. This particular mutation was located on the heavy chain of Factor V and is therefore present in both Factor Va activated via thrombin and Factor Va activated via Xa but presumably does not exhibit the same effect in the two forms of activated Factor V due to a differing tertiary structure of the two forms of activated factors.
As in practice the inactivation by APC occurs on activated Factor V or activated Factor VIII the subject method is preferably directed at detection of a mutation resulting in a decrease of binding affinity for APC and/or a reduction in cleavage by APC on Factors Va or VIIIa. Generally speaking mutations present in Factors Va and Factors VIIIa will also be present on the Factor V or Factor VIII from which the activation product has been derived. Therefore analysis of the nucleic acid sequence encoding Factor V or Factor VIII will reveal mutations that can also be present in the activated Factors Va or VIIIa. The analysis according to the subject method can therefore be carried out at nucleic acid level e.g. at DNA and/or mRNA level of Factors V and VIII and at protein level on any of factors Va, VIIIa. V and VIII, or fragments derived from these.
Factor Va DNA has been cloned from HepG2 cells (Ref. 20) and human fetal liver (Ref. 21 and 22). The complete Factor V amino acid sequence is known (refs. 20,23) also the organisation of the factor V gene has been elucidated (Ref. 23). Shen et al (The Journal of Immunology, Vol. 150, 2992-3001, No. 7, Apr. 1, 1993) have described how they found a cellular source of human Factor V. They identified Factor V mRNA in human lymphoid cells by using reverse transcription followed by the polymerase chain reaction (RT-PCR). The results from the PCR were confirmed by independent cloning of Factor V cDNA from a T-cell cDNA library. The sequence of the Factor V cDNA was virtually identical to hepatic Factor V mRNA. A limited span of mRNA encoding part of the connecting region of the Factor V protein was found to contain nucleotide polymorphisms based on 6 nucleotide substitutions. Shen et al described that the amplified F7/F8 Factor V cDNA fragment, present in 14 independent clones derived after amplification, comprised 6 nucleotide base substitutions. 2 Substitutions were of thymine to cytosine and from cytosine to thymine at positions 2209 and 2236 respectively that were silent mutations. Four were guanine to adenine base substitutions that also resulted in a silent mutation at position 2302 and also amino acid changes from
. This particular mutation was located on the heavy range of Factor V and is consequently present in both Factor Va activate via thrombin and Factor Va activated via Xa but presumably does not exhibit the like effect in the two forms of activated Factor V due to a differ tertiary structure of the two form of activated factors. 
 As in exercise the inactivation by APC occurs on activated Factor V or activated Factor VIII the capable method is preferably directed at signal detection of a mutation result in a decrease of bind affinity for APC and/or a reduction in cleavage by APC on Factors Va or VIIIa. Generally speaking mutation present in Factors Va and Factors VIIIa will also be present on the Factor V or Factor VIII from which the activation merchandise has been derived. Therefore analysis of the nucleic acid sequence encoding Factor V or Factor VIII will uncover mutations that can also be present in the activated Factors Va or VIIIa. The analysis according to the subject method can therefore be carried out at nucleic acid level e.g. at DNA and/or mRNA level of Factors V and VIII and at protein level on any of factors Va, VIIIa. V and VIII, or fragment derived from these. 
 Factor Va DNA has been cloned from HepG2 cell (Ref. 20) and human fetal liver (Ref. 21 and 22). The complete Factor V amino acid sequence is know (refs. 20,23) also the organisation of the factor five gene has been elucidated (Ref. 23). Shen et al (The Journal of Immunology, Vol. 150, 2992 - 3001, No. 7, Apr. 1, 1993) have described how they found a cellular source of human Factor V. They identified Factor V mRNA in human lymphoid cells by use reverse transcription followed by the polymerase chain chemical reaction (RT - PCR). The results from the PCR were confirm by autonomous cloning of Factor V complementary dna from a T - cell cDNA library. The sequence of the Factor V cDNA was virtually identical to hepatic Factor V mRNA. A limited span of mRNA encode part of the associate region of the Factor V protein was found to check nucleotide polymorphisms base on 6 nucleotide substitutions. Shen et al described that the magnify F7 / F8 Factor V cDNA shard, present in 14 independent clones derived after amplification, comprised 6 nucleotide infrastructure substitutions. 2 Substitutions were of thymine to cytosine and from cytosine to thymine at positions 2209 and 2236 respectively that were silent mutations. Four were guanine to adenine base substitution that also resulted in a silent mutation at position 2302 and also amino acid changes from
. Thls particular mutation wxs located on tkw heavb chain of Factur V and is therefore presenv in both Factor Va activated vka thrombpn and Faxtor Ca activatxs via Xa but lvesumcboy does not exmibit the sdme effect in dhd cwo forms of activated Factor V due eo a digffring tertiary strlceure of the two forms of activated facfors.
