Prothrombin Perija: A new congenital dysprothrombinemia in an Indian family

THROMBOSIS RESEARCH 44; 587-598, 1986 0049-3848/86 $3.00 t .OO Printed in the USA. Copyright (c) 1986 Pergamon Journals Ltd; All rights reserved.

PROTHROMBIN

PERIJA: A NEW CONGENITAL DYSPROTHROMBINEMIA IN AN INDIAN FAMILY

A. Ruiz-S6ez*,

J. Luengo**, A. Rodriguez*, A. Ojeda*** 0. G6mez**, 2. Acurero** * Departamento de Investigaciones. Banco Municipal de Sangre de1 D.F. Caracas, Venezuela. ** Banco de Sangre de1 Estado Zulia. Maracaibo, Venezuela. *** Medicina Experimental. I.V.I.C., Venezuela. (Received 21.3.1986; Accepted in orginal form 11.8.1986 by Editor C.L. Arocha-Pinango)

ABSTRACT A Western Venezuelan indigenous Yukpa(Irapa) family with moderate bleeding and discrepancy in the prothrombin values by biological and The propositus presented biologiimmunological methods is described. cal activityof 2?kby the methods in one-stage including StaphyloElectrophoretic migration of the procoagulase end Echis carinatus. thrombin in plasma was normal, while the serum only showed one precipitation arc instead of the three of normal serum. The propositus appeared to be homozygous for the abnormal prothrombin which we have denominated Prothrombin PerijB.

INTRODUCTION Prothrombin is a vitamin K-dependent protein, synthesized by the liver parenchimal cells and composed of a single polypeptide chain with a molecular weight of 72,500 daltons(1). Its activation results in the formation of an enzyme, thrombin, which is composed of two polypeptide chains. Congenital prothrombin deficiency is an infrequent problem in the molecular True deficiencies (hypoprothrombinemias) and pathology of coagulation factors. the production of abnormal molecules (dysprothrombinemias) have been reported. The differential diagnosis between the two is based on the finding of normal or considerably higher quantities of prothrombin by immunological procedures with a decreased biological activity which defines an abnormal molecule. Up to now, 15 families with functional alterations of prothrombin (2-16) have been reported, whose inheritable patterns are considered to be autosomal recessive, affecting both sexes. In some reported cases,a double heterozygosity, hypoprothrombinemia/dysprothrombinemia has been demonstrated, as in the cases of prothrombins Quick (7), Molise (8), Houston (II), Metz (13) and Habana (14). It has been possible to locate the functional defect in some prothrombins. In prothrombins Barcelona (17) and Madrid (18) the defect is related to an imKey words:

Prothrombin,

Congenital

Dysprothrombinemia, 587

indians.

588

CONGENITAL DYSPROTHROMBINEMIA

Vol. 44, No. 5

pairmentof proteolysisat Arg 273-Thr.In prothrombinCardeza (19),factorXa can not bind to the prethrombin2 region and initiatecleavageat Arg 322-Ile. There is a defect in the active site of the thrombinin prothrombinsQuick(20) Metz (211, Salakta (15) and Molise (22). This report deals with a new case of abnormalprothrombinin four members of an indigenousYukpa (Irapa)family,residentsof a forestalregion in Western Venezuela. CASE REPORT

A seventeen-yearold male Yukpa indian showed a mild bleeding tendency characterizedby epistaxis.Physicalexaminationwas normal and there was no clinicalor laboratoryevidenceof hepaticdisease.Family historyshowed epistaxisin the father,mother, two sister and a brother and hypermenorrhea in the mother and two sister. No evidenceexists of consanguinity,but they belong to a small isolatedethnic group. Questioningwas difficultbecause of their dialect.The family tree is shown in Fig. 1.

