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Identity of contact activation cofactor and Fitzgerald factor
vol. 6, ppe 451-454, 1975
THROMBOSIS RESEARCH Printed
in the United
Pergamon
States
press,
Inc.
COMMUNICATION
BRIEF
IDEM!I!lY OF COBITACT ACTIVA!l’IONCOFAC!lDRAND FI!EGERALDFACJQR
SandraSchiffuan,PearlLee,andRobertWaldmann Departmentsof Medicine and Biochemistry,University of southern CsJLforniaMedical School,IosAngeles,California9033, and HenryFord Rospital,Detroit,Michigan 48202
(Received
10.3.1975.
Accepted
by Editor K.M. Brinkhous)
The contactactivationof intrinsicclottingis classically describedas the interaction of factorsZ and III in the prSSenCaof an activatingsurface. ~owemr, in 1965 auotheractivity,Fletcherfactor,wa8 describsdwhich participates in this phase of clotting(1,2),andrecentlythe 8ystemha8been In194 twonewactivitiesweredescribed shomtobeevenmorecou@ex. whichparticipatein contactactivation:co&a&activation cofactor(CAC) (3) ahd Fitzgeraldfactor (4,5). CAC is the name ¥ to anactivityofrrarsrlplrsPrwhich~ici~te8 in the activationof factorII in the presenceof Platchetr factor,factorXII,
and kaolin. Fitzgeraldfactoris an activitymissingfrom the plasm of a patientwhose coagulation testsdemnstrateda defectin the contactphase of intrinsic cl&ting. This defectcouldnot be attributedto the absenceof anykuowh reactrntin contactactivation - factorII, factorIII,orFletcher factor- 8oFitsgeraldfactormustbe an additioualcontact,factor. It seemed possiblethat CAC and Fitcgtraldfactorore two n8sms for the 8am8 actidescribedbelowwere designedto test thispossibility. vity* The experiments MATRRIAIgARDm!l!HoDS FactorII-F+letcherfactorreagent. Factor= combinedtithl?letcherfactorwa8 s-ted fmmotherknown clottingfactorsinthe firstproteinpeak elutingfrom DRAR-cellulose on chmmatogrrgphy of Al(0R 3 adsorbednomealplasma at pR 7.2 in 0.05 M salt as describedpreviously(6 . The fractionused belowcontained45$factorXI and 3O$Fletcherfactoractivity. FaotorlUI reageht. FactorXII was s-ted fknnotherknown cl&ting factors by a-y Of Al(OH)3adsorbednomal plmm8 on DlW-cellulose using a sli>t modification of the previou8lydescribedtechnique(6). this factorXII fractionuasbroughtto25$amnniumsulfate saturation,thepmcipitatewas dbwrded,ti the supernatantwasbroughtto5O$asm~niumsulfate saturation.The 50% precipitate, cont&ning the factorXII, was dissolved and rechromatographedon DEAg-cellulose.ThepooledfactorxII fraction had about 4% factor XII activity. 451
CONTACT ACTIVATION
452
v01.6,~0.5
CAC reagent was prepared from Al(OH)3 adsorbed normal plasma by DEAEcellulose chromatography, auwnium sulfate precipitation, and chromatography on BioGel A-l. 5m (Bio-Rad Laboratories, Richmond, Calif. ). Details of these procedures have been submitted for publication elsewhere (7). The CAC fraction used in these experiments when tested at about 30% normalplasmaCAC activity had no detectable factor II, Ix, X, XI, XIII, or fibrinogen; 0.s factorVIII; 0.6 factorV; 0.1s factor XII; and much less than 0.15 Fletcher It was purified about 40 fold. factor activity.
