Activity of antithrombin III and effect of heparin on coagulation in shock

THROMBOSIS RESEARCH 19; 775-782, 1980 Printed in the USA. All rights reserved.

ACTIVITY OF ANTITHROMBIN

III

0049-3848/80/180775-08$02.00/O Copyright (c) 1980 Pergamon Press Ltd

AND EFFECT OF HEPARIN ON COAGULATION IN SHOCK

B. Blauhut, S. Necek, H. Kramar, H. Vinazzer and H. Bergmann Department of Anaesthesiology/Surgical Intensive Care Unit, General Hospital, Linz, and Ludwig Boltzmann Institute for Experimental Anaesthesiology and Intensive Care Research (Head: Prof.Dr.H.Bergmann) and Blood Coagulation Laboratory, Linz (Head: Prof.Dr.H.Vinatzer)

(Received 29.4.1980; in revisea form 7.8.1980. Accepted by Editor A. Henschen)

ABSTRACT

In 16 patients admitted for DIC and shock,series of coagulation testswere carriedout prior to and during heparin therapy. Plasma heparin concentrationsmeasuredwerecorrespondingtoexpected levels. In some cases however,considerably higher or lower levels were found. Thiscouldbepartlyexplained by release of PF 4 from platelets and by a decreased turnoverofheparinbecauseofamalfunction-of the kidneysoroftheliver. Arelation betweentheeffectof heparinon coagulation and the activityofantithrombin III couldbe established.Adiminution of this activity resultedinadiminishedorevenmissing effectof heparin. In someinstances,thePTTandthethrombin clotting time were considerably prolongedwhen FDPwere presentorwhen procoagulant factorswerediminished.These tests therefore,did not reflect true heparin concentrations in shock. For this reason,regular assays of antithrombin III and of heparin are proposed.

INTRODUCTION It is a well known fact that the effect of heparin on coagulation largely depends on the activity ofantithrombin III (AT III). An increased heparin toleranceofthe clottingmechanismwas found in congenital AT III deficiency (1,2,3,4,5,6,7,8). Moreover,adiminutionof the effectofheparin could also be demonstrated in comparativelymild cases of diminishedAT III activity (9,lO). In cases of shockand DIC,adiminutionof the activityofAT IIIwas repeatedly observed,too (10,11,12,13). Thelatterisofspecial clinical importance since hepariniswidely usedin shock in order to prevent or to control DIC. For this reason,a series of parameters of coagulation was evaluated in 16 consecutive patients who were admitted to the Intensive Care Unit. Key words:

Heparin, Antithrombin III,

DIC, shock 775

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MATERIALS AND METHODS Heparin was obtained by Immune AG,Vienna. The solution contained 1,000 I.U. sodium heparin per ml,mean MW 15,000 daltons. Thromboplastin was from DADE, Berne,Switzerland. PTT reagent was Actin activated cephaloplastin from DADE,Berne,Switzerland. Thrombin: Topostasin from Hoffmann-La Roche,Basle,Switzerland.One vial contained 3,000 NIH units of the lyophilized substance. This was dissolved in 30 ml sterile distilled water to obtain a stock solution which was frozen in 1 ml portions at -230C. The undiluted stock solution was used for fibrinogen assays whilst thrombin clotting times were carried out after dilution of lpart of the stock solution with 49 parts isotonic saline pH 7.35. Thrombin was thawed and diluted immediately prior to its use. Thrombin coagulase was from Boehringer Mannheim, FRG. Reagents for assays of factors II,V,VIII,IX and X were from DADE,Berne,Switzer land. Reagents for assays of AT III and of heparin (Coatest AT III and Coatest Heparin) were a gift from AB KABI Diagnostica, Stockholm,Sweden. Coagulometer: by Depex,De Bilt,Netherlands Photometer: S 100 by Labtronic,Zurich,Switzerland.This photometer was connected to a Diehl Certatronic calculating and printing unit. Platelet count: Brecher and Cronkite (14) Thromboplastin time: Quick (15) activated PTT: Proctor (16) Thrombin clotting time: Perlick (17) Thrombin coagulase time: Soulier (18) Fibrin monomer complexes: Godal (19) Fibrinogen: Clauss (20) Assays of factors II,V,VII,IX,and X: One-stage assays based on thromboplastin time, PTT,and Stypven time respectively. Antithrombin III: Wegaard (21) Heparin assays: Teien and Lie (22) PF 4 in plasma: Vinazzer (23). PF 4 in plasma was expressed as a percentage of the he arin neutralizing capacity of sonicated platelet rich plasma containing 3 x 101 platelets per ml. PATIENTS All patients were admitted in shock caused by a severe trauma,by septicemia or by a surgical procedure. Details are listed in table I. Immediately after admission,blood samples were collected for coagulation studies. Incipient or For this reason,heparin was progressed DIC was found in all cases (table II). included into the therapeutic procedures.The drug was administered in three different schemes of dosage depending on the individual circumstances. After an initial i.v. dose of 2,500 ( 2,000 ; 1,300) I.U.,heparin was given by continuous intravenous infusion of 500 ( 334 ; 250 ) I.U. per hour. Further blood samples were collected 1 hour after the initial heparin dose and at least twice per day as long as heparin was continued. The following tests were carried out in all samples: Platelet count, thromboplastin time (TPT), activated partial thromboplastin time (aPTT), thrombin clotting time (TCT), fibrinogen, thrombin coagulase time for the detection of split products ( TC), ethanol gelation test (EGT), antithrombin III (AT III), and heparin assays. In addition,plasma antiheparin levelswereassayed in the first samples. Factors II, V, VIII, IX and X were assayed when the results of aPTT's were not explainable by the level of heparin nor by the presence of FDP.

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TABLE I Patients and Diagnoses Name

Sex

H.L.

f f m f

:*;. M:H: J":w":

Cause of Shock thoracic and abdominal trauma,ruptured liver right hemicolectomy,peritonitis multiple fractures,hemopneumothorax subdural hematoma,cerebral edema acute pancreatitis fracture of base of skull abdominal trauma,ruptured spleen ruptured spleen and liver,septicemia hemangioma thoracic wall,intraoperative shock craniotomy for astrocytoma,postoperative shock serial rib fractures,ruptured spleen and liver septicemia nd severe burn, 80% of skin, 2 and 3rd degree gastric resection for cancer,splenectomy,shock ruptured spleen and liver,pancreatitis liver cirrhosis,hemorrhage from esophagus

f' m m f m m m m m m m

;1+? v:s: K J:D: if?;. E:P: W.B.

TABLE II Parameters of Coagulation prior to Onset of Therapy Name

H"+* E:G: J":;: :i?!. A:M: V.S.

TPT %

Plat. x 103 4"; 140 188 247 85 82

K:

;: 118 125

z:

;:

:*;* W:B:

1;: 58

Fibr. mg/dl

phase of shock

540 620 180 165 240 310 160 450 265 530 140 390

:: IV III II III III

4:: 325 195

I; III IV

III

I

f :I

Abbreviations are explained on preceding page. Phases of shock: I initial; II progressive; III advanced; IV irreversible. Parameters of coagulation, limits of 95% confidence: PTT 26 - 33 set; TPT 78122%; platelets (normal range) 150 - 350 x 103; fibrinogen 240 - 380 mg/dl; TCT 16 - 19 set; TC 16 - 18 set; PF 4: normally absent in plasma; AT III 84 - 116%.