Non Studied

symptomatic

Heterorygotes Homzpgotes

FIG. 1 Family Tree MATERIALS

AND METHODS

Studieswere performedon the propositus,father,motherand a sister, on two differentopportunitiesand comparedwith values obtainedin our laboratory from blood samples of at least 20 normal donors. Blood sample: Blood sampleswere collectedwith plasticsyringesin a l/IO volume of 3.2% sodium citrate.Plateletpoor plasma was obtainedby centrifugation at 2,500g and was tested immediatelyor kept frozen at -SO%, being

Vol. 44, No. 5

CONGENITAL DYSPROTHROMBINEMIA

589

thawed at 37% before use. An additionalsample from the proposituswas collected in a l/IO volume of O.lM sodium oxalate and adsorbedwith barium sulfate. Serum was obtainedfour hours after clottingat 37QC. Coagulationstudies: These includedbleeding time, plateletcount, prothromthrombintime (ThT),activatedpartial thromboplastintime bin time (PTI, (PW, assays for coagulationfactorsV, VII, VIII, IX, X, XI, XII by the one stage method, factor XIII screen and the screeningfor the presenceof inhibitors by standardprocedures(23). Fibrinogenwas measuredby the Cullen-van Slike method (24). The thromboplastingenerationtest by the Biggs and Douglas method (25) utilizinga normal generationmixture and as substrate,plasma from the propositus,a sister and a normal control. Prothrombinstudies:A.- The biologicalactivityof prothrombinwas measured by the followingmethods:l.- Stypven-cephalin time with Russel'sviper venom in Cephalin (SigmaChemicalCo, USA). 2.- One stage classic (26). 3.- One stage with Taipan viper venom (DiagnosticReagents,LTD. Thame Oxon, England) by the Denson method (27). 4.- Staphylocoagulase (DiagnosticaStago. Asniers, France) accordingto the Souliermethod (28). 5.- Echis carinatusvenom using Ecarine (DiagnosticaStago. Asniers,France).6.- Two stage method according to Biggs and Douglas (29). 7.- Amidolyticactivitywith chromogenicsubstrate S-2238 (AB Kabi Diagnostic.Stockholm,Sweden) accordingto the method of Girolami (30). In the propositus'plasma a possibleinhibitoryeffect on the normal prothrombinactivationwas studied using a I:1 mixture of normal and propositusplasma in the followingbiologicalassays: one stage classic,one stage with Taipan viper venom, Staphylocoagulase and with Echis carinatus venom. B.- Immunologicalstudies.Using a human antiprothrombinantiserum (AtlanticAntibodies.Scarborough,Maine, USA) the followingtests were performed:l.- Quantitativeimmunoelectrophoresis which includedbarium sulfateadsorbedplasma of the propositus,accordingto Laurel1 (31). 2.- Immunodiffusion accordingto Ouchterlony(32). 3.- Single radial immunodiffusionaccording to Mancini (33). 4.- Crossed Immunoelectrophoresis accordingto Laurel1 (34) of the plasma and serum, with and without the additionof 2mM calcium in the buffer of the first dimension.5.- Immunoelectrophoresis according to the Girolamimodification(6) of the techniqueof Scheidegger(35). RESULTS The coagulationstudy of the propositusand his family is shown in Table 1. An importantprolongationof the PT and PTT was observedin the propositusand a moderate prolongationof both tests in the sister. An inhibitoryeffect could not be demonstrated.The specificassays of factors I, V, VII, VIII, IX, X, XI, XII and XIII were normal in all members of the family. The results of the Thromboplastingenerationtest are demonstratedin Fig. 2 where the normal intrinsicprothrombinactivationcomplexwas unable to convert the prothrombinof the propositusto thrombin.In the sister, the formationof thrombin in the same study was retarded. The results of the quantificationof prothrombinby biologicaland immunological methods are shown in Table 2. The propositusshowed a very low activity by all methods includingStaphylocoagulase and Echis carinatus.A discrepancy exists with the chromogenicmethod. The parents showed abnormalvalues, but not as low as the propositus.The sister behaved differentlybut nevertheless her results resemblemore her parentsthan the propositus. The quantitativeimmunoelectrophoresis (Laurell)results are shown in Fig. 3, where the presenceof antigenicmaterial is observed:70% of the normal range in the propositusand from 75 to 84% in the other members of the family. There is no immunologicalprotein detectablein the propositus'oxalatedplas-

CONGENITAL DYSPROTHROMBINEMIA

590

Vol. 44, No. 5

ma after adsorptionwith barium sulfate.

