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THE QUANTITATIVE REDUCTION BY ACUTE-DOSE WARFARIN OF VENOUS THROMBOSIS IN NORMAL AND HYPERTHROMBOTIC RATS
Tkomhosis Research, Vol. S4, No. 6, pp. 411417, 1996 Copyright O 1996Ekevier Science Ltd Printed in the USA. All rights reserved 0049-3848/96$12.00 + .CO
Pergamon
PII S0049-3848(96)00209-5
THE QUANTITATIVE REDUCTION BY ACUTE-DOSE WARFARIN OF VENOUS THROMBOSIS IN NORMAL AND HYPERTHROMBOTIC RATS S.M.LavelleandMauraMhicIomhair, Departmentof ExperimentalMedicine,ClinicalScienceInstitute, UniversityCollegeGalway.
(Received 14August 1996 by EditorJ. Hladovec; revisetiaccepted22 October 1996)
ABSTRACT Warfarinwas testedfor its relativeabilityto reducethrombosisin two seriesof rats. In one series boththe warfarinand controlanimalswere madehyperthromboticby an infusion of ellagic acid just before thrombosiswas measured. In the second series(termednormothrombotic)no thromboticstimulantwas given.In all, 179test and 109controlanimalswereused. Warfarinwas givenin varyingdosesto the test animals. Ellagic acid was administeredas a singlestandarddose. At operation, two test animalsand one controlof similarweightwere operatedon concurrently. Thrombosiswas measuredby the weightof thrombusoccurringon a standard2cm intravenous platinum wire in one hour. In optimum dose, warfarin reduced thrombus formation by 74% in the unstimulated series and by 83% in the hyperthrombotic one, when the mean factor II levels were 1890 and 19% respectively. The residual thrombus under warfarin treatment was, however, greaterin the hyperthromboticthanin theunstimulatedanimalsat all levelsof factor II. Best correlationof thrombusweightwas with factor11in unstimulatedanimals. Copyright
(3 1996 Ektwier
Science L[d
A hypercoagl.il~.hle diathesisof the bloodmay accompanyor succeedan episodeof thrombosis (l-4). It is of imrest whetherthe drugscommonlyused againstthrombosisare as effectivein the presenceof kjqxixcoagulability as they are in its absence(5-7). The weightof thrombusformed on a standard wire, set in a vein for a fixed time, is a convenientmeasure of thrombosis. It respondsto both hypocoagulable(8,9) and hypercoagulable(10) states. Warfarin reducesthe activity of circulating clotting factors VII, X and 11. The factors have differing half-lives in plasma.Followinga singledose of warfarin,factorVII falls and recoversfirst and factor II last over 4 days. Thus on successivedays a different pattern of factor-depletionis present. The influenceof the factor-depletionon thrombusweightwas soughtin a seriesof normothrombotic and a seriesof hyperthromboticanimals,measuringthe relativeand absolutedecreasesin Key words:Warfarin,hypercoagulable,normocoagulable,experimentalthrombosis,factorII
411
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WARFARINANDVENOUSTHROMBOSIS
vol. 84, No.6
thrombusachievedby warfarinin the two series,for comparablereductionsof clottingfactors. Whichtestbestreflectedthe reductionin thrombosiswas alsoexamined. MATERIALS AND METHODS
Animals Sprague-Dawleyrats (200-450g)of both sexes were employed.Anesthesia was induced by intra-peritonealinjectionof pentoba-bitonesodium60mg/kg,andmaintainedby furtherinjection as required. Two test and one controlanimalof similarbody weightwere operatedon at a time by one operator. Normocoamdableseries In this, 88 test animals and 47 controls were used.Warfarin (3rngJkg) was injected intraperitoneallyinto groupsof 16at a time,to reducethe factorII levelto 12-24%. Otherdoses (0.1-3mg/kg)were used to producefactor II levelsoutsidethis range. One control and two test animalswere operatedon overthe following2 days,3 in the morningand 3 in the afternoon.The timingof the injectionsvariedfrom 17to 48 hoursbeforeoperation. Hvuercoa~ulableseries In this,91 test animals and 62 controlswereutilized.A rnillilitreof a solutionofellagic acid (5x10-5M in barbital-salinebuffer)was infusedper 100gmbody weightinto the femoralvein of test and of controlanimalsas soon as the wire was insertedin the vein.Warfarin 3mg/kgproved to be hemorrhagic in some of these animals and the general dose was reduced to 2.5mg/kg intraperitoneally.Otherwisetheyweretreatedas werethe normothromboticseries. Experimentalvenousthrombosis The inferiorvenacavawas exposedthrougha mid-lineincisionand a 3cmlengthof platinumwire, 0.5mm in diameter, sharpenedat one tip, was inserted2cm cephaladinto it, through a constant branch