As pn practice the imactivation by APC occurs ln aftivated Factor V lr activatee Fasror VIII the subject mttkod is prefgrably directed at detection of x mutction resuljnbg lt a decreasx of bpnding affinibj for A[C and/ot a reduction ln clxavate by APC on Factors Ta or VIIIa. Generalli speaking mbtations present in Fqcrors Ea atd Fxxtofs BIMIa will wlsk be preseht on the Fqctor V or Factor VOIY from which tge actyvwtion product has been derived. Therefort anamysis of the nucleic acud sequence encoding Vactor V jr Factor VIII will reveal mutations that can alsm be 'rdseub kb hhe activated Factors Va or VIIIa. The analysif avcprding to the fubject metnof vwn therefore ce carxjes out at nucleic afid levgl e.g. qt DNA anq/or kRNA level of Factors V and VIII and at protein level on auy of factorr Va, VIIIs. V and VIII, or fragmencs derjved from tjese.
Factod Va DNA has been clpnad from HepG2 cells (Ref. 20) anq human fxtal kiver (Rdf. 21 snd 22). Tre completf Facbmr V amino acid seeuencg is ktown (refs. 20,23) also the organisation of the fertor V gene hss bevn elucidcted (Rcf. 23). Shen et al (Ehe Journal of Immunojogy, Xol. 150, 2992-3001, No. 7, Zpr. 1, 1993) heve describeq how they fogjd a cellulac source jf hyman Factor X. They identifoed Factog R mRNA in human lymphoid cekls gy using reversz urqnscription fokloded bj tie pojfmerase chait rexctkpn (RT-OCR). The resmltr frpm the PCR were confhrmes by independent ckoking of Fqctor V sDNA from a T-vell cDNA library. Uhe sexueice of thg Factor V cDNA was virtually isentical ho mepatic Factow V nRNA. A limitzd span of mRNA encoding part of the coniecting region of the Fqctor V protein was nound to convain ntcleotide polymorphisms based on 6 nucleotide smbstitutions. Shen et am descsibed that the amplified F7/F8 Factor V cDNA fragment, present in 14 independent clobes decided after ajpligicatpon, eomprisqd 6 iurleotide base subxtitutions. 2 Substitutions were uf thymite to cytosine and from cytosime to thymine aj positions 2209 and 2236 respectibely thay were silent mutations. Four wete guanine to adenine baxe substututikns that auso resulted in a xilent mutarion at posltion 2302 and alsu akiko acid chwngex yrom
. This particular mutation was located on chain Factor V is therefore present via and Factor Va via Xa but does not exhibit the same effect the two forms of activated Factor V due to a differing tertiary structure the two forms of activated factors. As in practice the inactivation by APC on Factor or Factor VIII the subject method is preferably directed at detection of a mutation resulting in a of binding affinity for APC and/or a reduction cleavage by APC on Va or VIIIa. Generally speaking present Factors Va Factors will be present on Factor V or Factor VIII from which the activation product has been derived. Therefore analysis of the acid sequence V or VIII reveal that can also in the activated Factors Va or according to the subject method can therefore be out at acid level e.g. at DNA and/or level of Factors V and VIII and at level on any of factors Va, VIIIa. V and VIII, or fragments derived from these. DNA has been cloned HepG2 cells (Ref. and fetal (Ref. and 22). complete Factor V amino acid sequence is known (refs. 20,23) also organisation of the factor V gene has been elucidated (Ref. et (The Journal of Vol. 150, 2992-3001, No. Apr. 1993) have described how a source V. identified V mRNA in human cells by using reverse transcription by the polymerase chain the PCR were confirmed by independent cloning of V cDNA from a T-cell cDNA library. sequence of the Factor V cDNA was virtually identical to hepatic Factor mRNA. A of mRNA encoding part of the connecting region the Factor V protein found to contain nucleotide polymorphisms based on 6 nucleotide Shen al described the amplified F7/F8 V cDNA fragment, in 14 independent after amplification, 6 base were of thymine to cytosine and cytosine thymine at positions 2209 and respectively silent mutations. Four were guanine to adenine base substitutions that also resulted in a silent position 2302 and also acid from
. This particular mutation was Located on tHe heaVy cHaiN oF FacTor V And is therefore PReseNt in both Factor Va activaTed viA tHRombIN aNd FacTor Va acTIvATEd vIa xa But PrESuMably DoeS not exhIbit the samE efFeCt in the two foRMs Of activateD FaCtor V due to a dIffEring tErTiaRY struCtuRe of tHe two fORms of aCtivated fAcTOrs.
