Tissue-type plasminogen activator (tPA) homozygous Tyr471His mutation associates with thromboembolic disease.

Tissue-type plasminogen activator (tPA) encoded by PLAT is a major mediator that promotes fibrinolysis and prevents thrombosis. Pathogenetic mutations in PLAT associated with venous thromboembolism have rarely been reported. Here, we report the first case of a homozygous point mutation c.1411T>C (p.Y471H) in PLAT leading to thromboembolic events and conduct related functional studies. The corresponding tPA mutant protein (tPA-Y471H) and wild-type tPA (tPA-WT) were synthesized in vitro, and mutant mice (PLATH/H mice) were constructed. The molecular docking and surface plasmon resonance results indicated that the mutation impeded the hydrogen-bonding interactions between the protease domain of tPA and the kringle 4 domain of plasminogen, and the binding affinity of tPA and plasminogen was significantly reduced with a difference of one order of magnitude. mRNA half-life assay showed that the half-life of tPA-Y471H was shortened. The inferior vena cava thrombosis model showed that the rate of venous thrombosis in PLATH/H mice was 80% compared with 53% in wild-type mice. Our data suggested a novel role for the protease domain of tPA in efficient plasminogen activation, and demonstrated that this tPA mutation could reduce the fibrinolysis function of the body and lead to an increased propensity for thrombosis.

Gene editing of human iPSCs rescues thrombophilia in hereditary antithrombin deficiency in mice.

Hereditary antithrombin deficiency is caused by SERPINC1 gene mutations and predisposes to recurrent venous thromboembolism that can be life-threatening. Therefore, lifelong anticoagulation is required, which has side effects and may not be effective. In this study, peripheral blood mononuclear cells from a patient with severe antithrombin deficiency were reprogrammed into induced pluripotent stem cells (iPSCs). The mutation was corrected using CRISPR-Cas9 and Cre/LoxP genome editing. iPSCs were differentiated into hepatocytes, which were injected into the spleen of antithrombin knockout mice to restore the activity of antithrombin and reduce the thrombophilic state. Human iPSC-differentiated hepatocytes colonized mice and secreted antithrombin stably, normalizing antithrombin in plasma (activity: from 46.8 ± 5.7% to 88.6 ± 7.6%, P < 0.0001; antigen: from 146.9 ± 19.5 nanograms per milliliter to 390.7 ± 16.1 nanograms per milliliter, P < 0.0001). In venous thrombosis model, the rate of thrombosis in mice treated with edited hepatocytes, parental hepatocytes, and wild-type mice were 60, 90, and 70%, respectively. The thrombus weight was much lighter in mice treated with edited hepatocytes compared with parental hepatocytes (7.25 ± 2.00 milligrams versus 15.32 ± 2.87 milligrams, P = 0.0025) and showed no notable difference compared with that in wild-type mice (10.41 ± 2.91 milligrams). The activity and concentration of antithrombin remained high for 3 weeks after injection. The liver and kidney function markers showed no obvious abnormality during the observation period. This study provides a proof of principle for correction of mutations in patient-derived iPSCs and potential therapeutic applications for hereditary thrombophilia.

Characterization of a Missense Mutation in the Catalytic Domain and a Splicing Mutation of Coagulation Factor X Compound Heterozygous in a Chinese Pedigree.

BACKGROUND: Congenital coagulation factor X (FX) deficiency is a rare bleeding disorder with an incidence of one in one million caused by mutations in the FX-coding gene(F10), leading to abnormal coagulation activity and a tendency for severe hemorrhage. Therefore, identifying mutations in FX is important for diagnosing congenital FX deficiency. RESULTS: Genetic analysis of the proband identified two single-base substitutions: c.794T > C: p.Ile265Thr and c.865 + 5G > A: IVS7 + 5G > A. His FX activity and antigen levels were < 1% and 49.7%, respectively; aPTT and PT were prolonged to 65.3 and 80.5 s, respectively. Bioinformatics analysis predicted the two novel variants to be pathogenic. In-vitro expression study of the missense mutation c.794T > C: p.Ile265Thr showed normal synthesis and secretion. Activation of FXs by RVV, FVII/TF, and FVIII/FIX all showed no obvious difference between the variant and the reference. However, clotting activity by PT and aPTT assays and activity of thrombin generation in a TGA assay all indicated reduced activity of the mutant FX-Ile265Thr compared to FX-WT. Minigene assay showed a normal splicing mode c.865 + 5G > A: IVS7 + 5G > A, which is inconsistent with clinical phenotype. CONCLUSIONS: The heterozygous variants c.794T > C: p.Ile265Thr or c.865 + 5G > A: IVS7 + 5G > A indicate mild FX deficiency, but the compound heterozygous mutation of the two causes severe congenital FX deficiency. Genetic analysis of these two mutations may help characterize the bleeding tendency and confirm congenital FX deficiency. In-vitro expression and functional study showed that the low activity of the mutant FX-Ile265Thr is caused by decrease in its enzyme activity rather than self-activation. The minigene assay help us explore possible mechanisms of the splicing mutation. However, more in-depth mechanism research is needed in the future.

Simulation of a Novel Tubular Microalgae Photobioreactor with Aerated Tangent Inner Tubes: Improvements in Mixing Performance and Flashing-Light Effects.

At present, large-scale and high-efficiency microalgal cultivation is the key to realizing the technology for carbon capture and storage (CCS) and bioresource recovery. Meanwhile, tubular photobioreactors (PBRs) have great potential for microalgal cultivation due to their high productivity. To improve the mixing performance and flashing-light effect, a novel tube PBR with the inner tube tangential to the outer tube was developed, whose radial aeration pores are situated along the length of the inner tube. The direction of aeration, aeration rate, light/dark cycle period (L/D), light-time ratio, average turbulent kinetic energy (TKE), and degree of synergy between the velocity and direction of the light field in the PBR were optimized by a computational fluid dynamics (CFD) simulation and field synergy theory. The results show that a downwards aeration direction of 30° and an aeration rate of 0.7 vvm are the most conducive to reducing the dead zone and improving the light/dark cycle frequency. Compared to the concentric double-tube PBR, the light/dark cycle frequency and light time of the tangent double-tube PBR increased by 78.2% and 36.2% to 1.8 Hz and 47.8%, respectively, and the TKE was enhanced by 48.1% from 54 to 80 cm2·s-2. Meanwhile, field synergy theory can be extended and applied to the design of tubular microalgae PBRs, and the average synergy of the light and velocity gradients across the cross-section increased by 38% to 0.69. The tangential inner tube aeration structure generated symmetrical vertical vortices between the light and dark areas in the PBR, which significantly improved the mixing performance and flashing-light effect. This novel design can provide a more suitable microenvironment for microalgal cultivation and is promising for bioresource recovery applications and improving the yield of microalgae.