which enters below the renal vein, leavinga lcm shaftprotruding. The wire was left in place for 60 minutes. A 3 ml sampleof bloodwas aspiratedfromthe distalinferiorvenacava and 6 ml of glutaraldehydefixative (3g/dlin phosphatebuffer pH 7.3) was injectedinto the vein to fix the thrombusin situ. The wirewas weighedbeforeimplantationand againafterremoval. The weightof the thrombusformedon the wirewas foundby subtraction Clottimztests The activated whole blood clotting time (ABC) was measured by adding 0.5 ml of kaolin suspension (40 mg/rnl)to 1 ml of venousblood in a plastic tube and timing it to clot at 37°C. FactorII(11) VII (12) and X (13) were measured. Their resultsare expressedas percentageof normal obtained from dilutioncurves made on pooled plasmas of untreatedcontrol rats. The ABC test resultsare expressedin seconds. RESULTS
In the control animals given ellagic acid, mean thrombus weight was four times that of the untreatedcontrols.The animalsweredividedintofivegroupsaccordingto theircoagulationfactor test results. As mean factor II levels progressivelyfell, mean thrombus progressively fell coherentlybothin unstimulatedandhypercoagulableanimals,withno erraticresults(TableI). In the therapeuticrange of factor II, 12-24%,in which hemorrhage is rarely observed,venous thrombusweightwas reducedby 74% in the unstimulatedrats and 83Y0in the hyperthrombotic, but the absoluteamountof thrombusremainedgreaterin the hyperthromboticby 13Y0.
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TABLE I Relationof VenousThrombu$Weightto FactorII Depletionby Single-DoseWarfarin at 24 to 72 Hoursin NormothromboticandHyperthromboticSeriesof Rats Normothromboticseries HvDerthromboticseries FactorII N Mean Thrombus Reduction N Mean Thrombus Reduction FII % m&SEM TW Yo m@EM FII % Tw% band % --------------------------------------------------------------------------------------------------------------o-5 6-11 12-24 25-49 50-99
4 :7 8 30 17 21 34
11 83
Controls 45
**o.72 ~ **1.08 ~ **1.78 ~ **3.08 ~
o,29 oo16 oo26 o.49
*3.74~ 0.56
89 84 74 54
44
18 15 14 45
; 16 34
**2008@68 **3.42*1. 17 **4.41@80 **7.73*1 .32
93 87 83 70
15
82
22.95~3.20
12
6.73 + 0.43
::”07+1”46
------------Wilcoxonrank sum test versuscontrols* p >0.05, ** p> 0.01 FH = Factor 11 SEM = Standarderrorof the mean TW = thrombusweight ----------------------------------------------------------------::------
When,however, the factorVII testresultswereusedto effectthe partitions,mean thrombus weightdid not fallprogressivelywith depletionof thefactor,and the relationbetweenthem was erratic(TableII). In the normothromboticseries,meanthrombusweightwas maximally reduced(89’%0) when factorII levelswerein theO-5Y0 rangeand leastreduced(44Yo)when factor II valueswere in the 50-99%range(TableI). However,whenthe resultswere similarly partitioned accordingto factorVII values(TableIII), or by factorX values(TableIV), the relationbetweenreductionin thrombusweightand depletionof thesefactorswas poor .Partition of the resultsaccordingto FactorX depletionshowedfairlyprogressivereductionof thrombusas factorX fell,in both the normo-and hyperthromboticseries,with erraticresultsin the 12-24% band (TableIII). In the hyperthromboticseries, a reductionof factorII to about30%(intermediateanticoagulation) abolished the hyperthrombosis, while reduction of factor II to a mean of 17% (high-dose anticoagulation)reduced thrombus3090 (4.41mg)below that of the normalcontrols(6.73mg). However, a mean factor II of 8390 (low-dose anticoagulation)had negligible effect on the hyperthrombosis. TABLE II Relationof ThrombusWeightto FactorVII Reductionby Warfarinin Normothrombotic and HyperthromoticSeriesof Rats. HvDerthromboticseries Normothromboticseries Mean Thrombus Reduction N Mean Thrombus Reduction FactorVII N FVIIYOmg+SEM TW % F VII% mg + SEM TW ~0 band% --------------------------------------------------------------------------------------------------------------o-5 6-11 12-24 25-49 50-99
43 20 4
4 8 17
1;
%
**1082~ o.28 **1.63 ~o.27 1.09t 0.46 *2.27 LO.62 *4.00 * 0.51
73 76 84 66 41
43 3 **3.56&o.61 27 8 H4.30 ~0.88 10 19 **5.44* 1.54 4 45 26.45t 4.03 16 74 25.69f 3.18
86 ;: 0 2
62 26.07 ~ 1.46 6.73 ~ 0.43 Controls 47 --------------------------------------------------------------------------------------------------------------Wilcoxonrank sumtest *p<0.05, ** p< 0.01 FVII = FactorVII TW = Thrombusweight SEM = Standard error of the mean.