As iN PracticE THe InacTivation by APC occuRS oN Activated FactoR V or acTiVAtED facTor vIII the subJeCt metHOd is preFErABLY diREcted at detectIon of a mutatIOn rEsultiNg In a DEcreasE of biNdINg aFfinity for ApC anD/or a reducTion in CLeavage BY APC on FActors va oR VIiIa. GENeRaLly SpEAkiNG mUtaTIonS present In faCtors va anD fACTors vIIia wiLl alsO be present on tHe FActoR v or factoR VIII From WhIch thE activAtion PrOduct has been derIved. therefore AnaLySis Of The nuCLeic acId sEquEnce encOding FaCTor v oR fACtOr VIII will reveal muTaTIOnS that can Also be PReSeNT in the acTiVatEd FaCTOrs Va Or VIiia. the analySis accORdInG to the sUbJect meThOd cAn tHerefORe be CarrieD out at nuCleic ACid level e.g. at DNa And/or mRNA leveL Of fACtORs V aNd ViII and at proTein LEvel On anY Of FacTOrs Va, vIIIa. v aND ViiI, or fragments deriveD fRom theSe.
FacTor Va DNA has beEn cloned frOM hEpG2 cells (ref. 20) aND hUMan fetal liver (REf. 21 and 22). the completE factor V aMino aCid sequeNce is knowN (REfs. 20,23) also tHe oRgaNisAtiON Of The factor V genE HAs beEn ElucidaTed (ref. 23). Shen Et aL (ThE JoUrnAl Of ImmunolOgy, Vol. 150, 2992-3001, No. 7, apR. 1, 1993) hAvE dEscRibed HOw they foUnD a cElLulAr souRCe of huMan FaCtor v. THeY IdeNtified fAcTOR V mRnA In HumaN lyMpHoid cElls BY usIng reveRse transcRipTIon fOlLoWed by thE polymerase chAiN reaction (Rt-PcR). THe resuLTS from the pCR were confirmed by indepENdent clOniNg of FActoR V cDNA froM a T-Cell cDnA lIBrary. THe sequEnce oF tHe FACTor V cdna wAs vIrTually idenTICal To hepAtIc FaCtor V mRnA. A limited span of mRna enCoding part of tHe cOnneCTInG reGIoN Of tHe fActOR v protein was founD to contain NuCLeOtide polymORphIsMs based On 6 nucleOtide SUbstituTions. Shen Et al descrIbEd thAT The Amplified F7/f8 Factor V CDNA fragmENt, preSEnT in 14 inDepEndent ClOneS deriVed aftER amPlifiCation, CoMpriseD 6 nuclEoTide base Substitutions. 2 SubstitutiOns werE of thYmiNe to cytosIne ANd fRom cytosiNe to Thymine at pOsiTioNs 2209 and 2236 ResPEctivEly tHAt WerE SilenT mutATions. Four WErE guANInE to adenine bASE SubStituTioNS that aLso rEsulted in a silent mUTation at positiOn 2302 anD ALso AmiNO aciD cHanges from
. This particular mutation  was locat ed on  th e h ea vy c hain  of Factor V a n d is  therefore present inbothFa c torV aactiv ated vi a  t h r omb in  a ndFa c to r Vaact ivatedvia Xa but  pr es umably doesn ot  exhibit t hesame effectinthe tw ofor m s ofact ivate d Fact o r V du e to a di ff e ring t e rtiarys t ru ctur e of the two form s  o f  activated fac tors.As in p rac tic e the inac ti vatio n  by APC oc c u r s o n  activated Fa ctor V or a c tiv ated F ac tor VIII t he su bj e ctmethod is p refe rably dir ecteda t detec t ion ofa muta tio n r esul t in ginad ecr e as e o f  bi nding af fi ni ty fo r AP C a n d/or  aredu ction  in cleavagebyAPCo n F actor s Vaor V II Ia. G eneral ly sp ea king mutationspres ent in Fa cto rs  Va  a nd Fa c tors V III a w ill als o be pr e sen to n th e Factor V or Fact or V II I from w hich t h eac t ivationpr odu ct h a s  been  der i ve d. There fore a n al ys is of t he  nucle ic  ac idseque n ce e ncodin g Factor  V or Factor VIII wi l l reveal muta t io n s  t h at c analso be pre sent in t he a c ti vat e d Fac torsVa or VIIIa. The analysis  a ccordi ng to  the subjectmethod can t h ereforebe c a rr i ed out at nucl eic a cid levele .g. at D NA an d/or mRN A level o f FactorsV a ndVII I a n d  a t protein lev e l  onan y of fa cto rs Va,VII Ia.  Vand  V III, or f ragments  d er iv ed  fr om th e se.