Dicalite adsorbed plasma was prepared by chromatographing normal plasma on Dicalit 4200 (Grefco, Inc., Los Angeles, Calif. ) (1 g/ml plasma) equilibrated wit: TES (IV-tris (Hydroxymethyl) methyl-2-aminoethane Sulfonic Acid) buffer (0.05 M TES and 0.1 M IVaCl, pIi 7.35). The unbound protein fraction was pooled and incubated at 37% in glass tubes until the partial thromboplastin time (8) in the presence or absence of kaolin rose to about 150 seconds. Then the pH was adjusted to 7.4, and the adsorbed plasma was stored at -20°C in plastic vials. This reagent contained: 105 mg$ fibrinogen, 609 factor II, 32$ factor V, 60$ factorVIII, 91s factorIX, 749 factor X, 6$ factor XI, 15 factor XII, 0.79 Fletcher factor. The following reagents were added to a plastic clotting tube 0.01 ml cephalin (9) diluted l/l0 in veronal buffer pir 7.35, 0.05 ml factor XI-Fletcher factor reagent, 0.05 ml factor XII reagent, 0.05 ml Dicalite adsorbed plasma, 0.05 ml test substance, and 0.01 ml kaolin (20 mg/ml saline) (Kaolin, RF. Colloidal, Mallinckzodt). The tube was incubated at 37oC for precisely 10 minutes, then 0.025 ml of 25 IN CaCl2 was added and the clotting time noted. This system contained 30$ or more of all known clotting factors.
%Fer noted:
in the o
Fitzgerald factor assap The following reagents were added to a glass clotting tube in the order noted: 0.025 ml kaolin (20 mg/ml saline), O.G25 ml cephalin (9) l/30, 0.05 ml Fitzgerald deficient plasma, and 0.05 ml test substance. After 3 minutes incubation at 37oc, 0.05 ml 40 aM CM& was added and the clotting time noted. Other clotting factor assays. Assays for factors II, V, VIII, Ix, X, XI, XII and Fletcher factor have been described elsewhere (3). Fibrinogen Small amounts of was measured by the method of Blombkk and Blddck (10). fibrinogen were detected by i nnwnodiffusion against rabbit antihuman fibrinogen antiserum (Hyland, Div. Travenol Laboratories, Inc. ). Factor XIII was measured as the ability of a reagent to render a c&t formed in congenital factor XIII deficient plasma insoluble in 5 M urea. RESULTS The effect of various reagents on the CAC and Fitzgerald factor assays no is shown in Table 1. The limits of each system are defined by buffer test substance) and normal plasma as a source of the missing activity tlines l-3). Both assays are corrected by plasmas lacking factor XI, factor XII, or Therefore, the primary defect in the CAC and Fletcher factor (lines 4-6). Fitzgerald factor assayscannot be attributed to a deficiency of one of the In contrast, Fitzgerald deficient three known contact activation factors. plasma &es not correct either assay (line 7). Hence, the activity missing in Fitzgerald deficient plasma is also lacking in the CAC assay. Furthermore, partially purified CAC reagent, which lacks other known clotting factors, corrects the defective clotting in both the CAC and Fitzgerald assays (lines 8
and 9).
Vo1.6,No.5
CONTACT
ACTIVATION
453
TABLE I. CAC and FitzgeraldFactorAssays ClottingTime (set) Fitzgerald CAC
Test Material Buffer 2. Normalplasmal/10 Normalplasmal/20 Z: XI def. pl. l/10 XII def. pl. l/10 2: Fletcherdef. pl. l/10 Fitzgeralddef. pl. l/10 87: CAC reagent-lO$ 9* CAC reagent- 5%
1.
101 :; 44 45 45 96 45 52
284 46 '$ 55: 218 49 59
DISCUSSION The conceptthat the contactphase of intrinsicclottingutilizesadditionalactivitiesbesidesfactorsXI and XII has evolvedover the past 15 betweencrudeplasmafracyears. In 1960 no reactioncouldbe dearonstrated tions containingfactorXI or factor XII, and it was suggestedthat noxvnal plasmacontainedan additionalactivitylackingin the partiallypurifiedreagents (6). When Fletcherfactorwas described,it was postulatedthat Fletcher factormight be the activitylackingin the earlierexperiments(2). However,laterexperiments showedthat this was not the case and that plasma must containa fourthcontactactivationcofactor(3). Furthersupportfor an additionalactivityparticipating in contactactivationwas suppliedby the identification of Fitzgeraldtraitplasmawhichlackeda previouslyunidentifiedcomponentof the contactactivationsystem(4,5). The experiments presentedaboveprovidetwo linesof evidencesupporting the identityof CAC and Fitzgeraldfactor: 1) both assay systemslack a common activity,and 2) a partiallypurifiedreagentseparatedfrom otherknown clottingfactorscorrectsthe defectin both substrates.Therefore,it appears that thereare at leastfour activities whichparticipatein the contact activationof intrinsicclotting: factorXII, factorII, Fletcherfactor,and WC-Fitzgeraldfactor. A role for CAC-Fitzgerald factorin contactactivationhas been establishedboth in a syntheticsystem(MC!)and in an expe%?iment of nature (Fitzgeraldtrait). The mechanismof actionof this activityremainsto be defined. Many reports,includingtwo very recentones (XL&?), appearto demonstratedirectactivationof factorXI by activatedfactorIII, withoutparticipationof MC-Fitzgeraldfactor. Two possibleexplanations of the apparent contradiction come to mind: 1) WC-Fitzgeraldfactormay participatein a preliminaryactivationof factorXII or factorXI, whichhas alreadytaken place in reagentsused for other experiments, or 2) MC-Fitzgeraldfactormay influencethe rate of interaction of factorsXI and XII - a very slow reaction occurringin its absence. Thesepossibilities remainto be tested.