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WARFARINAND VENOUSTHROMBC)SIS
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TABLEIII Relationof ThrombusWeightto Factor X Reductionby Warfarinin Normothrombotic and HyperthromboticSeriesof Rats Hv~erthromboticseries Normothromboticseries Mean Thrombus Reduction N Mean Thrombus Reduction F X% mg + SEM Tw% FX % mg+ SEM Tw% -----------------------------------------------------------------------------------------------------------
FactorX
N
o-5 6-11 12-24 25-49 50-99
54 13 22 17 19
9 18 35 81
1.02~ 0.38 ** 1.99~ o.69 **161 f o,35 **2 11~o$35 **3.34 + o.42
85 70 76 69 50
12 7 32 17 2
2 8 15 29 74
**1.56~o.92 **2.63~o.13 **6. 19* o,74 **4.89*1.65 **7.20tl.65
94 90 76 81 72
6.73~ 0.43 62 26.07~1.46 Controls 47 --------------------------------------------------------------------------------------------------------------Wilcoxonrank sumtest versuscontrols *p
N
Hvperthromboticseries Normothromboticseries Thrombus Reduction Mean Thrombus Reduction Mean ABc mg+ SEM TW ?ZO ABC mg+SEM Tw %
---------------------------------------------------------------------------------------------------------------
120+
12
125.91A2.42
**1. ]3 ~ o.31 83
5
149.0f6.96 0.5~0.25
99
119-100 20
106.76~1.27 **1.18* 0.23 83
5 112.4~2,04 0.45~0.29
98
99-90
23
92.95~ 0.56
**2.35~ 0.45 66
6 93.5*1.59 *1.09t0.29
96
89-80
16
83.68~ 0.78 **1.87~ 0.24 73
79-70
6
73.33* 0.55
69-60
6
64.5f 1.11
4.33 ~0.55 36
8 65.50~1.24 **1O.79+2.72 59
66.65Y 1.19
6.75 ~ 0.43
61 64.24*1.19 26.07+1.46
Controls 44
*4.23 f 0.96 38
10 84.7~1.28 **4.54+1.45 83 21 74.67f0.63 **6.28t0.95
76
----------------------- _____________________________________________________________________
Wilcoxonrank sum *p< 0.05, **P
vol. 84, No. 6
WARFARINANDVENOUSTHROMBOSIS
415
TABLEV CorrelationCoefficient(r) of CoagulationTestResultsto Thrombusweight in NormalandHyperthromboticSeriesof Rats ,’
Normothrombotic r value N
~ N
r value
2: 66 45
*0.37 **0.44 **O-48 **O-34
------------------------------------------------------------,,-.-------------------------.----------------=------
FactorII% FactorVII YO FactorX % ABCsecs.
80 ;; 80
**0.53 *0.41 **0.38 **0.4
-
---------------------------------------------------------------------------------------------------------------
*=p
Our present studies indicate that warfarin is an effective agent in combatting this excess thrombosis.Previousexperimentshave shownthat ellagicacidlncreases thrombosis up to fivefold in the rat (10). In the hyperthromboticseries,warfarinreducedthrombosisby some 83% in safe dosage. These animalswere in the so-calledtherapeuticrange, and had a mean factor IIof 19Y0. This is in contrast to a reductionof 74% for a comparabledecrease of factor II in the normothrornboticsefieslThe differenceswere statisticallysignificant. The antithromboticaction of warfarin seems to be relativelygreater againstan activatedcoagulationsystemthan againsta quiescentone. While the dose of warfarin was slightlyless in the hyperthromboticseries, the effect as reflectedin levelsof factorsII and VII was.equivalentin the two series (TableI) while factorX levelswereislightlylowerin the hyperthmmbotics,whichwere a littlemore sensitiveto warfarin. Ellagic acid itself displayed minor coagulationchanges in twenty-two control animals. Tests carried out after one hour showed a slight reduction in factors 11(69~o,range 34-1OO7O), VII (72%, range46-95%) and X (71%,range38-1~%). Otherauthorsobserved shorteningof the siliconeclottingtimeand accelerationof thethrombc@astingenerationtestby the substancein both animalsand man (14-16). The correlation(r) between thrombusweightand the level of the severalfactors was not strong, being best for factorII (0.53)in the-normothromboticseries,whileXand VII relatedbetter in the hyperthromboticseries.The criticalissuefor a testusedto controlanticoagulanttherapy,however, is not the overallcorrelationwith thrombusdeposition,but whetherit can denotereliablya zonein whichantithromboticeffectis maximalbut!haemorrhage is rare. FactorII has such a zone,factorX less so, and factorVII lacksit, havingmerelya statistical correlationwith thrombosisreduction,as it has with hemorrhage (9-17). Yet the prothrombin time, widelyused for cohtrolof anticoagulanttherapy,measuresprimarilyFactorVII, whetherit has been calibratedwith other thromboplastinsor not. The clinicalcontrolit permits would be expectedto be deficient, as is th~case (18-20). In conclusion,warfarin showedmarkedactivityin reducingthrombosisin hypercoagulable animals,but was unable to restore thrombusto the level that warfarin reduced it to in normal animals. FactorII mostreliablyindicatedthe non-haemorrhagicantithromboticeffectof warfarin in both normothromboticand hyperthromboticseries of animals. Factor VII was the least reliable.
