Molecular Bases of Pseudo-homozygous APC Resistance: The Compound Heterozygosity for FV R506Q and a FV Null Mutation Results in the Exclusive Presence of FV Leiden Molecules in Plasma

Summary

Pseudo-homozygous APC resistance, the condition resulting from compound heterozygosity for FV R506Q (FV Leiden) and quantitative FV deficiency, provides a natural model to study the interaction between procoagulant and anticoagulant defects. This paper reports a complete FV characterization of a pseudo-homozygous APC resistant thrombotic patient. The expression of the patient’s non-Leiden gene was found to be severely impaired both at the mRNA and protein levels. In particular, only FV Leiden molecules were detected in the patient’s plasma by immunoblotting, which accounts for the observed marked APC resistance. Analysis of the FV cDNA obtained by reverse transcription of platelet RNA revealed that the mRNA of the non-Leiden gene was extremely reduced in amount. A PAC clone containing the whole FV gene was used to design primers for a complete FV exon scanning. A 2-bp insertion at nucleotide 3706 in the large exon 13 of the non-Leiden gene, predicting a frame-shift and premature termination of protein synthesis, was identified as responsible for the FV defect. Failure to find any case of pseudo-homozygous APC resistance in a large sample (6,804) of blood donors suggests that this condition is extremely rare among normal controls and that its detection is favoured by the thrombotic risk that it may confer.

• References

• 1 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-8.
• 2 Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-22.
• 3 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
• 4 Kalafatis M, Rand MD, Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-879.
• 5 Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular defect in factor V R506. J Biol Chem 1995; 270: 4053-7.
• 6 Bernardi F, Faioni EM, Castoldi E, Lunghi B, Castaman G, Sacchi E, Mannucci PM. A Factor V genetic component differing from factor V R506Q contributes to the activated protein C resistance phenotype. Blood 1997; 90: 1552-7.
• 7 Laffan MA, Manning R. The influence of FVIII on measurement of activated protein C resistance. Blood Coagul Fibrinol 1996; 7: 761-5.
• 8 Griffin JH, Heeb MJ, Kojima Y, Fernandez J, Kojima K. Activated protein C resistance: molecular mechanisms. Thromb Haemost 1995; 74: 444-8.
• 9 Greengard JS, Alhenc-Gelas M, Gandrille S, Emmerich J, Aiach M, Griffin J. Pseudo-homozygous protein C resistance due to coinheritance of heterozygous factor V-R506Q and type I factor V deficiency associated with thrombosis. Thromb Haemost 1995; 73: 1361 (Abstract).
• 10 Simioni P, Scudeller A, Radossi P, Gavasso S, Girolami B, Tormene D, Girolami A. “Pseudo homozygous” activated protein C resistance due to double heterozygous factor V defects (factor V Leiden mutation and type I quantitative factor V defect) associated with thrombosis: report of two cases belonging to two unrelated kindreds. Thromb Haemost 1996; 75: 422-6.
• 11 Guasch JF, Lensen RP, Bertina RM. Molecular characterization of a type I quantitative factor V deficiency in a thrombosis patient that is “pseudo homozygous” for activated protein C resistance. Thromb Haemost 1997; 77: 252-7.
• 12 Ioannou PA, Amemiya CT, Garnes J, Kroisel PM, Shizuya H, Chen C, Batzer MA, de Jong PJ. A new bacteriophage P1-derived vector for the propagation of large human DNA fragments. Nature Genetics 1994; 6: 84-9.
• 13 Castoldi E, Lunghi B, Mingozzi F, Ioannou P, Marchetti G, Bernardi F. New coagulation factor V gene polymorphisms define a single and infrequent haplotype underlying the factor V Leiden mutation in Mediterranean populations and Indians. Thromb Haemost 1997; 78: 1037-41.
• 14 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84: 4846-50.
• 15 Kalafatis M, Haley PE, Lu D, Bertina RM, Long GL, Mann KG. Proteolitic events that regulate factor V activity in whole plasma from normal and activated protein C (APC)-resistant individuals during clotting: an insight into the APC-resistance assay. Blood 1996; 87: 4695-707.
• 16 Gewirtz AM, Keefer M, Doshi K, Annamalai AE, Chong Chiu H, Colman RW. Biology of human megakaryocyte factor V. Blood 1986; 6: 1639-48.
• 17 Zivelin A, Griffin JH, Xu X, Pabinger I, Samama M, Conard J, Brenner B, Eldor A, Seligsohn U. A single genetic origin for a common Caucasian risk factor for venous thrombosis. Blood 1997; 89: 397-402.
• 18 Egan JO, Kalafatis M, Mann KG. The effect of Arg306→Ala and Arg506→Gln substitutions in the inactivation of recombinant human factor Va by activated protein C and protein S. Protein Science 1997; 6: 2016-27.
• 19 Kalafatis M, Rand MD, Mann KG. Factor Va-membrane interaction is mediated by two regions located on the light chain of the cofactor. Biochemistry 1994; 33: 486-93.
• 20 Cripe LD, Moore KD, Kane WH. Structure of the gene for human coagulation factor V. Biochemistry 1992; 31: 3777-85.
• 21 Cooper DN, Krawczak M. Human gene mutation. BIOS Scientific Publishers, Oxford,; 1993
• 22 Shen NLL, Fan S, Pyati J, Graff R, LaPolla RJ, Edgington TS. The serine protease cofactor factor V is synthesized by lymphocytes. J Immunol 1993; 150: 2992-3001.
• 23 Lunghi B, Iacoviello L, Gemmati D, Di Iasio MG, Castoldi E, Pinotti M, Castaman G, Redaelli R, Mariani G, Marchetti G, Bernardi F. Detection of new polymorphic markers in the FV gene: association with FV levels in plasma. Thromb Haemost 1996; 75: 45-8.
• 24 Guasch JF, Cannegieter S, Reitsma PH, van’t Veer-Korthof ET, Bertina RM. Severe coagulation factor V deficiency caused by a 4-bp deletion in the factor V gene. Brit J Haematol 1998; 101: 32-9.
• 25 Hockin MF, Kalafatis M, Shatos M, Mann KG. Protein C activation and factor Va inactivation on human umbilical vein endothelial cell. Arterioscler Thromb Vasc Biol 1997; 17: 2765-75.