Functional consequences of the THBD c.1611C>A (p.Cys537Stop) mutation. (A) THBD haplotype analysis. Four haplotype-tagging SNPs (MAF ≥ 0.05, r2 threshold 0.8) capturing most of the common genetic variation in the THBD gene in the Caucasian population were identified using the Genome Variation Server of SeattleSNPs (http://gvs.gs.washington.edu/GVS138/). All available members of the British (n = 3) and French (n = 9) families with the c.1611C>A mutation were genotyped for these SNPs and the haplotype underlying the mutation in each family was reconstructed by allele segregation analysis. (B) Plasma concentrations of soluble TM in normal controls (n = 8) and in carriers of the p.Cys537Stop mutation (n = 3, proband and 2 family members). (C) Expression of membrane-bound TM in ovarian tissue. Histological sections of the patient’s ovarian tissue (left and middle panels) prepared at the time of her ovariectomy (1995) were stained for the endothelial marker CD31 and for TM, as indicated. Archival ovarian tissue obtained from a woman undergoing surgical resection at the same time as the patient, processed in the same way and stored in the same room for a comparable time, served as a normal control (right panels). (Left panels) A large venous vessel (*), associated with numerous smaller arterial and venous vessels, is visible in the ovarian stroma near a follicular ovarian cyst (arrow). CD31 is readily discernible along the endothelial lining of all visible vessels, whereas TM is barely detectable. (Middle panels) A large vessel (*) in the ovarian stroma is cross-sectioned. Although CD31 is detectable all along the endothelial lining, TM shows heterogeneous expression and is not detectable in the smaller adjacent vessels. (Right panels) CD31 and TM show strong and comparable apparent expression levels and their distribution is homogeneous along the endothelial linings. Immunoperoxidase technique followed by nuclear counterstaining with Mayer’s hematoxylin. Original magnifications: left and middle panels, ×250; right panels, ×350. (D) APC generation. Control plasma (gray circles) and the patient’s plasma (black circles) were activated with 1 pM TF, 4 μM phospholipids, and 16.6 mM CaCl2. At timed intervals, aliquots were removed and assayed for APC as described in “Materials and methods”. (E) FVa generation. Control plasma (gray circles) and the patient’s plasma (black circles) were activated with 1 pM TF, 4 μM phospholipids, and 16.6 mM CaCl2. At timed intervals, aliquots were removed and assayed for FVa as described in “Materials and methods”. Addition of a neutralizing anti-protein C antibody (not shown) abolished the difference in FVa generation between the patient’s and control plasma. (F) Intrinsic FXa generation. Control plasma (gray diamonds) and patient’s plasma (black diamonds) were activated with 2 nM FIXa, 4 μM phospholipids, and 16.6 mM CaCl2. At timed intervals, aliquots were removed and assayed for FXa as described in “Materials and methods”. Because the experiment was conducted at limiting FVIIIa concentrations, the generated FXa is proportional to the FVIIIa concentration.
