted which is flatbetween these two points. This is not actually thecase in most instances. In normal individuals thereis no peak at the 8th or 20th minute.

A clot is always formed in the recalcified plasmabefore the maximum thrombin yield is obtained,except with very pathological specimens. Wefeel that the initial descending portion of thethrombin generation curve is the most importantfor diagnostic purposes since its course is deter-mined predominantly by the rapidity and quantityof thrombin formed, and to some extent by re-moval of thrombin. The diagnostic significance ofthe subsequent ascending portion, which is de-termined predominantly by the rate of disappear-ance of the generated thrombin, is less clear atthe present time. We have noted, however, thatthe disappearance rate is frequently much fasterin samples in which a high yield of thrombin isgenerated quickly.

We do not interpret rapid removal of thrombinfrom the generation mixture, after a large throm-bin yield has been generated previously, as anindication of hypocoagulability. The high throm-bin concentration is present for a certain length oftime (permitting its assay) during which it in-teracts with fibrinogen. Its subsequent removalhas no bearing on the process of fibrin formation.In contrast, thrombin removal, occurring so rapid-ly that its concentration remains low, could bethe cause of hypocoagulability.

Effect of Dilution

The thrombin generation test is most sensitivewhen the sample is not diluted. With the methoddescribed here, the dilution of the sample by re-calcification is only 10%, in contrast to previousmethods in which the dilution was 50% or greater.The differences in thrombin generation are quitemarked; more thrombin is generated faster in thediluted sample. Two examples shown in figure 2demonstrate that the dilution effect on thrombingeneration masks the one important deviation fromthe normal which the thrombin generation test

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FIGURE 2

Acceleration and enhancement of thrombin generation by dilution of the plasma sample, solidline = diluted 10% by recalcification; broken line = diluted 50% by recalcification. A: plasmawith normal thrombin generation. B: plasma ivith reduced thrombin generation. For furtherlegend see figure 1.



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should detect, — the faster and greater thrombingeneration with hypercoagulability.

Effect of Storage

Blood or plasma samples for thrombin genera-tion cannot be stored, even in siliconized tubes.The changes in the thrombin generation patternwhich occur on standing of the sample vary con-siderably from plasma to plasma but, in any case,are markedly enhanced by contact with glass. Ingeneral, there is more rapid thrombin generation,except for longer preincubation at 37°C. Freezing,followed by thawing, induces extreme shifting ofthe generation pattern. This particular conditionis shown in figure 3 which represents the delayedand deficient thrombin generation pattern of athrombocytopenic (platelet count of 40,000) plas-ma and the marked alterations induced by freez-ing and thawing of the sample. The fresh samplereflects correctly the defective thrombin genera-tion as compared with the frozen sample in whichthe defect is entirely masked.

Reproducibility

The reproducibility of the thrombin generationpattern is excellent both with regard to duplicatesfrom the same plasma sample and tests done atintervals in the same individual with a stable clot-ting system. Figure 4 shows the thrombin genera-tion curve obtained from two normal individualson separate occasions. The graph at the left in-cludes duplicate tests performed on the same daywhich are practically identical.

Hypercoagulability

The combination of accelerated generation andincreased yield of thrombin as a form of hyperco-agulability results in a very characteristic pattern.The accelerated thrombin generation is manifestedby a relatively short fibrinogen clotting time at the4th minute and the peak values (shortest clottingtimes of fibrinogen solution) at the 8th minute.This is in contrast to normal samples in whichvery long (over 100 seconds) or no values are ob-

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FIGURE 3

Thrombocytopenic plasma. Thrombin generation. I:fresh sample. II: sample frozen for 24 hours. Forfurther legend see figure 1.

tained at the 4th minute and in which the peakvalues occur at the 12th or 16th minute. Figure 5represents some characteristic thrombin genera-tion patterns of patients with suspected in vivo hy-percoagulability associated with various diseases.The patterns are distinctly different from normalcurves. The reproducibility of thrombin genera-tion is again demonstrated by the curves la andlb obtained on different days from a patientwith idiopathic pulmonary hypertension.

