Role of the B domain for factor VIII and factor V expression and function

Factor V and factor VIII are homologous cofactors in the blood coagulation cascade that have the domain structure A1-A2-B-A3-C1-C2, of which the B domain has extensively diverged. In transfected COS-1 monkey cells, expression of factor VIII is approximately 10-fold less efficient than that of factor V, primarily because of inefficient protein secretion and, to a lesser extent, reduced mRNA expression. To study the functional significance and effect of the B domain on expression and activity, chimeric cDNAs were constructed in which the B domains of factor V and factor VIII were exchanged. Expression of a factor VIII chimera harboring the B-domain of factor V yielded a fully functional factor VIII molecule that was expressed twofold more efficiently than wild-type factor VIII because of increased mRNA expression. Thus, sequences within the factor VIII B domain were not responsible for the inefficient secretion of factor VIII compared with factor V. Expression of a factor V chimera harboring the B domain of factor VIII was slightly reduced compared with wild-type factor V, although the secreted molecule had significantly reduced procoagulant activity correlating with dissociated heavy and light chains and resistance to thrombin activation. Interestingly, the factor V chimera containing the factor VIII B domain was efficiently activated by Russell's viper venum (RVV). A factor V B domain deletion (residues 710- 1545) molecule also exhibited significantly reduced procoagulant activity caused by resistance to thrombin cleavage and activation, although this molecule was activatable by RVV. These results show that, in contrast to factor VIII, thrombin activation of factor V requires sequences within the B domain. In addition, thrombin activation of factor V occurs through a different mechanism than activation by RVV.

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The Structure and Function of Murine Factor V and Its Inactivation by Protein C

Blood ◽

10.1182/blood.v91.12.4593.412a02_4593_4599 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4593-4599 ◽

Cited By ~ 1

Author(s):

Tony L. Yang ◽

Jisong Cui ◽

Alnawaz Rehumtulla ◽

Angela Yang ◽

Micheline Moussalli ◽

...

Keyword(s):

Amino Acid ◽

Protein C ◽

Mammalian Species ◽

Structure And Function ◽

Procoagulant Activity ◽

Factor V ◽

Cleavage Sites ◽

Wild Type ◽

Coding Region ◽

And Function

Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated protein C (APC). To examine the evolutionary conservation of FV procoagulant activity and functional inactivation by APC, we cloned and sequenced the coding region of murine FV cDNA and generated recombinant wild-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,183-amino acid protein. Sequence comparison shows that the A1-A3 and C1-C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino acid identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly conserved B domain. The arginines at all thrombin cleavage sites and the R305 and R504 APC cleavage sites (corresponding to amino acid residues R306 and R506 in human FV) are invariant in all three species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas recombinant murine FV carrying both mutations (R305Q/R504Q) is nearly completely APC resistant. Thus, the structure and function of FV and its interaction with APC are highly conserved across mammalian species.

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Residues Surrounding Arg372, Arg740, and Arg1689 Contribute to the Rates of Thrombin-Catalyzed Cleavage of Factor VIII.

Blood ◽

10.1182/blood.v114.22.847.847 ◽

2009 ◽

Vol 114 (22) ◽

pp. 847-847

Author(s):

Jennifer L. Newell ◽

Amy E. Griffiths ◽

Philip J. Fay

Keyword(s):

