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Human induced pluripotent stem cells (hiPSCs) are an invaluable tool to study molecular mechanisms on a human background. Culturing stem cells at an oxygen level different from their microenvironmental niche impacts their viability. To understand this mechanistically, dermal skin fibroblasts of 52 probands were reprogrammed into hiPSCs, followed by either hyperoxic (20 % O 2 ) or physioxic (5 % O 2 ) culture and proteomic profiling. Analysis of chromosomal stability by Giemsa-banding revealed that physioxic -cultured hiPSC clones exhibited less pathological karyotypes than hyperoxic (e.g. 6 % vs. 32 % mosaicism), higher pluripotency as evidenced by higher Stage-Specific Embryonic Antigen 3 positivity, higher glucose consumption and lactate production. Global proteomic analysis demonstrated lower abundance of several subunits of NADH:ubiquinone oxidoreductase (complex I) and an underrepresentation of pathways linked to oxidative phosphorylation and cellular senescence. Accordingly, release of the pro-senescent factor IGFBP3 and β-galactosidase staining were lower in physioxic hiPSCs. RNA- and ATAC-seq profiling revealed a distinct hypoxic transcription factor-binding footprint, amongst others higher expression of the HIF1α-regulated target NDUFA4L2 along with increased chromatin accessibility of the NDUFA4L2 gene locus. While mitochondrial DNA content did not differ between groups, physioxic hiPSCs revealed lower polarized mitochondrial membrane potential, altered mitochondrial network appearance and reduced basal respiration and electron transfer capacity. Blue-native polyacrylamide gel electrophoresis coupled to mass spectrometry of the mitochondrial complexes detected higher abundance of NDUFA4L2 and ATP5IF1 and loss of incorporation into complex IV or V, respectively. Taken together, physioxic culture of hiPSCs improved chromosomal stability, which was associated with downregulation of oxidative phosphorylation and senescence and extensive re-wiring of mitochondrial complex composition., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: TE is a shareholder and a member of the scientific advisory board of DiNAQOR, which is not relating to this manuscript. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Engineered heart tissue (EHT) transplantation represents an innovative, regenerative approach for heart failure patients. Late preclinical trials are underway, and a first clinical trial started recently. Preceding studies revealed functional recovery after implantation of in vitro-matured EHT in the subacute stage, whereas transplantation in a chronic injury setting was less efficient. When transplanting matured EHTs, we noticed that cardiomyocytes undergo a dedifferentiation step before eventually forming structured grafts. Therefore, we wanted to evaluate whether immature EHT (EHTIm) patches can be used for transplantation. Chronic myocardial injury was induced in a guinea pig model. EHTIm (15×106 cells) were transplanted within hours after casting. Cryo-injury led to large transmural scars amounting to 26% of the left ventricle. Grafts remuscularized 9% of the scar area on average. Echocardiographic analysis showed some evidence of improvement of left-ventricular function after EHTIm transplantation. In a small translational proof-of-concept study, human scale EHTIm patches (4.5×108 cells) were epicardially implanted on healthy pig hearts (n=2). In summary, we provide evidence that transplantation of EHTIm patches, i.e. without precultivation, is feasible, with similar engraftment results to those obtained using matured EHT., Competing Interests: Competing interests C.v.B., T.E. and F.W. contribute in a structured partnership between Evotec AG and the UKE to originate an EHT-based remuscularization approach. They have no financial interests and did not obtain consultation fees. A patent relating to the generation of human-scale EHT patches has been filed., (© 2023. Published by The Company of Biologists Ltd.)

Background: Transplantation of pluripotent stem cell-derived cardiomyocytes represents a promising therapeutic strategy for cardiac regeneration, and the first clinical studies in patients with heart failure have commenced. Yet, little is known about the mechanism of action underlying graft-induced benefits. Here, we explored whether transplanted cardiomyocytes actively contribute to heart function., Methods: We injected cardiomyocytes with an optogenetic off-on switch in a guinea pig cardiac injury model., Results: Light-induced inhibition of engrafted cardiomyocyte contractility resulted in a rapid decrease of left ventricular function in ≈50% (7/13) animals that was fully reversible with the offset of photostimulation., Conclusions: Our optogenetic approach demonstrates that transplanted cardiomyocytes can actively participate in heart function, supporting the hypothesis that the delivery of new force-generating myocardium can serve as a regenerative therapeutic strategy.