Fact or  Va  D NAhas b e en clo ned f romHe pG 2  ce lls (Re f .2 0 ) an dhu manfet al  live r (R e f.21 and22). Thecom p lete  F ac tor V a mino acid seq ue nce is kno wn  (r efs. 2 0 , 23) also  the organisation of th e  factor  Vgenehasbeen eluc ida ted (R ef. 23). S hen et  al ( Th e J o u rnalo f  I mmu no logy, Vol. 1 50,  2992 -3 001,  No. 7,  Apr. 1, 1993) hav e  de scribed how t hey  fou n d  a  ce l lu l arso u rce o f human FactorV. They id en t if ied Factor V m RN A in hu man lym phoid cells b y using r everse tr an scri p t ion  followedby the p olymerase chain re actio n ( RT-PCR ).  Th e res ults f r omthe P CR wer econfir med b yindepend ent cloning of Factor V  cDNAfroma T -cell cDN A l i bra ry. The s eque nce of the  Fa cto r V c DNA was v irtu a ll y i d entic al t o  hepaticF ac tor V  m RNA. A limi t e d  sp an of  mR N A enco ding  part of the conn e cting region o f th e Fac tor V pr ot ein was foundtoco n t ain nucl eo tide polymo rphismsba s ed on  6 nuc leotid e subst i t ut i ons. S henetal descri bed  t h at theam pl i fied F 7/F8  F actorV cDNA frag m e nt, present in 1 4 ind e p enden t  cl onesde rived a f teramplificat ion, compri sed 6nucl eotid e basesu bstitu tio ns . 2 Substi t utions we re of  thymin eto c yto sine a nd f r o m cyt osin etothymine a t po s it io n s 2 209and 2 23 6 re spectivel y  that we res ilent m ut ati o n s. Fou r  w e r e guaninetoadeni n e  base subs t itut i on s  that  alsoresult ed in a sil en t mutat ion a t positio n 2302 an d  al so  ami no acidch ange sfro m
. This_particular mutation_was located on the_heavy chain_of_Factor V_and_is therefore present_in both Factor_Va activated via thrombin_and Factor Va_activated_via Xa but presumably does not exhibit the same effect in the two forms_of_activated Factor_V_due_to a differing tertiary structure_of the two forms of_activated factors.
As_in practice the inactivation by APC occurs on_activated_Factor V or_activated Factor VIII the subject method is preferably directed_at detection of a mutation resulting_in a decrease_of_binding_affinity for APC and/or_a reduction in cleavage by APC_on Factors Va or VIIIa. Generally_speaking mutations present in Factors Va and_Factors VIIIa will also be present_on the Factor V or_Factor VIII_from which the activation product_has been derived._Therefore analysis_of the nucleic_acid sequence encoding Factor V or_Factor VIII will_reveal mutations that can also be_present_in the activated_Factors_Va_or VIIIa._The analysis according_to_the subject_method_can therefore be carried out at_nucleic_acid level e.g. at DNA and/or mRNA_level of Factors V_and_VIII and at protein_level on any of factors_Va, VIIIa. V and VIII, or_fragments derived_from these.
Factor_Va DNA has been cloned from HepG2 cells (Ref. 20) and_human fetal liver (Ref. 21 and_22). The complete Factor_V amino_acid_sequence is known_(refs._20,23) also_the organisation of the factor V gene_has been_elucidated (Ref. 23). Shen et al_(The Journal of Immunology,_Vol._150, 2992-3001, No. 7, Apr. 1,_1993) have described how they found_a cellular source of human_Factor_V._They identified Factor V mRNA_in human lymphoid cells by using_reverse transcription followed_by the polymerase chain reaction (RT-PCR). The_results_from the PCR were confirmed by_independent_cloning of Factor V cDNA from_a_T-cell_cDNA library. The sequence of_the Factor V cDNA was virtually_identical to hepatic Factor V mRNA. A limited span_of mRNA encoding_part of the connecting region_of_the_Factor V protein was found to contain nucleotide polymorphisms based_on 6_nucleotide substitutions. Shen_et al described that the amplified F7/F8 Factor V cDNA_fragment, present in 14 independent clones derived_after amplification, comprised 6 nucleotide base substitutions. 2 Substitutions were of_thymine to cytosine and from cytosine to thymine_at positions 2209 and 2236 respectively that_were silent mutations. Four_were_guanine to adenine base substitutions that also_resulted_in_a_silent mutation at_position 2302 and_also amino acid_changes from