Factor Viii ◽

Cleavage Site ◽

Conflicts Of Interest ◽

Specific Activity ◽

Factor Xa ◽

Wild Type ◽

Cleavage Rate ◽

Thrombin Cleavage ◽

Type Factor ◽

Bold Type

Abstract Abstract 847 Hemophilia A results from defects or deficiencies in the blood coagulation protein, factor VIII. Factor VIII circulates as an inactive procofactor that must be cleaved by thrombin or factor Xa at Arg740 (A2-B junction), Arg372 (A1-A2 junction), and Arg1689 (a3-A3 junction) to yield the active cofactor, factor VIIIa. Activation of factor VIII by thrombin is exosite-dependent yielding rates of cleavage at Arg740 ∼20-fold faster than Arg372, while cleavage at Arg1689 appears intermediary to Arg740 and Arg372. The contribution of P3-P3' residues flanking each cleavage site to the mechanism of thrombin-catalyzed cleavage of factor VIII has not been extensively studied. The P3-P3' residues for the 372, 1689, and 740 factor VIII sites are 370QIR*↓SVA375, 1687SPR*↓SFQ1692, and 738EPR*↓SFS743, respectively. Residues flanking Arg372 are considered non-optimal for thrombin cleavage with only two residues optimal (in bold type) for cleavage in the P3-P3' sequence, while residues flanking at the two other P1 sites are considered near-optimal with four out of six residues optimal (in bold type). Therefore, we investigated whether the P3-P3'residues surrounding Arg740, Arg372, and Arg1689 affect activation of factor VIII by thrombin. We constructed, stably transfected, and expressed four recombinant P3-P3' factor VIII mutants designated 372(P3-P3')740, 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740/1689(P3-P3')372. For example, the 372(P3-P3')740 variant has replaced the non-optimal P3-P3' residues flanking Arg372 with the near-optimal P3-P3' residues flanking Arg740. The specific activities of the 372(P3-P3')740 and 372(P3-P3')740/740(P3-P3')372 mutants were 98% and 122% the wild-type factor VIII value, respectively. In comparison, the 372(P3-P3')1689 and 372(P3-P3')740/1689(P3-P3')372 showed reductions in specific activity with values that were 14% and 17% of wild-type factor VIII, consistent with possible impaired rates of activation by thrombin. SDS-PAGE and Western blotting of the three variants possessing the 372(P3-P3')740 mutation showed cleavage rates at Arg372 increased 11- to 14-fold compared with wild-type factor VIII as judged by rates of generation of the A1 subunit. Furthermore, these variants revealed 11-21-fold rate increases in the generation of the A2 subunit as compared to wild-type factor VIII. The rates of A1 and A2 subunit generation were moderately increased from 2-3-fold for the 372(P3-P3')1689 mutant. These results indicate that replacing the non-optimal residues flanking Arg372 with near-optimal residues enhances rates of cleavage at this site. Furthermore, since the P2-P2' residues flanking Arg740 and Arg1689 are identical, these results also suggest that the P3 and/or P3' residues from the Arg740 cleavage site make a greater contribution to the enhanced cleavage rate when inserted at Arg372 than the equivalent residues from the Arg1689 site. Thrombin cleavage of light chain showing the largest effect was obtained for the 372(P3-P3')740/1689(P3-P3')372 mutant which yielded a reduced rate of A3-C1-C2 subunit generation by 33-fold. This result suggests that replacing near-optimal P3-P3' residues at Arg1689 with non-optimal residues at Arg372 significantly reduces the rate of thrombin cleavage at Arg1689, an effect that may contribute to its low specific activity. There was no observed defect in Arg1689 cleavage in the 372(P3-P3')740 mutant and moderate 2-3-fold reductions in thrombin-catalyzed cleavage rates at Arg1689 in the 372(P3-P3')1689, 372(P3-P3')740/740(P3-P3')372, and 372(P3-P3')740 variants. Overall, these results suggest that faster cleavage rates at Arg740 and Arg1689 can be attributed to more optimal residues in the P3-P3' region, while the relatively slower cleavage rate at Arg372 can be accelerated by replacement with more optimal residues for thrombin cleavage. Thus, the P3-P3' residues surrounding Arg740, Arg1689, and Arg372 in factor VIII impact rates of thrombin proteolysis at each site and contribute to the mechanism for thrombin activation of the procofactor. Disclosures: No relevant conflicts of interest to declare.