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III AN0 HEPARIN

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RESULTS Levels of heparinmeasuredwere comparedtolevels calculated from thei.v. dose, the plasma volumeandtheaveragehalflife ofheparin in circulation.Forcalculationsofheparinlevelsduringcontinuous infusions,thefollowingformula was used: Heparin/ml =

units/h x 75 x 2 60 x V

V: plasma volume in ml; 2: constant factor when the equilibrium is reached; 75:60 : correctionfactorforhalflifeof heparin incirculation which is 75 min. The results are shown in table III. TABLE III Heparin Levels Found and Heparin Levels Expected 1 hour after the First Intravenous Dose and During Continuous Intravenous Infusion.

lSt dose ,

I**U

2,500 2,000 1,000

n

: 4

continuous dose/h 500 334 250

59 2"':

heparin measured I.U./ml plasma 11 1 SD

heparin calculated I.U./ml plasma M 1 SD

0.59 0.47 0.30

0.18 0.12 0.08

0.60 0.48 0.26

0.08 0.07 0.04

0.34 0.18 0.13

0.22 0.05 0.09

0.31 0.21 0.15

0.04 0.03 0.02

For reasons of comparison between the activity of AT 111,the heparin level and the effect of heparin on coagulation,plasma samples were grouped according to their AT III activities. The following groups were formed: AT III,% 81 and above 71 to 80 61 to 70 51 to 60 50 and below

6;:

9: 8

12.0 1 SD

::

63.8 74:o

;::

::

40.1 54.8

:::

In some instances,the two groups with the lowest activities of AT III were evaluated together. The activity after this combination was 46.0 2 11.4%. the activity The dependence of TCT'sand PTT's on heparin levels as well as on ._ -__ of AT III is shown in figure 1. Thelinearity ofthedependence on AT III was examined by the followingmethod: Theminimal concentrations of heparin which were requiredtoprolongtheTCTandthe PTT justbeyondthe limits of confidence (i.e. a TCTof20 secanda PTT of 34 sec)were compared to the grouped activities of AT III. The results are demonstrated in figure 2. Though a linearity between AT III andtheeffectofheparin was demonstrable,a considerable number ofresultsdid notcorrespondto the heparin- and AT III levels. This phenomenon was most comnon when AT III activities were low,and it was most frequently observed when PTT'swereexamined. Thisobservationis shown in figure 3. The reasonsforthis phenomenonwerethe presence ofFDPand in one case an extremely lowleveloffibrinogenwhen TCT's wereexamined. A diminutionoffactors I, II,V,VIII,IXorXorthepresenceof FDPwerethereasonsfor irregular PTT's.

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III

TCT

SW.

PTT

AT III ?? i.

100 :i/’ ’

779

AND HEPARI N

AT III%

96

70 50 LO

20 30

/

5’

.i

/ 4s .3

,111,

.L .5

1

.7 I

.; Heparin I.U. per ml plasma

‘.i’

.; Y-5 ’.;

Heparin 111. per ml plasma

FIG. 1 Influence

of

AT III

Activity on the Effect of Heparin on the TCT and on the PTT

JO0 80 : 60 1 50 : LO -

.l

.i

’.i .i ‘.; ‘Y7

Heparin I.U. per ml plasma 0 TCT, A PTT FIG. 2 Minimal Concentration of Heparin which Prolongs the TCT and the PTT at Different Levels of AT III

The following clinical results were obtained in these patients: In 11 out of 16 cases,shock and DIC could be brought under control. One of these patients however,died from her severe injury of the brain after the circulation was stabilized ( M.H.). In five other cases,irreversible shock was diagnosed. All these patients died between the second and the fifth day after admission. It should be mentioned thdt AT III levels in these five cases further decreased during therapy whilst in all other cases an increase of AT III above 75% was observed after an average of 3.3 days.

ANTITHROMBIN

780

III

AND HEPARIN

TCT

.3

.L .5 .6

#

I

.

..‘.

.6 .05 pdna .2 .3 .L .5 Heprrin I.U. per ml A low clotting factors AT 111::46.0 *- 11.&V.,n = 28, @ FDP detected,

.l

NO.6

PTT

I ) .2

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FIG.

