632 Thromb Haemost 2002; 88: 632 – 8 © 2002 Schattauer GmbH, Stuttgart Protein C Deficiency caused by Homozygosity for a Novel PROC D180G Mutation – in vitro Expression and Structural Analysis of the Mutation Bent Lind 1 , Tobias Gedde-Dahl 2 , Geir Tjønnfjord 2 , Bruno O. Villoutreix 3 , Frank Brosstad 4 1 Department of Clinical Biochemistry KB 3-01-1, Rigshospitalet, Copenhagen, Denmark 2 Medical Department A and 4 Research Institute for Internal Medicine, Rikshospitalet Oslo, Norway 3 INSERM U428, University of Paris V, School of Pharmacy, Paris, France Keywords Protein C deficiency, mutation, venous thrombosis, skin necrosis, serine proteinase Summary Homozygosity for a novel D180G mutation in the protease domain of protein C, associated with plasma protein C activity and antigen levels of 8% of normal was identified in a thrombosis prone family. Transient expression of protein C in HK-293 cells and analysis of protein C antigen in culture media and cell lysates showed that the secretion of mutant protein as compared with wild-type protein was reduced by 79% while the intracellular contents were similar. Computer analysis of the X-ray structure of activated protein C and of a theoretical model of the zymogen predicts that the mutation destabilises the molecule locally. Our results are compatible with a relatively unstable mutant molecule that could be trapped inside the cell and degraded. However, if secreted the mutant molecule could have a relatively normal catalytic activity and structure consistent with the plasma levels of protein C activity and the late onset of thrombosis. Introduction The vitamin K-dependent plasma glycoprotein protein C is the precursor of the anticoagulant serine proteinase activated protein C that plays a role in regulating blood coagulation (1, 2). Activated protein C exerts its anticoagulant function by proteolytic inactivation of the blood coagulation cofactors V a and VIII a in the presence of protein S and of negatively charged membrane surface. Protein C (419 amino acid residues) is produced mostly in the liver as a single-chain inactive zymogen, which is proteolytically processed (removal of K156 and R157) to a two-chain molecule composed of a light and a heavy chain covalently bound through a disulphide bond (1). The light chain (residues 1 through 155) is organised in several structural domains. From the NH 2 -terminus to the C-terminus, they are the Gla-domain and two epidermal growth factor modules (EGF). The heavy chain (residues 170 to 419) consists of the serine proteinase domain. Under physiological conditions, activated protein C is generated after a specific proteolytic cleavage within its activation peptide by thrombin (between R169 and L170), preferentially when the latter is bound to thrombomodulin. Upon activation, the 12 amino acid activation peptide of protein C is released (residues 158 to 169). Protein C deficiency is an autosomally inherited disorder associated with an increased risk of venous thrombosis (1, 2). Homozygosity and compound heterozygosity for protein C deficiency is a rare condition (prevalence about 1 in 200,000-400,000 individuals). In patients with undetectable levels of protein C activity in plasma, massive thrombotic complications may occur during the neonatal period. In patients with very low but detectable levels of protein C activity in plasma the clinical manifestations are milder and include late onset of thrombotic symptoms and repeated episodes of skin necrosis. Heterozygosity for protein C deficiency is characterised by an increased risk of venous thrombosis in early adulthood. Anticoagulant treatment of protein C deficient individuals with vitamin K antagonists (warfarin, coumarin, phenprocoumon) may be complicated by warfarin-induced skin necrosis (3). In hereditary protein C deficiency more than 160 different mutations have been reported (4). The mutations responsible for homozygote and compound heterozygote protein C deficiency have been identified in 30 and 23 index patients, respectively (4 -16). In agreement with the distribution of mutation types in genetic diseases in general (17) more than 65% of the mutations identified in the protein C gene are missense mutations. In most cases, their influence on the biosynthesis and structure of protein C has not been studied. The present work describes homozygosity for a novel D180G mutation (D26 in the chymotrypsinogen nomenclature) in the protein C gene associated with protein C deficiency, late onset of recurrent venous thrombosis and recurrent warfarin induced skin necrosis. The influence of the mutation on the biosynthesis of protein C was studied in vitro in a transient expression system using human kidney (HK) 293 cells. Structural analysis of the activated protein C X-ray structure and of a theoretical model for the protein C zymogen was also performed to evaluate further the potential roles of the identified amino acid substitution on folding and stability of the mutant protein C. Materials and Methods Clinical Material Subjects under study were the proband, her parents and her 4 siblings (Table 1). The study was performed after informed consent. Correspondence to: Dr. Bent Lind, Department of Clinical Biochemistry KB 3-01-1, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen, Denmark – Tel.: + 45 35 45 30 11; Fax: + 45 35 45 25 24; E-mail: bli@kpll.dk For personal or educational use only. No other uses without permission. All rights reserved. Downloaded from www.thrombosis-online.com on 2018-05-13 | ID: 1001066444 | IP: 54.70.40.11