TABLE 1 CoagulationStudiesin Four Family Members Test

Control

Bleedingtime (min) Plateletcount x109/1 Thrombintime (set) Prothrombintime (set) PTT kaolin time (set) Fibrinogen mg/dl Inhibition Factor V (X) Factor VII (X) Factor VIII (X) Factor IX (X) Factor X (7;) Factor XI (X) Factor XII (X)

Propositus

Father

Mother

Sister

2-7

4

3

5

3

150-350

221

285

320

255

15-17

15.2

15.2

15.6

15.8

13-15

52

14.2

14.2

18.4

32-38

101

32

31

47

160-450

204

275

280

230

_71-150 58-125 48-150 52-130 68-128 50-155 60-150

neg 90 100 100 90 95 90 100

_100 95 100 90 a5 95 100

__ 95 97 100 95 90 100 100

neg 93 100 105 95 100 100 100

Mancini'ssingle radial immunodiffusion confirmedthe presenceof antigenic materialwith values between 80 and 90X of the normal range for the propositus and his family.Plasma immunoelectrophoresis was normal and the studies of qualitativedouble diffusionshowed identitybetween the plasma of the propositus, his family and the control.Similar resultswere found in the serum. Bidimensionalimmunoelectrophoresis of the plasma is shown in Fig. 4 where a precipitationarc similar in mobilityto that of the controlbut lower in height may be observedin the propositus.Similarresultswere found in the family (not shown). In Fig. 5, the bidimensionalimmunoelectrophoresis of serum is shown. In normal serum, three precipitationarcs are formed:one major and two minor. In the serum of the propositusand his parents,only one arc with mobility similar to the major arc of normal serum may be observed.Nevertheless,when the serum sample concentrationwas increasedin the bidimensionalsystem we observeda small faint arc in both parents,correspondingto the minor and more anodic arc of the control serum (not shown). In the serum of the sister, althoughthe minor ones are lower three precipitation arcs may be observed, than those of normal serum, but higher than those of her parents. The addition of calcium to the buffer of the first dimensiondoes not alter the immunoelectrophoretic

shown).

pattern

of the plasma and serum in the bidimensional

system

(not

Vol. 44, No. 5

CONGENITAL DYSPROTHROMBINEMIA

50

591

1

40.

‘ii * 30” : c

20-

z ._ = S 0

IO-

0.

1

6

2

Incubation

Time

(mid

FIG. 2 Thromboplastin Generation

Test

Generation mixture: 0.3 ml normal adsorbed plasma, 0.3 ml normal serum, 0.3ml normal platelet and 0.3 ml 0.025M CaC12. Tubes were incubated at 37%. At one minute intervals, 0.1 ml were removed and added together with 0.1 ml 0.025M CaCl to 0.1 ml of substrate plasma previously placed at 37%. Subs 2rate plasma: D---_-O control; 0-O propositus; e-0 sister

Prothrombin Test Stypven-Cephslin time (set) Prothrombin X Biological: one-stage two-stage Staphylocoagulase Echis carinatus Taipan venom S-2238 Immunological: Laurel1 Mancini

Levels

TABLE2 in the Family using

Control

10

Propositus

Different Father

Techniques Mother

Sister

34

__

2

30 __ 30 25 25 _-

15 -_ 25 20 16 -_

82 88

75 85

58-l 20 100 72-l 20 70-I 20 69-110 89-l 02

<:: 2 2 37

35 __ 30 30 27 64

63-117 68-l 25

70 80

84 90

CONGENITAL DYSPROTHROMBINEMIA

Vol. 44, No. 5

FIG. 3 Laurel1 Immunoelectrophoresis l-4: Control plasma (undiluted) and diluted l/2,1/4,1/8. 5: Sister plasma (undiluted) 6: Father plasma (undiluted) 7: Propositus plasma (undiluted) 8: Mother plasma (undiluted) 9: Control adsorbed plasma (undiluted) 10: Propositus adsorbed plasma (undiluted)

FIG. 4 Crossed Immunoelectrophoresis of Plasma C: Control; P: Propositus

FIG. 5 Crossed Immunoelectrophoresis of Serum C: Control; P: Propositus F: Father; M: Mother; S: Sister