These studieswere supportedby a grant from the AmericanHeartAasociation-Greater Los AngelesAffiliate,and were completedduringthe tenureof
454
CONTACT ACTIVATION
Vo1.6,No.5
8n Estsblished ~nvestigatorship of the AmericanHeart Associationto Dr. SchiffUl8b. -Es
1. HATHAWAY,W.E.,BEIliASEH, L.P. and HATHAWAY,H.S. Evidencefor 8 new plasm8 thmmbopl8stinfsctorI. Case report,coagulation studiesand physiocheuic8lproperties. Blood.26, 52l,1965. 2. HATHAWAY,W.E. 8nd ALSEVER,J. The relationship of "FletcherFactor” fsctorsXI and XII. Brit.J. Haemt. l8, 161, 1970.
to
S. 8nd LEE, P. Preparation, chsrscterization, and activ8tion 3. SC-, of a highlypurifiedfactorXI: Evidencethat a hithertounrecognized p&m8 activityparticipstes in the interaction of fsctorsXI and XII. Brit.J. Hacmrt.3, 101, 1974. 4. WALDMMN, R. 8nd ABRAHAM,J.P. Fitzgeraldfsctor:A heretoforeunrecognized co8gul8tion fsctor.AmericaaSocietyof Hematology,17th Annual Meeting,(Abstrrct), p* 74, 1974. O.D.,WALDMMN, R. and ABRAHAM,J.B. ImpairedHagems 5. SAIIQ,Ii.,-, isctor (FactorZI)-medi8tedre8ctionsin "Fitsgemild Trait".American Societyof Hesmtology,17th Annu8lMeeting,(Abstract), p. 74, 1974. 6. m, S., RAPApX!!,S.I.,WARE,A.G. and MEHL, J.W. Sepsrationof pl8sm8thmmbopl8stinantecedent(PTA)8nd Hagtman factdr (HF) from human pl8sm8.Pmt. Sot. Exper.Biol.Med. lC&. 453, 1960. and p8rtislpurifica7. SCIUFFMAW,S. and LEE, P. Assay,chsrscterization, tion of contact8ctiv8tioncof8ctor(CAC).Submittedfor publication. 0. PROCZQR,R.R. and RAPAXXQ, S.I. The partialthrombopl8stin time with kralin.AM. J. Clin. Psth.36, 212, 1961. P.A. A simple,specificone-stage 9. HJORT,P., RAPAPORT,S. I. 8nd OWREIV, pmthranbin 8ss8yusingRussell'sviper venomin ceph8linsuspension. J. I&J. & Clin.Med. 46, 89, 1955.
10. BKMB&K, B. and BKMlBkX, M. Purification A&. mmi. 2, 415, 1956.
of human 8nd bovinefibririogen.
ll. HICK, L.W. and KAPLAN,A.P. Substratesof H8gemn factor.I. Isolation 8nd chanrcterization of human factorXI (m) end inhibitionof the activ8ted enzymeby o(,antitrypsin. J. Exper. Med. 140, 1615, 1974. 12. MDVAT,H.Z. and &ZE-AEWAR, A.H. The cont8ctphase of blood cosgulation: clottingfactorsXt 8nd XII, theirisolationand interaction. J. U&b. & Cl&l.Med. 84, 861, 1974. Reprintrequeststo Dr. schiffam, USC MedicalSchool,Los Angeles,Calif.