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The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain

Blood ◽

10.1182/blood.v86.8.3026.3026 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3026-3034 ◽

Cited By ~ 20

Author(s):

KA Marquette ◽

DD Pittman ◽

RJ Kaufman

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Light Chain ◽

Factor Xa ◽

Factor V ◽

Acidic Amino Acid ◽

Homologous Proteins ◽

Rich Region ◽

Thrombin Cleavage ◽

Thrombin Activation

Abstract Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the B-domains of both molecules are released. Previous studies on factor VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant activity that was not cleaved by thrombin. However, this factor V B-domain deletion molecule was activated by factor Xa, although the fold-activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

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The Structure and Function of Murine Factor V and Its Inactivation by Protein C

Blood ◽

10.1182/blood.v91.12.4593 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4593-4599 ◽

Cited By ~ 25

Author(s):

Tony L. Yang ◽

Jisong Cui ◽

Alnawaz Rehumtulla ◽

Angela Yang ◽

Micheline Moussalli ◽

...

Keyword(s):

Amino Acid ◽

Protein C ◽

Mammalian Species ◽

Structure And Function ◽

Procoagulant Activity ◽

Factor V ◽

Cleavage Sites ◽

Wild Type ◽

Coding Region ◽

And Function

Abstract Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated protein C (APC). To examine the evolutionary conservation of FV procoagulant activity and functional inactivation by APC, we cloned and sequenced the coding region of murine FV cDNA and generated recombinant wild-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,183-amino acid protein. Sequence comparison shows that the A1-A3 and C1-C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino acid identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly conserved B domain. The arginines at all thrombin cleavage sites and the R305 and R504 APC cleavage sites (corresponding to amino acid residues R306 and R506 in human FV) are invariant in all three species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas recombinant murine FV carrying both mutations (R305Q/R504Q) is nearly completely APC resistant. Thus, the structure and function of FV and its interaction with APC are highly conserved across mammalian species.

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The factor V B-domain provides two functions to facilitate thrombin cleavage and release of the light chain

Blood ◽

10.1182/blood.v86.8.3026.bloodjournal8683026 ◽

1995 ◽

Vol 86 (8) ◽

pp. 3026-3034 ◽

Cited By ~ 2

Author(s):

KA Marquette ◽

DD Pittman ◽

RJ Kaufman

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Light Chain ◽

Factor Xa ◽

Factor V ◽

Acidic Amino Acid ◽

Homologous Proteins ◽

Rich Region ◽

Thrombin Cleavage ◽

Thrombin Activation

Blood coagulation factors V and VIII are homologous proteins that have the domain organization A1-A2-B-A3-C1-C2. Upon thrombin activation, the B-domains of both molecules are released. Previous studies on factor VIII showed that the B-domain was not required for thrombin cleavage or activity. In contrast, deletion of the factor V B-domain (residues 709 to 1545) yielded a molecule with sevenfold reduced procoagulant activity that was not cleaved by thrombin. However, this factor V B-domain deletion molecule was activated by factor Xa, although the fold-activation was 85% that of wild-type factor V. Thrombin cleavage of factor V occurs initially after residue 709 and subsequently after residues 1018 and 1545. The requirement for thrombin cleavage within the B-domain at residue 1018 was evaluated by mutagenesis of Arg1018 to Ile. In the resultant R1018I mutant, the rate of thrombin activation and appearance of maximal cofactor activity was delayed and was consistent with delayed cleavage of the light chain at residue 1545. In contrast, the rate of factor Xa activation in the R1018I mutant was not altered. This finding suggests that thrombin cleavage at 1018 facilitates subsequent thrombin cleavage at 1545. Further mutagenesis was used to study the requirement for sequences within the factor V B-domain for thrombin cleavage at residue 1545. Whereas the factor V deletion molecule removing residues 709 to 1545 was not cleaved by thrombin, a smaller B-domain deletion molecule (residues 709 to 1476) containing an acidic amino acid-rich region (residues 1490 to 1520) was effectively cleaved by thrombin. These results show that residues 1476 to 1545, which contain an acidic amino acid-rich region, were required for thrombin cleavage of the light chain. Introduction of an acidic amino acid-rich region from factor VIII (residues 337 to 372) into the factor V 709 to 1545 deletion also restored thrombin cleavage of the light chain. In contrast, similar replacement with the acidic region from the factor VIII light chain (residues 1649 to 1689) was significantly less effective in promoting thrombin cleavage of the light chain. This finding suggests that the different acidic regions in factors V and VIII are not functionally equivalent in their interaction with thrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