5 __

50

35

Barcelona

Padua

Brussels

10 -_

__

10

3

10

1

5

Madrid

Denver

Houston

10

17

5

15

1.7

2

Habana

Salakta

Poissy

Perij&

2 14 94

50 __ 50

__

<12

37

2

N A N

17

37

50 70

A2

__

__

-_

100

70

18

20

50 17

12

N

A

AC __

13 52

N

70 --

AC

N

50

23 __

N

CIP

_-

103

45

__

96

__

Chromogenic Substrates x

3 -_

10

34

71

__

2

100

32

93

100

100

Immunologic assay "m

45

__

9

3

22

A

105

50 --

100

80

__

__

__

100

_-

100

100

Taipan venom %

Echis carinatus x

Staphylocoagulase %

Al

Acl

A5

US

Modifiedfrom Shapiro and McCord (19)

of SeN; Normal; A: Abnormal;CIP: Crossed Immunoelectrophoresis of Plasma; CIS: Crossed Immunoelectrophoresis rum; c: cathodic;1: one precipitationarc; 2: two arcs; 5: five arcs.

10

42

Gainesville

24

Metz

11

12

Moliee

2

2

48

50

20

Quick

San Juan

50

--

Cardeza

12

two stage #

one stage x

Prothrombin

TABLE 3 ReportedCongenitalDyaprothrombinemias

594

CONGENITAL DYSPROTHROMBINEMIA

Vol. 44, No. 5

DISCUSSION In the family reportedhere, the diagnosisof Dysprothrombinemia is based on the significantdiscrepancybetween the biologicaland immunologicalactivity of prothrombinin the presenceof normal values of the other vitaminK dependentfactorswithout clinicaland laboratoryevidenceof hepatic disease. In the geneticalterationsof the prothrombin,true hypoprothrombinemias and dysprothrombinemias are included.In the former category,some cases have been reportedwith a lack of correlationbetween the clinicaland the laboratory findings(36) and the presenceof symptomaticheterozygous(37). In the case of dysprothrombinemias, the clinicalmanifestationsvary from asymptomatic patientsas in the case of prothrombinCardeza (2) and Salakta (15) to the very symptomaticas in the case of prothrombinPoissy (16). In the family describedhere, the proposituspresenteda lack of correlation between the bleedingtendencyand the laboratorytests showingmoderate clinicalmanifestationsand an importantprolongationof PT and PTT in the absenceof an inhibitoryeffect. The parentswere symptomaticwith normal PT and PTT and functionalprothrombinactivitybetween 25 and 35% of the normal range, althoughthe minimum value of biologicalactivityof prothrombinconsiderednecessaryfor normal hemostasisis 25% (19). The sister showed the same bleedingtendencyas the parentsbut presentedonly moderate alterations of PT and PTT. A great variationin laboratorytests has been found in the abnormalprothrombinsdescribed,which is demonstratedin Table 3, where the reported dysprothrombinemias are compared.In the majorityof these cases, normal values or values similar to those found by immunologicalmethods were obtained when an activatingagent such as Staphylocoagulase is used. This could indicate that these abnormalprothrombinsare capable of formingcomplexeswith Staphylocoagulase and developingnormal biologicalactivity (38). This new prothrombinshows some similarityto prothrombinQuick (7), Houston (11) and Salakta (15) such as low values of biologicalactivityfor all methods,and with prothrombinsPadua and Molise in which a discrepancyof some prothrombinassays with the chromogenicmethod have been described (30). However,it was not possibleto do a definitivecomparisionbetween them, because some tests were not performedon the other dysprothrombinemias, especially the serum bidimensionalimmunoelectrophoresis. ProthrombinMolise (8) presentsonly one precipitationarc in serum bidimensionalimmunoelectrophoresis, but the migrationis more cathodicthan the correspondingone in normal serum. We have denominatedthe describedabnormalprothrombin:PERIJA, keeping in mind the name of the mountainchain where the proposituslives. The propositusof the prothrombinPERIJA presentedvery low values with all one stage methods used includingStaphylocoagulase and Echis carinatus venom with a discrepancybetween these and the chromogenicmethod. This could indicatethat thrombinis formed at a slower rate or that there exists a defect in the active site of thrombin.Our inabilityto find antigenicmaterial in adsorbedplasma from the propositusas well as the lack of modificationin the electrophoreticpatternin the bidimensionalsystem in the presenceof calcium suggeststhat no calcium-bindingdefect exist, which has been shown to be the functionof fragment1 of the prothrombinmolecule (39). During blood coagulation,prothrombinis split in a complex manner resulting in the formationof thrombinand of three immunochemically detectable fragments:one major, which can be a double fragmentor which can suffer posterior fragmentation;and anotherminor one, with variablecharacteristics, which seems to be producedby posteriorconversionof the major fragments during coagulation(40,41).It is known that the major fragmentcorresponds to fragmentI-2 (42) describedpreviouslyas a "pro-piece"(43).