I-

.7

1.0

3

False "Heparin Effect" at Low AT III Activity Caused by FLIPand by Diminution of Coagulation Factors

DISCUSSION Levels of heparin were assayed by a method which measured the residual factor Xa by means of a chromogenic substrate. This method was chosen because of the independence of the results from fluctuations of clotting factors,of AT III and of FDP (24). The mean heparin levels found by this method were almost identical with the mean levels expected. Calculations were made from the initial and continuous doses of heparin,from the factor of dilution in plasma,and from the average half life of heparin in circulation which is 75 min (25). Some of the individual heparin levels were however,considerably lower or higher than expected. Lower levels could be partly explained by an increased inactivation of platelet factor 4 which was released from platelets altered or destroyed by the process of DIC. This kind of liberation of PF 4 has been described earlier (23). Heparin levels higher than expected were found in some patients who suffered from a severe malfunction of kidneys and/or liver. It is known that the short half-life of heparin in circulation is partly caused by its rapid passage through the kidneys (26,27), and partly by its destruction in the liver (28). A cumulation of heparin was therefore,expectable in cases of severe kidney and/or liver dysfunction. The effect of heparin on clotting was a function of the activity of AT III. Since a diminution of AT III in progressive or in advanced shock almost regularly occurred in these patients,the effect of heparin expectable in healthy persons was frequently diminished or even missing. It is noteworthy that at 75% AT III activity more than twice as much heparin was needed to get the same response of the TCT and of the PTT as at 100% AT III activity. An apparently mild decrease of AT III had a surprisingly strong influence on the effect of heparin. A similar result was found in a different group of patients (2). Furthermore,discrepanciesbetween heparin levels,heparin effects and AT III activities were found in severe cases of shock and DIC. Considerably prolonged PTT's and TCT's were observed at low activities of AT III. The reasons were diminutions of plasmatic clotting factors and/or the presence of FDP.

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Though PTT's and TCT's in shock and DIC are most useful tests to get an overall information on the clotting mechanism,these parameters do not reflect the concentrations of heparin under these special conditions. As a consequence of these results,administration of heparin in shock without the knowledge of the activity of AT III should only be carried out in incipient cases for reasons of prophylaxis. In further advanced stages of shock,assays of AT III would be extremely helpful in the management of the situation. Assays of heparin levels on the other hand would help to prevent an over- or underdosage. At low activities of AT III, substitution therapy with the aim to keep this inhibitor above 75% of its normal activity would be likely to improve the therapeutic effect. REFERENCES 1 EGEBERG,O.: Inherited antithrombin deficiency causing thrombophilia. Thrombos. Diathes. haemorrh. -13, 516-530, 1965 2 LECHNER,K.,THALER,E.,NIESSNER,H.,NOWOTNY,Ch. and PARTSCH,H.: Antithrombin III-Mange1 und Thromboseneigung. Wien. klin. Wschr. -89, 215-222, 1977 3 MARCINIAK,E.,FARLEY, Ch. and DESIMONE,P.A.: Familial thrombosis due to antithrombin III deficiency. -Blood 43, 219-231, 1974 4 NAGY,I. and LOSONCZY,H.: The significance of the chronic anticoagulant treatment in recurrent thromboembolism caused by hereditary antithrombin III deficiency. Thrombos.Diathes.haemorrh.(abstract)-34, 366, 1975 5 ROSENBERG,R.D.: Actions and interactions of antithrombin and heparin. New Engl.J.Med. 292, 146-151, 1961 6 SAS,G.,BLASKO,G.,BANHEGYI,D.,JAKO,J. and PALOS,L.A.: Abnormal antithrombin III (antithrombin III "Budapest") as a cause of familial thrombophilia. Thrombos.Diathes.haemorrh. 32,105-115, 1974 7 SHAPIRO,S.S.,PRAGER,D. and MARTINEZ,J.: Inherited antothrombin III deficiency associated with multiple thromboembolic phenomena.Blood (abstract) -42, 1001, 1973 8 VAN DER MEER,J.,STOEPMAN VAN DALEN,E.A. and JANSEN,J.M.S.: Antithrombin III deficiency in a Dutch family. J.Clin.Pathol. -26, 532-538, 1973 9 CELLA,G. and RUSSO,R.: Heparin neutralizing activity (HNA) and antithrombin III in coronary artery disease. Thrombos.Haemostas. -38,696-700, 1977 10 THALER,E.: Disseminierte intravaskulare Gerinnung: Antithrombin III und Heparin. Folia Haematol. Leipzig 104, 740-750, 1977