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Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

Biochemical Journal ◽

10.1042/bj2630187 ◽

1989 ◽

Vol 263 (1) ◽

pp. 187-194 ◽

Cited By ~ 41

Author(s):

A Leyte ◽

K Mertens ◽

B Distel ◽

R F Evers ◽

M J M De Keyzer-Nellen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Factor Viii ◽

Factor Xa ◽

Coagulation Factor ◽

Procoagulant Activity ◽

Coagulation Factor Viii ◽

Coagulation Cascade ◽

Restriction Enzyme Digestion ◽

Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

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The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis

Blood ◽

10.1182/blood-2003-04-1225 ◽

2003 ◽

Vol 102 (8) ◽

pp. 2856-2861 ◽

Cited By ~ 35

Author(s):

Hongmin Sun ◽

Tony L. Yang ◽

Angela Yang ◽

Xixi Wang ◽

David Ginsburg

Keyword(s):

Gene Expression ◽

Bacterial Artificial Chromosome ◽

Transgenic Mice ◽

Coagulation Factor ◽

Artificial Chromosome ◽

Coagulation Cascade ◽

Factor V ◽

Wild Type ◽

Plasma Factor ◽

Coagulation Factor V

Abstract Coagulation factor V (FV) is a central regulator of the coagulation cascade. Circulating FV is found in plasma and within platelet α granules. The specific functions of these distinct FV pools are uncertain. We now report the generation of transgenic mice with FV gene expression restricted to either the liver or megakaryocyte/platelet lineage using bacterial artificial chromosome (BAC) constructs. Six of 6 independent albumin BAC transgenes rescue the neonatal lethal hemorrhage of FV deficiency. Rescued mice all exhibit liver-specific Fv expression at levels ranging from 6% to 46% of the endogenous Fv gene, with no detectable FV activity within the platelet pool. One of the 3 Pf4 BAC transgenes available for analysis also rescues the lethal FV null phenotype, with FV activity restricted to only the platelet pool (approximately 3% of the wild-type FV level). FV-null mice rescued by either the albumin or Pf4 BAC exhibit nearly normal tail bleeding times. These results demonstrate that Fv expression in either the platelet or plasma FV pool is sufficient for basal hemostasis. In addition, these findings indicate that the murine platelet and plasma FV pools are biosynthetically distinct, in contrast to a previous report demonstrating a plasma origin for platelet FV in humans.

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Proteolytic inactivation of human factor VIII procoagulant protein by activated human protein C and its analogy with factor V

Blood ◽

10.1182/blood.v63.2.486.486 ◽

1984 ◽

Vol 63 (2) ◽

pp. 486-489 ◽

Cited By ~ 202

Author(s):

CA Fulcher ◽

JE Gardiner ◽

JH Griffin ◽

TS Zimmerman

Keyword(s):

Factor Viii ◽

Protein C ◽

Human Factor ◽

Activated Protein C ◽

Light Chains ◽

Factor V ◽

Human Factor Viii ◽

Thrombin Activation ◽

A Site ◽

Polypeptide Chains

Abstract Purified human factor VIII procoagulant protein (VIII:C) was treated with purified human activated protein C (APC) and the loss of VIII:C activity correlated with proteolysis of the VIII:C polypeptides. APC proteolyzed all VIII:C polypeptides with mol wt = 92,000 or greater, but not the doublet at mol wt = 79–80,000. These results and our previous thrombin activation studies of purified VIII:C, are analogous with similar studies of factor V and form the basis for the following hypothesis: activated VIII:C consists of heavy and light chain polypeptides [mol wt = 92,000 and mol wt = 79–80,000 (or 71–72,000), respectively] which are similar in Mr to the heavy and light chains of activated factor V. Thrombin activates VIII:C and V by generating these polypeptide chains from larger precursors and APC inactivates both molecules by cleavage at a site located in the heavy chain region of activated VIII:C and V.