Vol. 44, No. 5

CONGENITAL DYSPROTHROMBINEMIA

595

In the propositusof the prothrombinPERIJA, only the arc correspondingto the fragmentl-2 was observedin the serum bidimensionalimmunoelectrophoresis, which apparentlydoes not suffer posteriormodification.In the sister, the minor arcs correspondingto normal serum, althoughpresent,are abnormal in height. In this regard it is importantto note the fact that in the parents, two arcs were observedonly when the volume of the serum sample,was increasedby 50%. We may concludethat the propositusis homozygousand the parents and the sister heterozygousfor this abnormality.The differencebetween the sister'sand the parents'bidimensionalpatterncould be due to a differentexpressionof the abnormalgene assessedwith this method, or to the fact that the sister is not the geneticproduct of the two parents,or that there exists in the family a double abnormalcondition.It should be pointed out that the family studied belongs to a small isolatedindigenous group, where osteochondrodysplasia and several other metabolicdisordershad been describedby other investigators(44). Furthermore,the possibilityof a genetic polymorphismin certain proteinssuch as transferrinand albuminis being studied in this group. Studies are in progressto determinethe molecularstructureof this abnormal prothrombinand the presenceof geneticalheterogenicityof prothrombin in this population,as has been reportedfor populationsof the Pacific Islands,where the presenceof a varianceof prothrombindue to structural alterationlocalizedin a profragmentand not on the enzymaticallyactive thrombinmolecule,has been demonstrated(45).

ACKNOWLEDGEMENTS

We thank Dr. N-Bosch,Dr. C.L. Arocha-Piiiango and Dr. P. Taylor for their helpful discussionsand comments. REFERENCES

1. JACKSON,

C.M. Biochemistryof prothrcmbinactivation.In: Haemostasisand Thrombosis.A.L. Bloom and D.P. Thomas (Eds.) Edinburgh-London-Melbourne and New York: ChurchillLivingstone,1981, pp. 140-156.

2. SHAPIRO, S.S., MARTINEZ, J. and HOLBURN, R.R. Congenitaldysprothrcmbinemia: an inheritedstructuraldisorderof human prothrombin.J. Clin. Invest., 48, 2251-2259,1969. 3. JOSSO, F., MONASTERIO DE SANCHEZ,J., LAVERGNE,J.M., MENACHE,D. and SOULIER,J.P. Congenitalabnormalityof the prothrombinmolecule (factor II) in four siblings:prothrombinBarcelona.Blood, 38, 9-16, 1971. 4. SHAPIRO,S.S., MALDONADO,N.I., PRADERA,J., McCORD, S. ProthrombinSan Juan: A complex new dysprothrombinemia. J. Clin. Invest., 53, 73A, 1974. 5. KAHN, M.J.P. and GOVAERTS,A. ProthrombinBrussels:a new congenitaldefectiveprotein.Thromb. Res., 5, 141-156,1974. 6. GIROLAMI,A., BAREGGI,G., BRUNETTI,A. and STICCHI,A. ProthrombinPadua a "new" congenitaldysprothrombinemia. J. Lab. Clin. Med., 84, 654-666, 1974.

