Impaired human tissue factor-mediated activity in blood clotting factor VIINagoya (Arg304–>Trp). Evidence that a region in the catalytic domain of factor VII is important for the association with tissue factor.

T Matsushita,T Kojima,N Emi,I Takahashi,H Saito

doi:10.1016/s0021-9258(17)37292-7

Abstract

A 37-year-old man was found to have an inherited abnormal factor VII (FVII) (designated FVIINagoya) that has a lower FVII procoagulant activity when tested in a one-stage assay using rabbit, simian, or human tissue factor (TF), but surprisingly has a nearly normal activity using bovine TF. DNA sequence analysis of the propositus' FVII gene revealed a C to T mutation that results in a novel amino acid substitution of Arg304 to Trp. Restriction enzyme analysis of the amplified fragment obtained from each family member demonstrated that the patient is homozygous for the point mutation, and the mother and sister of the patient are heterozygous for this mutation. To elucidate the abnormality in FVIINagoya, we compared the recombinant mutant FVII with normal FVII. In the presence of human TF, kinetic analyses demonstrated that the apparent Km values of FVIINagoya for factor Xa-mediated FVII activation and for FVIIa-mediated factor X (FX) activation were 3.6- and 4.5-fold higher than those of normal FVII, respectively. The ligand binding assay to human TF also showed that FVIINagoya had a 5.2-fold larger apparent Kd than normal. In the presence of bovine TF, however, these values showed no difference between normal and mutant FVII. These findings indicate that the major abnormality in FVIINagoya is not a catalytic dysfunction but an impaired complex-formation of human TF.FVII.FX, suggesting the importance of Arg304 in the interaction with human TF.

Highlights

From the $First Department of Internal Medicine, Nagoya University School of Medicine, Nagoya 466, §Aichi Blood Center Japanese Red Cross, Seto, Aichi 489 and the Michi Juridical Foundation for Blood Disease Research, Nagoya 463, Japan
It was tion enzyme analysisof the amplified fragment obtainedproposed that FXa, formedin extrinsic or intrinsicoagulation pfiatrsinaoohrdnmeo.sdTmiesoattohceezherlyufgorcafeoimcduthosailmetyfeobptrmihanteetahimaenebntbpntemooriarunrmdtteaemanmhltiuteoFyttnaVeistnrItioIorFaznwVyt,eigItadIhonNudtnashogtathorfthtomhyeeraimasp, lamowFttiuVehetIenaIcrt-.omifp1-naa3cct)htr.oewFrasausyeMr,tshrtaaehprnemiddpolXyrroe(tFa,etcoIhXtlieyvatainccteodasmcFFtpViXvl)IeiItxbybyfoousorefnmvdFaetVtroiaoIlTInaFoorftdoforeFfroiVstrosmIfIsamFuVabwgsIintItraihat(uTt1edF2se, In the presence of human TF, kinetic analyses demon- [14,15]
Nested two rounds of polymerase chain reaction (PCR) were performed with each FVII-specific primer set (F7-1 and F7-5.1 in the firstround, F7-1.2 and F7-5.3in the second round).ADNAfragment of approximately 1.4 kilobase pairs was isolated from a 1%NuSieve GTG-agarose gel followed by digestionwith EcoRI and SalI.This fragment was cloned into M13mp18 or -19 and completely sequenced.The clone correspondedto positions 27-1481 of hHVII2463, that is thelargest full-length FVII cDNA obtained by Hagen et al [31],but the clone lacked 66 nucleotides[100-165] found in hHVII565 of Hagen etal

Summary

11 Towhom correspondence should be addressed

First Dept. of Internal Medicine, Nagoya University School of Medicine, 65-Tsurumai-cho, Showa-ku,Nagoya466 Japan. Nested two rounds of PCR were performed with each FVII-specific primer set (F7-1 and F7-5.1 in the firstround, F7-1.2 and F7-5.3in the second round).ADNAfragment of approximately 1.4 kilobase pairs was isolated from a 1%NuSieve GTG-agarose gel followed by digestionwith EcoRI and SalI.This fragment was cloned into M13mp or -19 and completely sequenced.The clone correspondedto positions 27-1481 of hHVII2463, that is thelargest full-length FVII cDNA obtained by Hagen et al [31],but the clone lacked 66 nucleotides[100-165] found in hHVII565 of Hagen etal. Becausethis fragment contained the mutation site in exon WII, it human or bovineTF were incubated with 1251-labeled FVII fo1r min at was used as a 3' megaprimer for generation of the mutant cDNA.

RESULTS

A G C TA G C T

DISCUSSION