Subjects

It is possible to demonstrate transitory hyper-coagulability patterns by thrombin generation,provided the samples are properly timed. In ourinitial attempt to study variations in coagulabilityin humans under physiologic conditions, stresswas applied in the form of a standardized treadmillexercise. Individuals with cardiac diseases wereselected because we have previously noted thatthey frequently have an unstable clotting system.Most of these patients were evaluated by theWork Classification Unit of the Colorado HeartAssociation. Their functional reserve was assessedby observing the effect of a mild treadmill stress

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FIGURE 4

Reproducibility of thrombin generation pattern in normal individuals. For further legend see figure 1.

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FIGURE 5

Hypercoagulahility patterns of thrombin generation ofpatients with severe primary (idiopathic) pulmonaryhypertension (la, lb); ihromhocythemia (800,000) withrecurrent thrombophlebitis (2); recurrent mijocardialinfarction (3); thrombocythemia (500,000) (4); throm-bocythemia (480,000) with recurrent pulmonary in-farction (5). Platelet count for la, lb, and 3 wasnormal. Note early onset of thrombin formation, earlypeak of maximum thrombin yield.

on cardiovascular function. Serial thrombin gen-eration tests were carried out simultaneously. Theduration and intensity of the treadmill exercisewere adapted to the individual's capacity, butthe original protocol was designed as follows:six minutes at three miles per hour at a gradient

of 4°; six minutes rest; six minutes at three milesper hour at a gradient of 8°; six minutes rest;six minutes at three miles per hour at a gradientof 12°. Thrombin generation tests were performedgenerally before the treadmill exercise, within oneminute after completion of each phase, and, inaddition, thirty or fifty minutes after performanceof the last phase.

ResultsTREADMILL; NORMAL INDIVIDUALS

Serial thrombin generation curves from anormal healthy male before, during, and aftertreadmill stress are shown in figure 6 as arepresentative example. There are at no timegross deviations from the normal as are foundin patients described below. There is a sug-gestion of an earlier onset of thrombin genera-tion after the second treadmill phase and onehour later. The rapid disappearance rate ofthrombin from the pretreadmill control sam-ple, which is occasionally seen in normalindividuals, is slowed down by stress.

TREADMILL; EBSTEIN'S ANOMALY

Serial thrombin generation curves from apatient with Ebstein's anomaly are shown infigure 7. In this instance a pattern of normalcoagulability which existed before the stress isconverted stepwise into patterns of a marked

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FIGURE 6

Normal male subjected to treadmill exercise. Serial thromhin generation tests. Control: beforetreadmill; treadmill 1: after the first two phases; treadmill 2: immediately after the third phase;1 hour later: 1 hour after the third phase. For further legend see figure 1.



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hypercoagulability during the stress; thechanges persisted for at least thirty minutesafter termination of the exercise.

TREADMILL; TETRALOGY OF FALLOT

Serial thrombin generation curves from apatient with tetralogy of Fallot during tread-mill stress are shown in figure 8. They providean instructive example of an unstable clottingsystem as reflected by thrombin generation.This patient initially exhibited a delayed andreduced thrombin generation. After the firsttreadmill phase, no measurable thrombin wasgenerated; after the second phase, thrombingeneration was nearly normal. After the thirdphase, there was a swing back to markedhypocoagulability which was more pro-nounced fifty minutes after the third phase.The patient also exhibited considerable pro-longation of the thrombin time and a marked-ly increased fibrinolytic activity throughoutthe test. Other patients with tetralogy ofFallot did not exhibit such marked alterationsof thrombin generation.