416
, WARFARINAND VENOUSTHROMBOSIS
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84, No.6
REFERENCES
1.
OWEN C.A. Hypercoagulabilityand thrombosis,Mayo Clin.Proc. 40,830-833, 1965.
2.
SCHAFERA.L The hypercoagulablestates. Ann. Intern.Med. 102,814-828, 1985.
3. EBY C.S. A review of the hypercoagulablestate. Oncology Clin. North AM. 7, 11211142, 1993. 4. BICK R.L., PEGRAM M. Syndromesof hypercoagulabilityand thrombosis:a review. Thrombos.Haemostas,20, 109-132,1994. 5. SEVITT S. Venousthrombosisand pulmonary,embolism.Their preventionby oral anticoagulants.Am. J. Med., 30, 703-716, 1962. 6.
HIRSH J. Oral anticoagulantdrugs. N. Eng. J. Med. 324,‘1865-1875,1991.
KAKKARV.V. Preventionand managementof venousthrombosis. Brit. Med. Bulletin 5i; 871-903, 1994 8. LAVELLE S.M.,LENIHANF. The antithromboticeffectof warfarin anticoagulant. Ir. J. Med. Sci. 140, 142, 1971. MAC IOMHAIR M., S.M. LAVELLE. The anticoagulant, antithrombotic, and 9. hemorrhagic effect of long-termwarfarin on experimentalvenous and arterial thrombosisin ~the rat. Ir. J. Med., Sci.,165, 213-218, 1996. 10. MAC IOMHAIR M., LAVELLE S.M. Thrombus weight as a measure of hypercoagulability induced by drugs. Thrombos. Haemostas.42, 10.1,8-1021, 1979. 11. OWREN P.A., AAS K. The control of dicoumarol therapy and the quantitative determinationof prothrombin and proconvertin. Scand. J. Clin. Lab. Invest. 3S 201-208, 1951. ADAMIS D., SISE H.S., KIMBALL D.M. Proconvertintest: simplifiedmethod and 12. its applicationto studyof anticoagulantprocesses.J. Lab .Clin.Med. 47, 320-325,1956. 13 SISE H.S., LAVELLE S.M. , BECKER R . A method for assay of Stuart factor. Proc. Soc. Exp. Biol. Med. 96, 662-664, 1957. 14. RATNOFF O.D., CRUM J.D. Activationof Hageman factor by solutionsof ellagic acid. J. Lab. Clin. Med. 63, 359-377,1964. 15. GIROLAMI A., AGOSTINO D., CLIFFTON E.E. The effect of ellagic acid on coagulationin vivo. Blood 27,93-102, 1966. 16. GIROLAMI A., CLIFFTONE.E. Hypercoagulablestate induced in humans by the intravenous administrationof purifiedellagicacid.Thromb.Diath.Haemorr.7,165-75,1967.
17. TALSTAD I. Which coagulationfactorsinterferewith the one stageprothrombintime. Haemostasis,23, 19-25, 1993. 18. SISE, H.S., LAVELLE S.M., MOSCHOS C., BECKER R. The relation of hemorrhage, thrombosis and prothrombinduringlong-termcoumarintherapy.New EnglandJ. Med. 259.266, 1958.
vol. 84, No. 8
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PETITTI D., B. Prothrombintime ratio and otbe$fqctorsassociatedwith bleeding.in ~~tientstreated with warfarin,J., Clin.,Epidemiology42,759-764, 1989.. 20 LEMOS MJ., SUTTEN D., HOZACK W. J., BALDERSON R.A., BOOTH R.E. Jnr., ROTHMANR.H. Pulmonaryembolismin total hip and knee arthroplasty. Risk factorsin patients on warfarin prophylaxis and analysis of the prothrombin time as an indicator of warfarin’sprophylaxis.Clin. Ortho.& RelatedResearch282, 158-163,1992.
