A thrombophilic allele of clotting Factor VII/VIIa promoting recurrent pulmonary emboli, clinical details, and a structural model of the altered protein: a case report

Abstract

Background: The clotting or hemostasis system is a meticulously regulated set of enzymatic reactions that occur in the blood and culminate in formation of a fibrin clot. The precisely calibrated signaling system that prevents or initiates clotting originates with the activated Factor Seven (FVIIa) complexed with tissue factor (TF) formed in the endothelium. Here we describe a rare inherited mutation in the FVII gene which is associated with pathological clotting.

Case presentation: The 52-year-old patient, with European, Cherokee and African American origins, FS was identified as having low FVII (10%) prior to elective surgery for an umbilical hernia. He was given low doses of NovoSeven (therapeutic Factor VIIa) and had no unusual bleeding or clotting during the surgery. In fact, during his entire clinical course he had no unprovoked bleeding. Bleeding instances occurred with hemostatic stresses such as gastritis, kidney calculus, orthopedic surgery, or tooth extraction, and these were handled without factor replacement. On the other hand, FS sustained two unprovoked and life-threatening instances of pulmonary emboli, although he was not treated with NovoSeven at any time close to the events. Since 2020, he has been placed on a DOAC (Direct Oral Anticoagulant, producing Factor Xa inhibition) and has sustained no further clots.

Possible mechanism of (unauthorized) fvii activation: FS has a congenitally mutated FVII/FVIIa gene, which carries a R315W missense mutation in one allele and a mutated start codon (ATG to ACG) in the other allele, thus rendering the patient effectively homozygous for the missense FVII. Structure based comparisons with known crystal structures of TF-VIIa indicate that the patient's missense mutation is predicted to induce a conformational shift of the C170's loop due to crowding of the bulky tryptophan to a distorted "out" position (Fig. 1). This mobile loop likely forms new interactions with activation loop 3, stabilizing a more active conformation of the FVII and FVIIa protein. The mutant form of FVIIa may be better able to interact with TF, displaying a modified serine protease active site with enhanced activity for downstream substrates such as Factor X.

Conclusions: Factor VII can be considered the gatekeeper of the coagulation system. Here we describe an inherited mutation in which the gatekeeper function is altered. Instead of the expected bleeding manifestations resulting from a clotting factor deficiency, the patient FS suffered clotting episodes. The efficacy of the DOAC in treating and preventing clots in this unusual situation is due to its target site of inhibition (anti-Xa), which lies downstream of the site of action of FVIIa/TF.

Keywords: DOAC; Factor VII; Factor VIIa; Hemostasis; Thrombophilia; Tissue Factor (TF); Zymogen.

Fig. 1

Structural changes predicted to be induced by the R315W mutation. The FVIIa-TF complex (PDB 1DAN) is depicted using UCSF Chimera X [14] as a ribbon structure (panel A, light gray) where the critical residues are labeled (numbering refers to the mature processed form) and shown as ball and stick representations with blue and red for basic and acidic chemical groups, respectively. The structure is oriented such that the R315 region faces out, and the critical features are overlayed with a space-filling representation, where the C170's loop (aa 310–329) shaded in light yellow is adjacent and partially overlapping with the alpha 2 helix (aa 306–313), the protruding R315 region is in light blue, and the activation loop 3 (aa 365–374) [11] is in pink. A similar structural model corresponding to the mutated FVIIa-TF is computationally constructed using the DUET [15], a predictive server that models missense mutations. We replaced the R315 with W to depict the mutation found in our patient (FS) (panel B). The two models are shown in the same orientation (with respect to R315) for easy comparison, and the specific residues and features are maintained as in panel A, except for the W315 and its surrounding region. We utilized the Dunbrack 2010 backbone-dependent rotamer library [16] to estimate the preferred rotamer conformation for our residue of interest. The missense tryptophan 315 was found to prefer an "outward" orientation. The substitution of the R315 with its basic chemical group by a neutral and bulkier W drastically affected the local conformation and electrostatics around the C170's loop due to loss of charge and increased side chain volume. In the bottom part of the figure (panels C and D), the local changes are better visualized using a space-filling model depicting the local surface charges (blue for positive and red for negative) of wild type and mutant FVIIa R315 (panel C) and mutant W315 (panel D). The insets depict the entire VIIa-TF complex for context, with the encircled regions highlighting the regions of interest for the wild type R315 and mutant W315 areas

Fig. 2

Clinical data. A Time line. Major events of the clinical history are arrayed on a time line calibrated in months, beginning in 2013 (T = 0) and ending with the present in 2022. Note in particular that bleeding episodes (ulcer T = 33, renal calculus T = 82) were provoked whereas clotting episodes (pulmonary emboli T = 39, pulmonary emboli T = 81) were unprovoked. DOAC stands for direct oral anticoagulant, a Xa inhibitor. FS has been on apixaban 5 mg twice a day continuously since T = 81, and he has not sustained any instances of clotting since then. B Special Coagulation Laboratory Data Citrated anticoagulated blood was analyzed by standard methods and PT was found to be prolonged from 10.7 to 13.8 to 20.6 s. Mixing studies revealed lack of correction with FVII deficient plasma, indicating that the patient has deficiency of FVII. No inhibitor was detected, since PT remained at 12.9 s after 1:1 mix with normal plasma and 30 min incubation at 37 degrees

Fig. 3

FVII Sequences A. Deoxyribonucleic acid sequencing of the FVII gene was performed by Fulgent Genetics using the buffy coat of a peripheral blood sample. Two mutations were detected, one c.2 T > C converted the ATG start codon to ACG, thereby abrogating the start and creating a null. A second mutation presumed to be on the other allele was detected, c.1123 C > T p. Arg 315 Trp, and this is most likely the FVII "activating" mutation. B. Multiple sequence alignment of the relevant region of FVII from different species. Amino acid sequences from Genbank were aligned using the multiple sequence alignment Tool Clustal W: Human Homo sapiens factor VII AAA51983.1; Fish Danio rerio factor VII precursor NP_571894.2; Mouse Mus musculus factor VII preproprotein NP_034302.2; Rabbit Coryctolagus cuniculus factor VII. Highlighted in bold capital letters is the alpha 2 helix ³⁰⁶MTQDCLQ³¹² that responds to TF binding by altering its conformation. The C170's loop extends from C310 to C329. The relevant mutation is shown by an arrow from the R315 indicated in red to the W315 above it. The beta 9 sheet ³²⁷MFCA³³⁰ is highlighted in bold capital letters