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Val34Leu polymorphism of plasma factor XIII: biochemistry and epidemiology in familial thrombophilia

Blood ◽

10.1182/blood.v96.7.2479 ◽

2000 ◽

Vol 96 (7) ◽

pp. 2479-2486 ◽

Cited By ~ 87

Author(s):

István Balogh ◽

Gabriella Szôke ◽

Levente Kárpáti ◽

Ulla Wartiovaara ◽

Éva Katona ◽

...

Keyword(s):

Protective Effect ◽

Venous Thrombosis ◽

Factor Xiii ◽

Coagulation Factor ◽

Wild Type ◽

Thrombin Cleavage ◽

Plasma Factor ◽

Thrombin Activation ◽

A Subunit ◽

The Mean

Abstract Val34Leu polymorphism of the A subunit of coagulation factor XIII (FXIII-A) is located in the activation peptide (AP) just 3 amino acids away from the thrombin cleavage site. This mutation has been associated with a protective effect against occlusive arterial diseases and venous thrombosis; however, its biochemical consequences have not been explored. In the current study it was demonstrated that the intracellular stability and the plasma concentration of FXIII of different Val34Leu genotypes are identical, which suggests that there is no difference in the rate of synthesis and externalization of wild-type and mutant FXIII-A. In contrast, the release of AP by thrombin from the Leu34 allele proceeded significantly faster than from its wild-type Val34 counterpart. By molecular modeling larger interaction energy was calculated between the Leu34 variant and the respective domains of thrombin than between the Val34 variant and thrombin. In agreement with these findings, the activation of mutant plasma FXIII by thrombin was faster and required less thrombin than that of the wild-type variant. Full thrombin activation of purified plasma FXIII of different genotypes, however, resulted in identical specific transglutaminase activities. Similarly, the mean specific FXIII activity in the plasma was the same in the groups with wild-type, heterozygous, and homozygous variants. Faster activation of the Leu34 allele hardly could be associated with its presumed protective effect against venous thrombosis. No such protective effect was observed in a large group of patients with familial thrombophilia.

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Molecular characterisation of Tyr530Ser and IVS16–1G>T mutations causing severe factor V deficiency

Thrombosis and Haemostasis ◽

10.1160/th09-10-0735 ◽

2010 ◽

Vol 104 (09) ◽

pp. 536-543 ◽

Cited By ~ 2

Author(s):

Weidong Zheng ◽

Yanhui Liu ◽

Ying Luo ◽

Zhihong Chen ◽

Yan Wang ◽

...

Keyword(s):

Deletion Mutant ◽

Coagulation Factor ◽

Underlying Mechanism ◽

Procoagulant Activity ◽

Site Directed Mutagenesis ◽

Factor V ◽

Wild Type ◽

Site Mutation ◽

Bioinformatics Analyses ◽

Mutant Transcript

SummaryOur previous study reported a missense mutation (Tyr530Ser) and a splicing site mutation (IVS16–1G>T) in blood coagulation factor V (FV) gene in a two-year-old Chinese boy. However, the linkage between the mutations and severe FV deficiency and the underlying mechanism has not been elucidated. The present study was designed to investigate the effect of the two mutations and the possible pathogenetic mechanism. FV procoagulant activity showed tremendous decrease in the patient with two mutations. The bioinformatics analyses predicted that IVS16–1G>T mutation may cause the entire exon 17 of FV to be skipped in transcription and thereby result in a deletion mutant. To confirm the predicted results, the fragment of exon 16 to exon 18 containing IVS16–1G>T mutation was obtained by PCR and site-directed mutagenesis. IVS16–1G>T mutant and wild-type constructs were transfected into COS-7 cells. Sequence analysis showed that mutant transcript lacked the entire 180-bp length of exon 17. Moreover, compared to wild-type, the expression of the two mutant proteins was decreased and the procoagulant activity was also reduced when the deletion mutant cDNA and Tyr530Ser site mutant cDNA were transfected into COS-7 cells, respectively. Our results indicate that Tyr530Ser and IVS16–1G>T could be separately responsible for severe FV deficiency, while the phenotype in the proband could be caused by the combination effect of the two defects.

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