Pergamon
PII S0049-3848(96)00209-5
THE QUANTITATIVE REDUCTION BY ACUTE-DOSE WARFARIN OF VENOUS THROMBOSIS IN NORMAL AND HYPERTHROMBOTIC RATS S.M.LavelleandMauraMhicIomhair, Departmentof ExperimentalMedicine,ClinicalScienceInstitute, UniversityCollegeGalway.
(Received 14August 1996 by EditorJ. Hladovec; revisetiaccepted22 October 1996)
ABSTRACT Warfarinwas testedfor its relativeabilityto reducethrombosisin two seriesof rats. In one series boththe warfarinand controlanimalswere madehyperthromboticby an infusion of ellagic acid just before thrombosiswas measured. In the second series(termednormothrombotic)no thromboticstimulantwas given.In all, 179test and 109controlanimalswereused. Warfarinwas givenin varyingdosesto the test animals. Ellagic acid was administeredas a singlestandarddose. At operation, two test animalsand one controlof similarweightwere operatedon concurrently. Thrombosiswas measuredby the weightof thrombusoccurringon a standard2cm intravenous platinum wire in one hour. In optimum dose, warfarin reduced thrombus formation by 74% in the unstimulated series and by 83% in the hyperthrombotic one, when the mean factor II levels were 1890 and 19% respectively. The residual thrombus under warfarin treatment was, however, greaterin the hyperthromboticthanin theunstimulatedanimalsat all levelsof factor II. Best correlationof thrombusweightwas with factor11in unstimulatedanimals. Copyright
(3 1996 Ektwier
Science L[d
A hypercoagl.il~.hle diathesisof the bloodmay accompanyor succeedan episodeof thrombosis (l-4). It is of imrest whetherthe drugscommonlyused againstthrombosisare as effectivein the presenceof kjqxixcoagulability as they are in its absence(5-7). The weightof thrombusformed on a standard wire, set in a vein for a fixed time, is a convenientmeasure of thrombosis. It respondsto both hypocoagulable(8,9) and hypercoagulable(10) states. Warfarin reducesthe activity of circulating clotting factors VII, X and 11. The factors have differing half-lives in plasma.Followinga singledose of warfarin,factorVII falls and recoversfirst and factor II last over 4 days. Thus on successivedays a different pattern of factor-depletionis present. The influenceof the factor-depletionon thrombusweightwas soughtin a seriesof normothrombotic and a seriesof hyperthromboticanimals,measuringthe relativeand absolutedecreasesin Key words:Warfarin,hypercoagulable,normocoagulable,experimentalthrombosis,factorII
411
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WARFARINANDVENOUSTHROMBOSIS
vol. 84, No.6
thrombusachievedby warfarinin the two series,for comparablereductionsof clottingfactors. Whichtestbestreflectedthe reductionin thrombosiswas alsoexamined. MATERIALS AND METHODS
Animals Sprague-Dawleyrats (200-450g)of both sexes were employed.Anesthesia was induced by intra-peritonealinjectionof pentoba-bitonesodium60mg/kg,andmaintainedby furtherinjection as required. Two test and one controlanimalof similarbody weightwere operatedon at a time by one operator. Normocoamdableseries In this, 88 test animals and 47 controls were used.Warfarin (3rngJkg) was injected intraperitoneallyinto groupsof 16at a time,to reducethe factorII levelto 12-24%. Otherdoses (0.1-3mg/kg)were used to producefactor II levelsoutsidethis range. One control and two test animalswere operatedon overthe following2 days,3 in the morningand 3 in the afternoon.The timingof the injectionsvariedfrom 17to 48 hoursbeforeoperation. Hvuercoa~ulableseries In this,91 test animals and 62 controlswereutilized.A rnillilitreof a solutionofellagic acid (5x10-5M in barbital-salinebuffer)was infusedper 100gmbody weightinto the femoralvein of test and of controlanimalsas soon as the wire was insertedin the vein.Warfarin 3mg/kgproved to be hemorrhagic in some of these animals and the general dose was reduced to 2.5mg/kg intraperitoneally.Otherwisetheyweretreatedas werethe normothromboticseries. Experimentalvenousthrombosis The inferiorvenacavawas exposedthrougha mid-lineincisionand a 3cmlengthof platinumwire, 0.5mm in diameter, sharpenedat one tip, was inserted2cm cephaladinto it, through a constant branch which enters below the renal vein, leavinga lcm shaftprotruding. The wire was left in place for 60 minutes. A 3 ml sampleof bloodwas aspiratedfromthe distalinferiorvenacava and 6 ml of glutaraldehydefixative (3g/dlin phosphatebuffer pH 7.3) was injectedinto the vein to fix the thrombusin situ. The wirewas weighedbeforeimplantationand againafterremoval. The weightof the thrombusformedon the wirewas foundby subtraction Clottimztests The activated whole blood clotting time (ABC) was measured by adding 0.5 ml of kaolin suspension (40 mg/rnl)to 1 ml of venousblood in a plastic tube and timing it to clot at 37°C. FactorII(11) VII (12) and X (13) were measured. Their resultsare expressedas percentageof normal obtained from dilutioncurves made on pooled plasmas of untreatedcontrol rats. The ABC test resultsare expressedin seconds. RESULTS
In the control animals given ellagic acid, mean thrombus weight was four times that of the untreatedcontrols.The animalsweredividedintofivegroupsaccordingto theircoagulationfactor test results. As mean factor II levels progressivelyfell, mean thrombus progressively fell coherentlybothin unstimulatedandhypercoagulableanimals,withno erraticresults(TableI). In the therapeuticrange of factor II, 12-24%,in which hemorrhage is rarely observed,venous thrombusweightwas reducedby 74% in the unstimulatedrats and 83Y0in the hyperthrombotic, but the absoluteamountof thrombusremainedgreaterin the hyperthromboticby 13Y0.