Objectives: Univentricular malformations are severe cardiac lesions with limited therapeutic options and a poor long-term outcome. The staged surgical palliation (Fontan principle) results in a circulation in which venous return is conducted to the pulmonary arteries via passive laminar flow. We aimed to generate a contractile subpulmonary neo-ventricle from engineered heart tissue (EHT) to drive pulmonary flow actively., Methods: A three-dimensional tubular EHT (1.8-cm length, 6-mm inner diameter, ca. 1-mm wall thickness) was created by casting human-induced pluripotent stem cell-derived cardiomyocytes (0.9 ml, 18 mio/ml) embedded in a fibrin-based hydrogel around a silicone tube. EHTs were cultured under continuous, pulsatile flow through the silicone tube for 23 days., Results: The constructs started to beat macroscopically at days 8-14 and remained stable in size and shape over the whole culture period. Tubular EHTs showed a coherent beating pattern after 23 days in culture, and isovolumetric pressure measurements demonstrated a coherent pulsatile wave formation with an average frequency of 77 ± 5 beats/min and an average pressure of 0.2 mmHg. Histological analysis revealed cardiomyocytes mainly localized along the inner and outer curvature of the tubular wall with mainly longitudinal alignment. Cell density in the center of the tubular wall was lower., Conclusions: A simple tube-shaped contractile EHT was generated from human-induced pluripotent stem cells and developed a synchronous beating pattern. Further steps need to focus on optimizing support materials, flow rates and geometry to obtain a construct that creates sufficient pressures to support a directed and pulsatile blood flow., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Association for Cardio-Thoracic Surgery.)

Myocardial injury leads to an irreversible loss of cardiomyocytes (CM). The implantation of human engineered heart tissue (EHT) has become a promising regenerative approach. Previous studies exhibited beneficial, dose-dependent effects of human induced pluripotent stem cell (hiPSC)-derived EHT patch transplantation in a guinea pig model in the subacute phase of myocardial injury. Yet, advanced heart failure often results from a chronic remodeling process. Therefore, from a clinical standpoint it is worthwhile to explore the ability to repair the chronically injured heart. In this study human EHT patches were generated from hiPSC-derived CMs (15 × 10 6 cells) and implanted epicardially four weeks after injury in a guinea pig cryo-injury model. Cardiac function was evaluated by echocardiography after a follow-up period of four weeks. Hearts revealed large transmural myocardial injuries amounting to 27% of the left ventricle. EHT recipient hearts demonstrated compact muscle islands of human origin in the scar region, as indicated by a positive staining for human Ku80 and dystrophin, remuscularizing 5% of the scar area. Echocardiographic analysis demonstrated no significant functional difference between animals that received EHT patches and animals in the cell-free control group (fractional area change 36% vs. 34%). Thus, EHT patches engrafted in the chronically injured heart but in contrast to the subacute model, grafts were smaller and EHT patch transplantation did not improve left ventricular function, highlighting the difficulties for a regenerative approach., (Copyright © 2022. Published by Elsevier Ltd.)

MYBPC3 is the most frequently affected gene in hypertrophic cardiomyopathy (HCM), which is an autosomal-dominant cardiac disease caused by mutations in sarcomeric proteins. Bi-allelic truncating MYBPC3 mutations are associated with severe forms of neonatal cardiomyopathy. We reprogrammed skin fibroblasts from a HCM patient carrying a heterozygous MYBPC3 truncating mutation into human induced pluripotent stem cells (iPSC) and used CRISPR/Cas9 to generate bi-allelic MYBPC3 truncating mutation and isogenic control hiPSC lines. All lines expressed pluripotency markers, had normal karyotype and differentiated into endoderm, ectoderm and cardiomyocytes in vitro. This set of three lines provides a useful tool to study HCM pathomechanisms., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose., Methods: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility., Results: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×10 6 cardiomyocytes) and pigs (5×7 cm, 450×10 6 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×10 6 , or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation., Conclusions: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

The reproducibility of stem cell research relies on the constant availability of quality-controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch-to-batch variation. Here, we provide a cost-efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1: Expansion of hiPSCs Basic Protocol 2: Cell banking of hiPSCs Support Protocol 1: Pluripotency assessment by flow cytometry Support Protocol 2: Thawing control: Viability and sterility Support Protocol 3: Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT-PCR Support Protocol 4: Identity: Short tandem repeat analysis., (© 2020 The Authors.)

Background: Human induced pluripotent stem (iPS) cells have revolutionised research and spark hopes for future tissue replacement therapies. To obtain high cell numbers, iPS cells can be expanded indefinitely. However, as long-term expansion can compromise cell integrity and quality, we set out to assess potential reduction of clonal diversity by inherent growth imbalances., Methods: Using red, green, blue marking as a lentiviral multi-colour clonal cell tracking technology, we marked three different iPS cell lines as well as three other cell lines, assigning a unique fluorescent colour to each cell at one point in culture. Subsequently, we followed the sub-clonal distribution over time by flow cytometry and fluorescence microscopy analysis in regular intervals., Results: In three human iPS cell lines as well as primary human fibroblasts and two widely used human cell lines as controls (K562 and HEK 293 T), we observed a marked reduction in sub-clonal diversity over time of culture (weeks). After 38 passages, all iPS cultures consisted of less than 10 residual clones. Karyotype and function, the latter assessed by cardiomyocyte differentiation and tissue engineering, did not reveal obvious differences., Conclusions: Our results argue for a quick selection of sub-clones with a growth advantage and flag a normally invisible and potentially undesired behaviour of cultured iPS cells, especially when using long-term cultured iPS cells for experiments or even in-vivo applications.