11 LASCH,H.G., KRECKE,H.J.,RODRIGUEZ-ERDMANN,F.,SESSNER,H.H.and SCHDTTERLE,G. Verbrauchskoagulopathien (Pathogenese und Therapie) Fol.haemat.(Frankfurt) 2, 325-330, 1961

12 HENSEN,A. and LOELIGER,E.A.: Antithrombin III. Its metabolism and its function in

blood coagulation. F.K.Schattauer, Stuttgart 1963, p. 42

13 VINAZZER,H.: Die postoperative Thrombosebereitschaft. F.K.Schattauer St.uttg.2. Aufl. 1973, p. 29

14 BRECHER,G. and CRONKITE,E.P.: Morphology and enumeration of human blood platelets. J. appl. Physiol. 3, 365-377, 1950

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15 QUICK,A.J.: The hemorrhagic diseases and the ohvsioloov of hemostasis. Thomas, Springfield, 111: 1942, p. 312-313 ’ “” 16 PROCTOR,R.R. and RAPAPORT,S.I.: The partial thromboplastin time with kaolin. A simple screening test for first stage plasma clotting deficiencies. Amer.J.clin.Path.-36, 212-219, 1961 17 PERLICK,E. and BERGMANN,A.: Gerinnungslaboratorium in Klinik und Praxis. G.Thieme,Leipzig 1971, pp. 273-274 18 SOULIER,J.P., PROU,O. and HALLE,L.: Further studies on thrombin coagulase. Thrombos.Diathes.haemorrh.23, 37-49, 1970 19 GODAL,H.C. and ABILDGAARD,UT Gelation of soluble fibrin in plasma by ethanol. Scand.J.Haemat. 3, 342-350, 1961 20 CLAUSS,A.: GerinnungsphysiologischeSchnellmethode zur Bestimmung des Fibrinogens. Acta haemat. -17, 237-246, 1957 21 ODEGAARD, O.R.,LIE,M. and ABILDGAARD,U.: Heparin cofactor activity measured with an amidolytic method. Thromb. Res. 6, 287-294, 1975 22 TEIEN,A.N. and LIE,M.: Evaluation of an amidolytic heparin assay method: Increased sensitivity by adding purified antithrombin III. Thromb. Res. -10, 399-410, 1977 23 VINAZZER,H.: A simplified assay method for platelet factor 4 in plasma and in platelets with a chromogenic substrate. Haemostasis 7, 352-358,1978 24 VINAZZER,H.: Zur Bestinwnungvon Heparin im Plasma. Qualitatsvergleich verschiedener Methoden. Anaesthesist 8,299-303, 1979 25 PERLICK,E.: Antikoagulantien. 3. Aufl. G.Thieme,Leipzig 1964, p. 93 26 MARBET,R. and WINTERSTEIN,A.: Probleme der Blutgerinnung. Helv. physiol. Acta 10,528-542, 1952 27 MONKHOUSE,F.C.: Physiological -Ifactors concerned with the removal of injected heparin from circulating blood. Amer.J.Physiol. 178,223-228,1954 28 JAQUES,L.B.: Heparinase. J.biol.Chem. 133, 445-451, 1940