THROMBOSIS RESEARCH Printed
in the United
Pergamon
States
press,
Inc.
COMMUNICATION
BRIEF
IDEM!I!lY OF COBITACT ACTIVA!l’IONCOFAC!lDRAND FI!EGERALDFACJQR
SandraSchiffuan,PearlLee,andRobertWaldmann Departmentsof Medicine and Biochemistry,University of southern CsJLforniaMedical School,IosAngeles,California9033, and HenryFord Rospital,Detroit,Michigan 48202
(Received
10.3.1975.
Accepted
by Editor K.M. Brinkhous)
The contactactivationof intrinsicclottingis classically describedas the interaction of factorsZ and III in the prSSenCaof an activatingsurface. ~owemr, in 1965 auotheractivity,Fletcherfactor,wa8 describsdwhich participates in this phase of clotting(1,2),andrecentlythe 8ystemha8been In194 twonewactivitiesweredescribed shomtobeevenmorecou@ex. whichparticipatein contactactivation:co&a&activation cofactor(CAC) (3) ahd Fitzgeraldfactor (4,5). CAC is the name ¥ to anactivityofrrarsrlplrsPrwhich~ici~te8 in the activationof factorII in the presenceof Platchetr factor,factorXII,
and kaolin. Fitzgeraldfactoris an activitymissingfrom the plasm of a patientwhose coagulation testsdemnstrateda defectin the contactphase of intrinsic cl&ting. This defectcouldnot be attributedto the absenceof anykuowh reactrntin contactactivation - factorII, factorIII,orFletcher factor- 8oFitsgeraldfactormustbe an additioualcontact,factor. It seemed possiblethat CAC and Fitcgtraldfactorore two n8sms for the 8am8 actidescribedbelowwere designedto test thispossibility. vity* The experiments MATRRIAIgARDm!l!HoDS FactorII-F+letcherfactorreagent. Factor= combinedtithl?letcherfactorwa8 s-ted fmmotherknown clottingfactorsinthe firstproteinpeak elutingfrom DRAR-cellulose on chmmatogrrgphy of Al(0R 3 adsorbednomealplasma at pR 7.2 in 0.05 M salt as describedpreviously(6 . The fractionused belowcontained45$factorXI and 3O$Fletcherfactoractivity. FaotorlUI reageht. FactorXII was s-ted fknnotherknown cl&ting factors by a-y Of Al(OH)3adsorbednomal plmm8 on DlW-cellulose using a sli>t modification of the previou8lydescribedtechnique(6). this factorXII fractionuasbroughtto25$amnniumsulfate saturation,thepmcipitatewas dbwrded,ti the supernatantwasbroughtto5O$asm~niumsulfate saturation.The 50% precipitate, cont&ning the factorXII, was dissolved and rechromatographedon DEAg-cellulose.ThepooledfactorxII fraction had about 4% factor XII activity. 451
CONTACT ACTIVATION
452
v01.6,~0.5
CAC reagent was prepared from Al(OH)3 adsorbed normal plasma by DEAEcellulose chromatography, auwnium sulfate precipitation, and chromatography on BioGel A-l. 5m (Bio-Rad Laboratories, Richmond, Calif. ). Details of these procedures have been submitted for publication elsewhere (7). The CAC fraction used in these experiments when tested at about 30% normalplasmaCAC activity had no detectable factor II, Ix, X, XI, XIII, or fibrinogen; 0.s factorVIII; 0.6 factorV; 0.1s factor XII; and much less than 0.15 Fletcher It was purified about 40 fold. factor activity.