TREADMILL; RHEUMATIC HEART DISEASE

Changes in the coagulability of humanblood induced by mild treadmill stress arewell demonstrated in individuals with a re-duced ability to generate thrombin. The fol-

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FIGURE 7

Ebstein's anomaly. Treadmill stress. Serial thrombingeneration tests. Control: before treadmill; treadmill1: after the first treadmill phase; treadmill 2: imme-diately after the second phase; 30 minutes later: 30minutes after the second and last phase. For furtherlegend see figure 1.

lowing two examples were obtained from pa-tients with rheumatic heart disease. Figure 9shows serial thrombin generation curves froma patient with aortic insufficiency. Thrombingeneration was delayed markedly before thetreadmill exercise. After each of the twophases the thrombin generation curve wasprogressively shifted toward normal; thirtyminutes after treadmill exercise the curve wasentirely normal. The thrombin generation pat-tern in this patient during and after treadmill

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FIGURE 8

Tetralogy of Fallot. Treadmill stress. Serial thrombin generation tests. Control: before tread-mill. After first treadmill phase (treadmill 1) no measurable thrombin was generated. Tread-mill 2: after second phase. Treadmill 3: after third phase. Fifty minutes later: fifty minutesafter third phase. For further legend see figure 1.



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FIGURE 9

Rheumatic heart disease with aortic insufficiency. Treadmill stress. Serial thrombin generationtests. Control: before treadmill. Treadmill 1: after first phase. Treadmill 2: immediately aftersecond phase; 30 minutes later: 30 minutes after second phase. For further legend see figure 1.

stress suggests that a reduced ability to gen-erate thrombin during rest does not preventhim from rapid adaptation with exercise ofhis clotting system toward normal or even tohypercoagulability.

This observation applies also to patients be-ing treated with prothrombin-depressingagents (fig. 10). Serial thrombin generationcurves were determined on a patient withmitral valvulitis and pulmonary hypertension.The patient's prothrombin activity was re-

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FIGURE 10

Coumadin-treated patient with mitral valvulitis andpulmonary hypertension. Prothrombin 17%. Treadmillstress. Serial thrombin generation tests. I: beforetreadmill; II: after two minutes on treadmill; N: aver-age of 25 normal thrombin generations (for compari-son). For further legend see figure 1.

duced therapeutically to 17% of normal (one-stage method). This reduction is reflected ina diminished thrombin generation ( I ) . Thepatient walked only two minutes on the tread-mill before he was forced to stop because ofa pounding heart. These two minutes ofmoderate exercise, however, were enough toinduce a marked shift of the thrombin gener-ation pattern toward normal values ( I I ) .Treatment with Coumadin did not prevent theshift.

TREADMILL; MYOCARDIAL INFARCTION

In figure 11 are reproduced serial thrombingeneration curves obtained during treadmillstress from two patients who had recoveredfrom myocardial infarctions. Neither wastreated with prothrombin-depressing agents.Patient A, on the left side of the figure, did notexhibit marked changes of his thrombin gen-eration pattern following exercise. This is incontrast to the thrombin generation pattern ofpatient B, on the right side, who exhibited amoderate delay of thrombin generation beforetreadmill exercise; the curve shifted alreadywith the first treadmill phase into the normalrange and remained there after the second andfinal phase.

Patients with previous myocardial infarction



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FIGURE 11

Two patients with previous myocardial infarction. Treadmill stress. Serial thromhin generationtests. 1: control before treadmill; 2: after first phase; 3: after second phase; 4: after thirdphase. For further legend see figure 1.

who are treated with prothrombin-depressingagents, such as Coumadin, are definitely notprotected, in all instances, against the develop-ment of hypercoagulability as defined in theintroduction. Figure 12 shows serial thrombingeneration curves during treadmill stress in apatient with previous myocardial infarctionwho was treated inadequately with Coumadin.The prothrombin activity was 42% and, con-sequently, thrombin generation was not verymarkedly reduced. Thrombin generation wasnot altered to any extent during the threetreadmill phases; in particular, no hypercoag-ulability was induced by the stress despiteinadequate reduction of prothrombin activity.