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TABLE I Relationof VenousThrombu$Weightto FactorII Depletionby Single-DoseWarfarin at 24 to 72 Hoursin NormothromboticandHyperthromboticSeriesof Rats Normothromboticseries HvDerthromboticseries FactorII N Mean Thrombus Reduction N Mean Thrombus Reduction FII % m&SEM TW Yo m@EM FII % Tw% band % --------------------------------------------------------------------------------------------------------------o-5 6-11 12-24 25-49 50-99
4 :7 8 30 17 21 34
11 83
Controls 45
**o.72 ~ **1.08 ~ **1.78 ~ **3.08 ~
o,29 oo16 oo26 o.49
*3.74~ 0.56
89 84 74 54
44
18 15 14 45
; 16 34
**2008@68 **3.42*1. 17 **4.41@80 **7.73*1 .32
93 87 83 70
15
82
22.95~3.20
12
6.73 + 0.43
::”07+1”46
------------Wilcoxonrank sum test versuscontrols* p >0.05, ** p> 0.01 FH = Factor 11 SEM = Standarderrorof the mean TW = thrombusweight ----------------------------------------------------------------::------
When,however, the factorVII testresultswereusedto effectthe partitions,mean thrombus weightdid not fallprogressivelywith depletionof thefactor,and the relationbetweenthem was erratic(TableII). In the normothromboticseries,meanthrombusweightwas maximally reduced(89’%0) when factorII levelswerein theO-5Y0 rangeand leastreduced(44Yo)when factor II valueswere in the 50-99%range(TableI). However,whenthe resultswere similarly partitioned accordingto factorVII values(TableIII), or by factorX values(TableIV), the relationbetweenreductionin thrombusweightand depletionof thesefactorswas poor .Partition of the resultsaccordingto FactorX depletionshowedfairlyprogressivereductionof thrombusas factorX fell,in both the normo-and hyperthromboticseries,with erraticresultsin the 12-24% band (TableIII). In the hyperthromboticseries, a reductionof factorII to about30%(intermediateanticoagulation) abolished the hyperthrombosis, while reduction of factor II to a mean of 17% (high-dose anticoagulation)reduced thrombus3090 (4.41mg)below that of the normalcontrols(6.73mg). However, a mean factor II of 8390 (low-dose anticoagulation)had negligible effect on the hyperthrombosis. TABLE II Relationof ThrombusWeightto FactorVII Reductionby Warfarinin Normothrombotic and HyperthromoticSeriesof Rats. HvDerthromboticseries Normothromboticseries Mean Thrombus Reduction N Mean Thrombus Reduction FactorVII N FVIIYOmg+SEM TW % F VII% mg + SEM TW ~0 band% --------------------------------------------------------------------------------------------------------------o-5 6-11 12-24 25-49 50-99
43 20 4
4 8 17
1;
%
**1082~ o.28 **1.63 ~o.27 1.09t 0.46 *2.27 LO.62 *4.00 * 0.51
73 76 84 66 41
43 3 **3.56&o.61 27 8 H4.30 ~0.88 10 19 **5.44* 1.54 4 45 26.45t 4.03 16 74 25.69f 3.18
86 ;: 0 2
62 26.07 ~ 1.46 6.73 ~ 0.43 Controls 47 --------------------------------------------------------------------------------------------------------------Wilcoxonrank sumtest *p<0.05, ** p< 0.01 FVII = FactorVII TW = Thrombusweight SEM = Standard error of the mean.
