Abnormal Propeptide Processing Resulting in the Presence of Two Abnormal Species of Protein C in Plasma

Summary

A heterozygous GT transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg^-1 to a Leu (PC_R-1L) was identified in a thrombophilic patient. The PC_R-1L was purified from the patient’s plasma by immunoaffinity chromatography using Ca⁺⁺-independent and Ca⁺⁺-dependent monoclonal antibodies. NH₂-terminal sequencing of the light chain of PC_R-1L revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PCR-1L/propeptide exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PCR-1L/propeptide the propeptide cleavage at Lys^-2 is less efficient since approximately 60% of PC variant molecules present in patient’s plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.

Keywords

Dysfunctional protein C - type II defect - propeptide clearage - thrombophilia - factor V

• References

• 1 Zhang L, Castellino FJ. The contribution of individual gamma-carboxyglutamic acid residues in the calcium-dependent binding of recombinant human protein C to acidic phospholipid vesicles. J Biol Chem 1993; 268: 12040-5.
• 2 Jhingan A, Zhang L, Christiansen WT, Castellino FJ. The activities of recombinant gamma-carboxyglutamic-acid-deficient mutants of activated protein C toward human coagulation factor Va and factor VIIIa in purified systems and in plasma. Biochemistry 1994; 33: 1869-75.
• 3 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane bound cofactor for thrombin catalyzed activation of protein C. J Biol Chem 1982; 257: 859-64.
• 4 Esmon CT. The roles of protein C and thrombomodulin in the regulation of blood coagulation. J Biol Chem 1989; 264: 4743-6.
• 5 Lu D, Kalafatis M, Mann KG, and Long GL. Loss of Membrane-dependent factor Va cleavage: a mechanistic interpretation of the pathology of protein C Vermont. Blood 1994; 84: 687-90.
• 6 McClure DB, Walls JD, Grinnell BW. Post translational processing events in the secretion pathway of human protein C, a complex vitamin-dependent antithrombotic factor. J Biol Chem 1992; 267: 19710-7.
• 7 Reitsma PH, Bernardi F, Doig RG, Gandrille S, Greengard JS, Ireland H, Krawczak M, Lind B, Long GL, Poort SR, Saito H, Sala N, Witt I, Cooper DN. Protein C deficiency: a database of mutations, 1995 update. On behalf of the subcommittee on plasma coagulation inhibitors of the scientific and standardization committee of the ISTH. Thromb Haemost 1995; 73: 876-89.
• 8 Girolami A, Simioni P, Girolami B, Marchiori A, Millar DS, Bignell P, Kakkar VV, Cooper DN. A novel dysfunctional protein C (protein C Padua 2) associated with a thrombotic tendency: substitution of Cys for Arg-1 results in a strongly reduced affinity for binding of Ca⁺⁺ . Br J Haematol 1993; 85: 521-7.
• 9 Gandrille S, Alhenc-Gelas M, Gaussem P, Aillaud MF, Dupuy E, Juhan-Vague I, Aiach M. Five novel mutations located in exon III and IX of the protein C gene in patients presenting with defective protein C anticoagulant activity. Blood 1993; 82: 159-68.
• 10 Kalafatis M, and Mann KG. Role of the membrane in the inactivation of factor Va by activated protein C. J Biol Chem 1993; 268: 27246-57.
• 11 Kalafatis M, Rand MD, and Mann KG. The mechanism of inactivation of human factor V and human factor Va by activated protein C. J Biol Chem 1994; 269: 31869-80.
• 12 Wojcik EGC, Simioni P, van den Berg M, Girolami A, Bertina RM. Mutations which introduce free cysteine residues in the Gla-domain of vitamin K dependent proteins result in the formation of complexes with α₁-microglobulin. Thromb Haemost 1996; 75: 70-5.
• 13 Miyata T, Zheng YZ, Sakata T, Kato H. Protein C Osaka 10 with aberrant propeptide processing: loss of anticoagulant activity due to an amino acid substitution in the protein C precursor. Thromb Haemost 1995; 74: 1003-8.
• 14 Lind B, Johnsen AH, Thorsen S. Naturally occurring Arg-1 to His mutation in human protein C leads to aberrant propeptide processing and secretion of dysfunctional protein C. Blood 1997; 89: 2807-16.
• 15 Sanson BJ, Simioni P, Tormene D, Moia M, Friederich PW, Huisman MV, Prandoni P, Bura A, Rejto L, Wells P, Mannucci PM, Girolami A, Buller HR, Prins MH. The incidence of venous thromboembolism in asymptomatic carriers of antithrombin, protein C or protein S: a prospective cohort study. Blood 1999; 94: 3702-6.
• 16 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.
• 17 Simioni P, Tormene D, Manfrin D, Gavasso S, Luni S, Stocco D, Girolami A. Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant. Br J Haematol 1998; 103: 1045-50.
• 18 Simioni P, Kalafatis M, Millar DS, Henderson SC, Luni S, Cooper DN, Girolami A. Compound heterozygous protein C deficiency resulting in the presence of only the β-form of protein C in plasma. Blood 1996; 88: 2101-8.
• 19 Kalafatis M, Bertina RM, Rand MD, Mann KG. Identification of the molecular defect in factor V^R506Q . J Biol Chem 1995; 270: 4053-7.
• 20 Kalafatis M, Haley PE, Lu D, Bertina RM, Long GL, Mann KG. Proteolytic events that regulate factor V activity in whole plasma from normal and a protein C-resistant individuals during clotting: an insight into the APC-resistance assay. Blood 1996; 87: 4695-707.
• 21 Grundy CB, Chisholm M, Kakkar VV, Cooper DN. A novel homozygous missense mutation in the protein C (PROC) gene causing recurrent venous thrombosis. Hum Genet 1992; 89: 683-4.
• 22 Diuguid DL, Rabiet M-J, Furie BC, Liebman HA, Furie B. Molecular basis of hemophilia B: a defective enzyme due to an unprocessed propeptide is caused by a point mutation in the factor IX precursor. Proc Natl Acad Sci USA 1986; 83: 5803-7.
• 23 Busby SJ, Berkner KL, Halfpap LM, Gambee JE, Kumar AA. Alteration of the propeptide sequence impairs biological activity of human factor VII. In: Suttie JW. ed. Current advances in vitamin K research. New York, NY: Elsevier Science; 1988: 173-81.
• 24 Bristol JA, Furie BC, Furie B. Propeptide processing during F. IX biosynthesis. J Biol Chem 1993; 268: 7577-84.
• 25 Gandrille S, Borgel D, Eschwege-Gufflet V, Aillaud M, Dreyfus M, Matheron C, Gaussem P, Abgrall JF, Jude B, Sie P, Toulon P, Aiach M. Identification of 15 different candidate causal point mutations and three polymorphisms in 19 patients with protein S deficiency using a scanning method for the analysis of the protein S active gene. Blood 1995; 85: 130-8.
• 26 Furie B, Boudchard BA, Furie BC. Vitamin K-dependent biosynthesis of γ-carboxyglutamic acid. Blood 1999; 93: 1798-808.