The patient whose thrombin generationcurves are shown in figure 13 behaved quitedifferently during exercise. He also had hada previous myocardial infarction and wastreated with Coumadin, the prothrombin activ-ity being reduced to 33%. As a result, the pa-tient's thrombin generation was moderatelydelayed and lessened. With the treadmillstress, however, the thrombin generation isaccelerated and the thrombin yield increased.Immediately after the third treadmill phase(IV), there was a clearcut picture of hyper-coagulability with the peak thrombin activityoccurring at the 8th minute; at this time, thepatient experienced angina. Thirty minutesafter the third treadmill phase, there was ashifting back of the thrombin generation pat-tern toward the pretreadmill hypocoagulability

values. It should be emphasized that, in con-trast, serial prothrombin determinations withthe conventional one-stage method did notshow any changes of the prothrombin timebefore, during, and after this treadmill experi-ment, not even at the time of the acceleratedand increased thrombin generation. This is agood example showing that the prothrombintime does not reflect the actual clotting condi-tion.

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FIGURE 12

Coumadin-treated patient with previous myocardialinfarction. Prothrombin 42%. Treadmill stress. Serialthrombin generation tests. I: before treadmill; II:after first treadmill phase; HI: after second phase; IV:immediately after third phase; V: 30 minutes afterthird phase. For further legend see figure 1.

Discussion

Thrombin generation is a screening test,



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FIGURE 13

Coumudin-treated patient with previous myocardialinfarction. Prothrombin 33%. Treadmill stress. Serialthrombin generation tests. 1: before treadmill; 11:after first treadmill phase; 111: after second phase; IV:immediately after third phase; V: 30 minutes afterthird phase. N: Average normal thrombin generationcurve from 25 individuals. For further legend seefigure 1.

with the outcome determined by the activitiesof many clotting factors. One of its distinctadvantages is that the results are expressedas a curve based upon a series of measure-ments. Thus, a more comprehensive analysisof an individual clotting situation is obtainedthan when a single measurement is used. An-other decisive advantage of the thrombin gen-eration test is that the relationship betweenthe factors promoting and inhibiting clot for-mation is not changed. The test circumventsthe potential physiochemical alterations ofclotting factors which might occur when plas-ma has to be diluted, incubated, stored, ab-sorbed, and otherwise handled. Consequently,variations in coagulability can be measuredwhich cannot be detected by other methodsin which the very technique employed mayartificially produce hypercoagulability. Wehave repeatedly observed that uncontrolledglass contact, dilution, and storage inducealterations which make comparison of minorchanges, from sample to sample, difficult.Similar observations have already been report-ed with a less sensitive thrombin generationtechnique.20 The acceleration of coagulationby dilution of the sample is a well establishedfact; the enhancement of thrombin genera-tion by dilution is a particular example ofthis general rule. Dilution of plasma with

equal amounts of saline increases the thrombinyield of the mixture two- to threefold.21 It hasbeen suggested that the protein concentrationin undiluted plasma prevents maximum throm-bin generation, thereby acting as a nonspecificprotection against intravascular coagulation.-1

Our study has shown by serial thrombingeneration tests that fluctuations of the clot-ting system occur in certain cardiac patients.Causes for the fluctuations are unknown, butmight be clarified in the future by additionalstudies during the course of the disease.

Hypercoagulability in patients with previ-ous myocardial infarctions is of great practicalinterest. Based on our limited experience,there are two kinds of hypercoagulability inthese patients: a) that which is already pres-ent at the time of the test (which is performedunder resting conditions in most instances),and b) that not present at the time of the testbut which is induced by physical exercise (orother stimuli). Hypercoagulability at the timeof the test (which was not always performedunder resting conditions, but in absence ofexercise) was found in patients with myocar-dial disease by several investigators who wereusing primarily the heparin-retarded clottingtime or the thrombelastograph.3 Hypercoagu-lability, however, was not present in all in-stances. The use of other methods, such asthe thromboplastin generation test, was lessrevealing.22 The coagulability of patients withprevious myocardial infarction after standard-ized physical exercise has not been studiedpreviously. Yet there are compelling reasonsfor intensive investigations in this regard.First, it is known that the antihemophilicglobulin, AHG, might be present in abnormal-ly higher concentrations in patients with cor-onary artery disease, thus predisposing themto thrombosis at the site of local vasculardisease.2" The average higher level of AHG inpatients with coronary atherosclerosis is notinfluenced by prothrombin-depressing agents.24