596

CONGENITAL DYSPROTHROMBINEMIA

Vol. 44, No. 5

7. OWEN, C.A., HERRIKSEN, R.A., McDUFFIE, F.C. and MANN, K.G. Prothrombin Quick: a newly identified dysprothrombinemia. Mayo Clin. Proc., 53, 29-33, 1978 8. GIROLAMI, A., COCCHERI, S., PALARETI, G., POGGI, M., BURUL, A. and CAPPELLATO, G. Prothrombin Molise: a new congenital dysprothrombinemia, double heterozygosis with an abnormal prothrombin and "true" prothrombin deficiency. Blood, 52_, 115-125, 1978. 9. MONTGOMERY, R., CORRIGAN, J., CLARKE, S. and JOHNSON, J. Prothrombin Denver. A new Dysprothrombinemia. Circulation. %,279,1980 (abstr.) IO. BEZEAUD, A., GUILLIN, M.C., OLMEDA, F., QUINTANA, M. and GOMEZ, N. Prothrombin Madrid: a new familial abnormality of prothrombin. Thromb.Res. 16, 47-58,1979. 11. WEINGER, R.S., RUDY, C., MOAKE, J.L., OLSON, J.D. and CIMO, P.L. Prothrombin Houston: a dysprothrombinemia identifiable by crossed immunoelectrofocusing and abnormal Echis carinatus venom activation. -Blood, 55, 811816, 1980 12. SMITH, L.G., COONE, L.A.H. and KITCHENS, C.S. Prothrombin Gainesville. A dysprothrombinemia in a pair of identical twins. Am. J. Hematol. 11, 223231, 1981. 13. JOSSO, F., RIO, Y. and BEGUIN, S. A new variant of human prothrombin: prothrombin Metz. Demonstration in a family showing double heterozygosity for congenital hypoprothrombinemia and dysprothrombinemia.Haemostasis,l&309316, 1982. 14. RUBIO, R, ALMAGRO, D., CRUZ, A. and CORRAL, J.F. Prothrombin Habana: a new dysfunctional molecule of human prothrombin associated with a true prothrombin deficiency. Brit. J. Haematol. 54, 553-560,198O. 15. BEZEAUD, A., DROUET, L., SORIA, C. and GUILLIN, M.C. Prothrombin Salakta: An abnormal prothrombin characterized by a defect in the site active of thrombin. Thromb.Res., 34, 507-518, 1984. 16. TAPON BRETAUDIERE, DUMONT, M.D., FISCHER, A.M., BROS, A., CHASSEVENT, J. and AUFEUVRE, J.P. Prothrombin Poissy: A new variant of human prothrombin. Thromb. Haemostas., 50, 250, 1983. 17. RABIET, M.J., ELION, J., BENAROUS, R., LABIE, D. and JOSSO, F. Activation of prothrombin Barcelona. Evidence for active high molecular weight intermediates. Biochem. Biophys. Acta, 584, 66-75, 1979. 18. GUILLIN, M.C. and BEZEAUD, A. Characterization of a variant of human prothrombin, prothrombin Madrid. Ann. N.Y. Acad. Sci., 370, 414-425, 1981. 19. SHAPIRO, S.S., McCORD, S. Prothrombin. In: Progress in Hemostasis and 1978, 5, 177-210. Thrombosis. T.H. Spaet (Ed.)New York: Grune Stratton, 20.

HENRRIKSEN, R.A., OWEN, W.C., NESHEIM, M.E. and MANN, K.G. Identification of a congenital dysthrombin, Thrombin Quick. 3. Clin. Invest., 66, 934-940 1980.

Vol. 44, No. 5

CONGENITAL DYSPROTHROMBINEMIA

597

21. RABIET,M.J., ELION, J., LABIE, D. and JOSSO, F. Purificationand partial characterization of a new variant of human prothrombin:prothrombinMetz. FEES Letters,108, 287-291,1979. 22. RABIET,M.J. ProthrombinMolise: A muttant prothrombincharacterizedby a defect in the thrombindomain. Thromb.and Haemostas.54, 46, 1985. 23. CAEN, J., LARRIEU,M.J. and SAMAMA,M. L'hemostaseMBthodesd'exploration et diaqnosticpratique.Paris: 1'ExpansionScientifique,1975. 24. CULLEN,G. and VAN SLIKE, D. Determinationof the fibrin,globulinand albuminnitrogenof'bloodplasma. J. Biol. Chem.,41, 587, 1930. 25. RIGGS, R and DOUGLAS, A.S. Path.,6, 15-22, 1953.