Dicalite adsorbed plasma was prepared by chromatographing normal plasma on Dicalit 4200 (Grefco, Inc., Los Angeles, Calif. ) (1 g/ml plasma) equilibrated wit: TES (IV-tris (Hydroxymethyl) methyl-2-aminoethane Sulfonic Acid) buffer (0.05 M TES and 0.1 M IVaCl, pIi 7.35). The unbound protein fraction was pooled and incubated at 37% in glass tubes until the partial thromboplastin time (8) in the presence or absence of kaolin rose to about 150 seconds. Then the pH was adjusted to 7.4, and the adsorbed plasma was stored at -20°C in plastic vials. This reagent contained: 105 mg$ fibrinogen, 609 factor II, 32$ factor V, 60$ factorVIII, 91s factorIX, 749 factor X, 6$ factor XI, 15 factor XII, 0.79 Fletcher factor. The following reagents were added to a plastic clotting tube 0.01 ml cephalin (9) diluted l/l0 in veronal buffer pir 7.35, 0.05 ml factor XI-Fletcher factor reagent, 0.05 ml factor XII reagent, 0.05 ml Dicalite adsorbed plasma, 0.05 ml test substance, and 0.01 ml kaolin (20 mg/ml saline) (Kaolin, RF. Colloidal, Mallinckzodt). The tube was incubated at 37oC for precisely 10 minutes, then 0.025 ml of 25 IN CaCl2 was added and the clotting time noted. This system contained 30$ or more of all known clotting factors.
%Fer noted:
in the o
Fitzgerald factor assap The following reagents were added to a glass clotting tube in the order noted: 0.025 ml kaolin (20 mg/ml saline), O.G25 ml cephalin (9) l/30, 0.05 ml Fitzgerald deficient plasma, and 0.05 ml test substance. After 3 minutes incubation at 37oc, 0.05 ml 40 aM CM& was added and the clotting time noted. Other clotting factor assays. Assays for factors II, V, VIII, Ix, X, XI, XII and Fletcher factor have been described elsewhere (3). Fibrinogen Small amounts of was measured by the method of Blombkk and Blddck (10). fibrinogen were detected by i nnwnodiffusion against rabbit antihuman fibrinogen antiserum (Hyland, Div. Travenol Laboratories, Inc. ). Factor XIII was measured as the ability of a reagent to render a c&t formed in congenital factor XIII deficient plasma insoluble in 5 M urea. RESULTS The effect of various reagents on the CAC and Fitzgerald factor assays no is shown in Table 1. The limits of each system are defined by buffer test substance) and normal plasma as a source of the missing activity tlines l-3). Both assays are corrected by plasmas lacking factor XI, factor XII, or Therefore, the primary defect in the CAC and Fletcher factor (lines 4-6). Fitzgerald factor assayscannot be attributed to a deficiency of one of the In contrast, Fitzgerald deficient three known contact activation factors. plasma &es not correct either assay (line 7). Hence, the activity missing in Fitzgerald deficient plasma is also lacking in the CAC assay. Furthermore, partially purified CAC reagent, which lacks other known clotting factors, corrects the defective clotting in both the CAC and Fitzgerald assays (lines 8
and 9).
Vo1.6,No.5
CONTACT
ACTIVATION
453
TABLE I. CAC and FitzgeraldFactorAssays ClottingTime (set) Fitzgerald CAC
Test Material Buffer 2. Normalplasmal/10 Normalplasmal/20 Z: XI def. pl. l/10 XII def. pl. l/10 2: Fletcherdef. pl. l/10 Fitzgeralddef. pl. l/10 87: CAC reagent-lO$ 9* CAC reagent- 5%
1.
101 :; 44 45 45 96 45 52
284 46 '$ 55: 218 49 59
DISCUSSION The conceptthat the contactphase of intrinsicclottingutilizesadditionalactivitiesbesidesfactorsXI and XII has evolvedover the past 15 betweencrudeplasmafracyears. In 1960 no reactioncouldbe dearonstrated tions containingfactorXI or factor XII, and it was suggestedthat noxvnal plasmacontainedan additionalactivitylackingin the partiallypurifiedreagents (6). When Fletcherfactorwas described,it was postulatedthat Fletcher factormight be the activitylackingin the earlierexperiments(2). However,laterexperiments showedthat this was not the case and that plasma must containa fourthcontactactivationcofactor(3). Furthersupportfor an additionalactivityparticipating in contactactivationwas suppliedby the identification of Fitzgeraldtraitplasmawhichlackeda previouslyunidentifiedcomponentof the contactactivationsystem(4,5). The experiments presentedaboveprovidetwo linesof evidencesupporting the identityof CAC and Fitzgeraldfactor: 1) both assay systemslack a common activity,and 2) a partiallypurifiedreagentseparatedfrom otherknown clottingfactorscorrectsthe defectin both substrates.Therefore,it appears that thereare at leastfour activities whichparticipatein the contact activationof intrinsicclotting: factorXII, factorII, Fletcherfactor,and WC-Fitzgeraldfactor. A role for CAC-Fitzgerald factorin contactactivationhas been establishedboth in a syntheticsystem(MC!)and in an expe%?iment of nature (Fitzgeraldtrait). The mechanismof actionof this activityremainsto be defined. Many reports,includingtwo very recentones (XL&?), appearto demonstratedirectactivationof factorXI by activatedfactorIII, withoutparticipationof MC-Fitzgeraldfactor. Two possibleexplanations of the apparent contradiction come to mind: 1) WC-Fitzgeraldfactormay participatein a preliminaryactivationof factorXII or factorXI, whichhas alreadytaken place in reagentsused for other experiments, or 2) MC-Fitzgeraldfactormay influencethe rate of interaction of factorsXI and XII - a very slow reaction occurringin its absence. Thesepossibilities remainto be tested.