414
WARFARINAND VENOUSTHROMBC)SIS
vol. 84, No. 6
TABLEIII Relationof ThrombusWeightto Factor X Reductionby Warfarinin Normothrombotic and HyperthromboticSeriesof Rats Hv~erthromboticseries Normothromboticseries Mean Thrombus Reduction N Mean Thrombus Reduction F X% mg + SEM Tw% FX % mg+ SEM Tw% -----------------------------------------------------------------------------------------------------------
FactorX
N
o-5 6-11 12-24 25-49 50-99
54 13 22 17 19
9 18 35 81
1.02~ 0.38 ** 1.99~ o.69 **161 f o,35 **2 11~o$35 **3.34 + o.42
85 70 76 69 50
12 7 32 17 2
2 8 15 29 74
**1.56~o.92 **2.63~o.13 **6. 19* o,74 **4.89*1.65 **7.20tl.65
94 90 76 81 72
6.73~ 0.43 62 26.07~1.46 Controls 47 --------------------------------------------------------------------------------------------------------------Wilcoxonrank sumtest versuscontrols *p
N
Hvperthromboticseries Normothromboticseries Thrombus Reduction Mean Thrombus Reduction Mean ABc mg+ SEM TW ?ZO ABC mg+SEM Tw %
---------------------------------------------------------------------------------------------------------------
120+
12
125.91A2.42
**1. ]3 ~ o.31 83
5
149.0f6.96 0.5~0.25
99
119-100 20
106.76~1.27 **1.18* 0.23 83
5 112.4~2,04 0.45~0.29
98
99-90
23
92.95~ 0.56
**2.35~ 0.45 66
6 93.5*1.59 *1.09t0.29
96
89-80
16
83.68~ 0.78 **1.87~ 0.24 73
79-70
6
73.33* 0.55
69-60
6
64.5f 1.11
4.33 ~0.55 36
8 65.50~1.24 **1O.79+2.72 59
66.65Y 1.19
6.75 ~ 0.43
61 64.24*1.19 26.07+1.46
Controls 44
*4.23 f 0.96 38
10 84.7~1.28 **4.54+1.45 83 21 74.67f0.63 **6.28t0.95
76
----------------------- _____________________________________________________________________
Wilcoxonrank sum *p< 0.05, **P
vol. 84, No. 6
WARFARINANDVENOUSTHROMBOSIS
415
TABLEV CorrelationCoefficient(r) of CoagulationTestResultsto Thrombusweight in NormalandHyperthromboticSeriesof Rats ,’
Normothrombotic r value N
~ N
r value
2: 66 45
*0.37 **0.44 **O-48 **O-34
------------------------------------------------------------,,-.-------------------------.----------------=------
FactorII% FactorVII YO FactorX % ABCsecs.
80 ;; 80
**0.53 *0.41 **0.38 **0.4
-
---------------------------------------------------------------------------------------------------------------
*=p
Our present studies indicate that warfarin is an effective agent in combatting this excess thrombosis.Previousexperimentshave shownthat ellagicacidlncreases thrombosis up to fivefold in the rat (10). In the hyperthromboticseries,warfarinreducedthrombosisby some 83% in safe dosage. These animalswere in the so-calledtherapeuticrange, and had a mean factor IIof 19Y0. This is in contrast to a reductionof 74% for a comparabledecrease of factor II in the normothrornboticsefieslThe differenceswere statisticallysignificant. The antithromboticaction of warfarin seems to be relativelygreater againstan activatedcoagulationsystemthan againsta quiescentone. While the dose of warfarin was slightlyless in the hyperthromboticseries, the effect as reflectedin levelsof factorsII and VII was.equivalentin the two series (TableI) while factorX levelswereislightlylowerin the hyperthmmbotics,whichwere a littlemore sensitiveto warfarin. Ellagic acid itself displayed minor coagulationchanges in twenty-two control animals. Tests carried out after one hour showed a slight reduction in factors 11(69~o,range 34-1OO7O), VII (72%, range46-95%) and X (71%,range38-1~%). Otherauthorsobserved shorteningof the siliconeclottingtimeand accelerationof thethrombc@astingenerationtestby the substancein both animalsand man (14-16). The correlation(r) between thrombusweightand the level of the severalfactors was not strong, being best for factorII (0.53)in the-normothromboticseries,whileXand VII relatedbetter in the hyperthromboticseries.The criticalissuefor a testusedto controlanticoagulanttherapy,however, is not the overallcorrelationwith thrombusdeposition,but whetherit can denotereliablya zonein whichantithromboticeffectis maximalbut!haemorrhage is rare. FactorII has such a zone,factorX less so, and factorVII lacksit, havingmerelya statistical correlationwith thrombosisreduction,as it has with hemorrhage (9-17). Yet the prothrombin time, widelyused for cohtrolof anticoagulanttherapy,measuresprimarilyFactorVII, whetherit has been calibratedwith other thromboplastinsor not. The clinicalcontrolit permits would be expectedto be deficient, as is th~case (18-20). In conclusion,warfarin showedmarkedactivityin reducingthrombosisin hypercoagulable animals,but was unable to restore thrombusto the level that warfarin reduced it to in normal animals. FactorII mostreliablyindicatedthe non-haemorrhagicantithromboticeffectof warfarin in both normothromboticand hyperthromboticseries of animals. Factor VII was the least reliable.