The thromboplastingenerationtest. J. Clin.

26. OWREN, P.A. A quantitativeone-stagemethod for the assay of prothrombin. Stand. J. Clin. Invest.,1_, 81, 1949. 27. DENSON, K.W., EARRET, R. and BIGGS, R. The specificassay of prothrombin using Taipan snake venom. Brit. J. Haematol.,2, 219-226,1971. 28. SOULIER,J.P., PROU WARLELLE,0. Etude comparativedes taux de cofacteur de la Staphilocoagulase (CRF) et des taux de Facteur II (Prothrobine) dans diversesconditions.Nouv. Rev. Fran. HBmatol.,6, 623-636,1966. 29. BIGGS, R. Human blood coaqulation.Hemostasisand Thrombosis.Blackwell ScientificPublications,Oxford, 1972, pp 612-613. F. and SAGGINI,L. Chromogenic 30. GIROLAMI,A., PATRASSI, G., TOFFANIN, Substrate(S-2238).Prothrombinassay in prothrombindeficienciesand abnormalities:lack of identitywith clottingassays in congenitaldysprothrombinemias.Am. J. Clin. Path.,74, 83-87, 1980.

31. LAURELL,C.B. Quantitativeestimationof proteinsby electrophoresisin agarosa gel containigantibodies.Anal. Biochem.,15, 45-52, 1966. 32. OUCHTERLONY,0. Immunodifussion and immunoelectrophoresis. In: Handbook of ExperimentalImmunology.D.N. Weir (Ed.) Oxford:BlackwellScientific Publications,1967, pp 655-706. 33. MANCINI,G., CARBONARA, 0. and HERMANS, JF. Immunochemicalquantification of antigensby single radial immunodiffusion. Immunochemistry, 2_,235-254 1965. 34. LAURELL, C.B. Antigen Antibodycrossed electrophoresis. Anal. Biochem.,lO 358-361,1975. 35. SCHEIDEGGER,J.J. Une microm&hode de l'immunoelectrophorese. Int. Arch. Allergy.,1, 103-110,1955. 36. GILL, F., SHAPIRO, S and SCHWARTZ, E. Severe congenitalhypoprothrombine1978. mia. J. of Ped., 93, 264-266, 37.

GIROLAMI, A. The hereditarytransmissionof CongenitalTrue Hypoprothrombinemia.Brit. J. Haematol.,2, 695-703,1971.

598

CONGENITAL DYSPROTHROMBINEMIA

Vol. 44, No. 5

38. MAMMEN, E. Congenital Coagulation disorders: Factor II abnormalities. Sem. Throm.Hemoa., 2, 13-16, 1983. 39. GITEL, S., OWEN, W., ESMON, CH. and JACKSON, C. A polypeptide region of bovine prothrombin specific for binding to phospholypids. Proc. Nat. Acad. Sci. USA, _70, 1344-1348, 1973. 40. GANROT, P.O., NILEHN, J.E. Prothrombin fragmentation during coagulation of whole blood and plasma. Stand. J. Clin. Lab. Invest., 24, 15-21, 1969. 41. GANROT, P.O., STENFLO, S. Prothrombin derivatives in Human serum. Isolation and some properties of the Non-Thrombin Fragments. Stand. J. Clin. Lab. Invest., 26_,161-168, 1970. 42. ARONSON, D.L., STEVAN, L., BALL, A. and FRANZA, B.R. Generation of the combined prothrombin activation peptide (F I-2) during the clotting of blood and plasma. J. Clin. Invest., 60, 1410-1418, 1977. 43. SHAPIRO, S.S. Human prothrombin activation: Immunochemical study. Science 162. 127-128. 1968. 44. ARIAS, S. Etiologia mtiltiplede1 enanismo entre 10s indios Yukpa(Irapa) de la Sierra de PerijB, llamados Pigmoides. Bol. Indigenista Venezolano, 17, 49-70, 1976. 45. BOARD, P.G., COGGAN, M. and PIDCOCK, M.E. Genetic heterogeneity of human prothrombin (F II). Ann. Hum. Genet., 46, l-9, 1982.