These studieswere supportedby a grant from the AmericanHeartAasociation-Greater Los AngelesAffiliate,and were completedduringthe tenureof
454
CONTACT ACTIVATION
Vo1.6,No.5
8n Estsblished ~nvestigatorship of the AmericanHeart Associationto Dr. SchiffUl8b. -Es
1. HATHAWAY,W.E.,BEIliASEH, L.P. and HATHAWAY,H.S. Evidencefor 8 new plasm8 thmmbopl8stinfsctorI. Case report,coagulation studiesand physiocheuic8lproperties. Blood.26, 52l,1965. 2. HATHAWAY,W.E. 8nd ALSEVER,J. The relationship of "FletcherFactor” fsctorsXI and XII. Brit.J. Haemt. l8, 161, 1970.
to
S. 8nd LEE, P. Preparation, chsrscterization, and activ8tion 3. SC-, of a highlypurifiedfactorXI: Evidencethat a hithertounrecognized p&m8 activityparticipstes in the interaction of fsctorsXI and XII. Brit.J. Hacmrt.3, 101, 1974. 4. WALDMMN, R. 8nd ABRAHAM,J.P. Fitzgeraldfsctor:A heretoforeunrecognized co8gul8tion fsctor.AmericaaSocietyof Hematology,17th Annual Meeting,(Abstrrct), p* 74, 1974. O.D.,WALDMMN, R. and ABRAHAM,J.B. ImpairedHagems 5. SAIIQ,Ii.,-, isctor (FactorZI)-medi8tedre8ctionsin "Fitsgemild Trait".American Societyof Hesmtology,17th Annu8lMeeting,(Abstract), p. 74, 1974. 6. m, S., RAPApX!!,S.I.,WARE,A.G. and MEHL, J.W. Sepsrationof pl8sm8thmmbopl8stinantecedent(PTA)8nd Hagtman factdr (HF) from human pl8sm8.Pmt. Sot. Exper.Biol.Med. lC&. 453, 1960. and p8rtislpurifica7. SCIUFFMAW,S. and LEE, P. Assay,chsrscterization, tion of contact8ctiv8tioncof8ctor(CAC).Submittedfor publication. 0. PROCZQR,R.R. and RAPAXXQ, S.I. The partialthrombopl8stin time with kralin.AM. J. Clin. Psth.36, 212, 1961. P.A. A simple,specificone-stage 9. HJORT,P., RAPAPORT,S. I. 8nd OWREIV, pmthranbin 8ss8yusingRussell'sviper venomin ceph8linsuspension. J. I&J. & Clin.Med. 46, 89, 1955.
10. BKMB&K, B. and BKMlBkX, M. Purification A&. mmi. 2, 415, 1956.
of human 8nd bovinefibririogen.
ll. HICK, L.W. and KAPLAN,A.P. Substratesof H8gemn factor.I. Isolation 8nd chanrcterization of human factorXI (m) end inhibitionof the activ8ted enzymeby o(,antitrypsin. J. Exper. Med. 140, 1615, 1974. 12. MDVAT,H.Z. and &ZE-AEWAR, A.H. The cont8ctphase of blood cosgulation: clottingfactorsXt 8nd XII, theirisolationand interaction. J. U&b. & Cl&l.Med. 84, 861, 1974. Reprintrequeststo Dr. schiffam, USC MedicalSchool,Los Angeles,Calif.