416
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84, No.6
REFERENCES
1.
OWEN C.A. Hypercoagulabilityand thrombosis,Mayo Clin.Proc. 40,830-833, 1965.
2.
SCHAFERA.L The hypercoagulablestates. Ann. Intern.Med. 102,814-828, 1985.
3. EBY C.S. A review of the hypercoagulablestate. Oncology Clin. North AM. 7, 11211142, 1993. 4. BICK R.L., PEGRAM M. Syndromesof hypercoagulabilityand thrombosis:a review. Thrombos.Haemostas,20, 109-132,1994. 5. SEVITT S. Venousthrombosisand pulmonary,embolism.Their preventionby oral anticoagulants.Am. J. Med., 30, 703-716, 1962. 6.
HIRSH J. Oral anticoagulantdrugs. N. Eng. J. Med. 324,‘1865-1875,1991.
KAKKARV.V. Preventionand managementof venousthrombosis. Brit. Med. Bulletin 5i; 871-903, 1994 8. LAVELLE S.M.,LENIHANF. The antithromboticeffectof warfarin anticoagulant. Ir. J. Med. Sci. 140, 142, 1971. MAC IOMHAIR M., S.M. LAVELLE. The anticoagulant, antithrombotic, and 9. hemorrhagic effect of long-termwarfarin on experimentalvenous and arterial thrombosisin ~the rat. Ir. J. Med., Sci.,165, 213-218, 1996. 10. MAC IOMHAIR M., LAVELLE S.M. Thrombus weight as a measure of hypercoagulability induced by drugs. Thrombos. Haemostas.42, 10.1,8-1021, 1979. 11. OWREN P.A., AAS K. The control of dicoumarol therapy and the quantitative determinationof prothrombin and proconvertin. Scand. J. Clin. Lab. Invest. 3S 201-208, 1951. ADAMIS D., SISE H.S., KIMBALL D.M. Proconvertintest: simplifiedmethod and 12. its applicationto studyof anticoagulantprocesses.J. Lab .Clin.Med. 47, 320-325,1956. 13 SISE H.S., LAVELLE S.M. , BECKER R . A method for assay of Stuart factor. Proc. Soc. Exp. Biol. Med. 96, 662-664, 1957. 14. RATNOFF O.D., CRUM J.D. Activationof Hageman factor by solutionsof ellagic acid. J. Lab. Clin. Med. 63, 359-377,1964. 15. GIROLAMI A., AGOSTINO D., CLIFFTON E.E. The effect of ellagic acid on coagulationin vivo. Blood 27,93-102, 1966. 16. GIROLAMI A., CLIFFTONE.E. Hypercoagulablestate induced in humans by the intravenous administrationof purifiedellagicacid.Thromb.Diath.Haemorr.7,165-75,1967.
17. TALSTAD I. Which coagulationfactorsinterferewith the one stageprothrombintime. Haemostasis,23, 19-25, 1993. 18. SISE, H.S., LAVELLE S.M., MOSCHOS C., BECKER R. The relation of hemorrhage, thrombosis and prothrombinduringlong-termcoumarintherapy.New EnglandJ. Med. 259.266, 1958.
vol. 84, No. 8
WARFARINAND VENOUSTHROMBOSIS
417
PETITTI D., B. Prothrombintime ratio and otbe$fqctorsassociatedwith bleeding.in ~~tientstreated with warfarin,J., Clin.,Epidemiology42,759-764, 1989.. 20 LEMOS MJ., SUTTEN D., HOZACK W. J., BALDERSON R.A., BOOTH R.E. Jnr., ROTHMANR.H. Pulmonaryembolismin total hip and knee arthroplasty. Risk factorsin patients on warfarin prophylaxis and analysis of the prothrombin time as an indicator of warfarin’sprophylaxis.Clin. Ortho.& RelatedResearch282, 158-163,1992.