Second, the antihemophilic globulin concen-trations in normal persons increase substan-tially after short but marked physical stress.25

The platelet count increases after physicalstress.26'27 The effect of exercise on otherclotting factors is known to be more erratic.20

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The increase of AHG and platelets might play arole in the hypercoagulability observed by uswith the thrombin generation test in patientswith myocardial infarction. The increase ineither or both of these clotting componentswould explain at least partially the absence ofprotection against the development of hyper-coagulability in some of these patients treatedwith prothrombin-depressing agents, becausethese agents have no influence on AHG andplatelets and their accelerating effect on pro-thrombin conversion. One might speculatewhether a clearcut hypercoagulability has tobe obtained in order to set the stage for theformation of intravascular clots or whether arapid shift of the coagulability from the lowrange to normal as in the patient of figure 10or the patient B of figure 11 has the same po-tential effects. We are inclined to believe inthe latter possibility.

Summary

A refined, reliable thrombin generation testwith good sensitivity for both hypo- and hyper-coagulability is described. When serial throm-bin generation curves were obtained in cardiacpatients subjected to standardized treadmillstress, fluctuations of the clotting systemwere observed in some and a marked tendencyto hypercoagulability in others. The relation-ship of in vitro hypercoagulability as reflectedby thrombin generation to an increased ten-dency to thrombosis remains to be determined.

References1. VON KAULLA, K. N., AND VON KAULLA, E.:

Thrombin generation. A sensitive test forphysiologic variations in the coagulabilty ofhuman blood. J. Lab. Clin. Med. 56: 955,1960.

2. vox KAULLA, K. N., AND VON KAULLA, E.: Spon-tane Schwankungen der Gerinnbarkeit men-schlichen Blutes in vivo. Med. Welt no. 36,1850, 1961.

3. BARBANO, G.: L'emocoagulazione nell'infartomiocardico recente. Therapeutikon 1961, p. 1.

4. MARCHAL, G., LEROUX, M. E., AND SAMAMA, M.:Atlas de Thrombodynamographie. Service dePropagande Edit., Inform. Paris.

5. VON KAULLA, K. N.: Continuous automatic re-cordings of fibrin formation and fibrinolysis: A

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valuable tool for coagulation research. J. Lab.Clin. Med. 49: 304, 1957.

6. OLLENDORFF, P.: Defects in and variability ofthe thromboplastic system in horse plasma.Thromb. Diath. Haemorrhag. 4: 45, 1959.

7. KEENEY, C. E., AND LARAMIE, D. \V.: Effect ofexercise on blood coagulation. Circulation Res.10: 691, 1962.

8. THOMPSON, J. H., OWEN, CH. A., SPITTEL, J. A.,AND PASCUZZI, C. A.: The "retarded" thrombo-plastin generation test. Its use in the study ofaccelerated blood coagulation. Am. J. Clin.Pathol. 37: 63, 1962.

9. MACFARLANE, R. G., AND BIGGS, R.: A thrombingeneration test. The application in hemophiliaand thrombocytopenia. J. Clin. Pathol. 6: 3,1953.

10. PITNEY, W. R., AND DACIE, J. V.: A simplemethod of studying the generation of thrombinin recalcified plasma. Application in the in-vestigation of haemophilia. J. Clin. Pathol. 6:9, 1953.

11. SJ0LIN, K. E.: Haemophilic Disease in Denmark.Oxford, Blackwell Scientific Publications, 1959.

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Circulation Research. Volume XIV, May 1964


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KURT N. VON KAULLA and EDITH VON KAULLAThrombin Generation in Normal Subjects and Cardiac Patients

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