Gene therapy is a promising option for severe forms of genetic diseases. We previously provided evidence for the feasibility of trans-splicing, exon skipping, and gene replacement in a mouse model of hypertrophic cardiomyopathy (HCM) carrying a mutation in MYBPC3, encoding cardiac myosin-binding protein C (cMyBP-C). Here we used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from an HCM patient carrying a heterozygous c.1358-1359insC MYBPC3 mutation and from a healthy donor. HCM hiPSC-CMs exhibited ∼50% lower MYBPC3 mRNA and cMyBP-C protein levels than control, no truncated cMyBP-C, larger cell size, and altered gene expression, thus reproducing human HCM features. We evaluated RNA trans-splicing and gene replacement after transducing hiPSC-CMs with adeno-associated virus. trans-splicing with 5' or 3' pre-trans-splicing molecules represented ∼1% of total MYBPC3 transcripts in healthy hiPSC-CMs. In contrast, gene replacement with the full-length MYBPC3 cDNA resulted in ∼2.5-fold higher MYBPC3 mRNA levels in HCM and control hiPSC-CMs. This restored the cMyBP-C level to 81% of the control level, suppressed hypertrophy, and partially restored gene expression to control level in HCM cells. This study provides evidence for (1) the feasibility of trans-splicing, although with low efficiency, and (2) efficient gene replacement in hiPSC-CMs with a MYBPC3 mutation., (Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.)

Since the advent of the generation of human induced pluripotent stem cells (hiPSCs), numerous protocols have been developed to differentiate hiPSCs into cardiomyocytes and then subsequently assess their ability to recapitulate the properties of adult human cardiomyocytes. However, hiPSC-derived cardiomyocytes (hiPSC-CMs) are often assessed in single-cell assays. A shortcoming of these assays is the limited ability to characterize the physiological parameters of cardiomyocytes, such as contractile force, due to random orientations. This protocol describes the differentiation of cardiomyocytes from hiPSCs, which occurs within 14 d. After casting, cardiomyocytes undergo 3D assembly. This produces fibrin-based engineered heart tissues (EHTs)-in a strip format-that generate force under auxotonic stretch conditions. 10-15 d after casting, the EHTs can be used for contractility measurements. This protocol describes parallel expansion of hiPSCs; standardized generation of defined embryoid bodies, growth factor and small-molecule-based cardiac differentiation; and standardized generation of EHTs. To carry out the protocol, experience in advanced cell culture techniques is required.

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30-40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5-10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research.

Various virus infections cause dysfunctional hemostasis and in some instances lead to the development of viral hemorrhagic fever syndrome. How do diverse viruses induce the expression of tissue factor on vascular cells? We hypothesize that a direct stimulation of pattern recognition receptors (PRR) by viral nucleic acids may be the key. Double-stranded RNA (dsRNA) is produced by many viruses and is recognized by various PRR, including Toll-like receptor-3 (TLR3). We have investigated whether poly I:C, a model for viral dsRNA, can influence cellular hemostasis. Poly I:C could up-regulate tissue factor and down-regulate thrombomodulin expression on endothelial cells but not on monocytes. The response to poly I:C was diminished upon small interfering RNA (siRNA)-mediated inhibition of TLR3, but not other PRR. In vivo, application of poly I:C induced similar changes in the aortic endothelium of mice as determined by enface microscopy. D-dimer, a circulating marker for enhanced coagulation and fibrinolysis, and tissue fibrin deposition was elevated. All the hemostasis-related responses to poly I:C, but not cytokine secretion, were blunted in TLR3(-/-) mice. Hence, the activation of TLR3 can induce the procoagulant state in the endothelium, and this could be relevant for understanding the mechanisms of viral stimulation of hemostasis.

The extrinsic coagulation system initiated by tissue factor (TF) appears to be a major procoagulant stimulus during cardiopulmonary bypass (CPB), although the precise mechanisms remain to be revealed. We recently reported the appearance of TF-bearing leukocytes during CPB and described their role in promoting coagulation. In this study, we visually identified the in-vivo appearance of TF-bearing leukocytes and platelet-derived particles on leukocytes in the pericardial blood during cardiac surgery with CPB, by flow cytometry and immunoelectron microscopy. Preliminary flow cytometric experiments showed that the proportion of TF-positive or both TF- and platelet antigen CD41a-positive leukocytes was increased markedly in pericardial blood obtained during CPB, compared with the proportions in preoperative circulating blood. Immunoelectron microscopic analysis revealed that both monocytes and polymorphonuclear leukocytes in the pericardial blood express TF. On the surfaces of these cells, CD41a-positive or both CD41a- and TF-positive platelet-derived particles were observed. Platelet-derived particles include not only microparticles, but also platelets themselves. Leukocytes from preoperative circulating blood contained far fewer of these particles. Our results demonstrate the in-vivo appearance of TF-bearing platelet-derived particles on leukocytes during cardiac surgery with CPB. These findings may be important for the development of strategies to control procoagulant activities during and after cardiac surgery.

An essential coagulation factor, tissue factor (TF), is rapidly expressed by human monocytes when exposed to a variety of agonists, such as lipopolysaccharide or tumor necrosis factor (TNF). We previously found that 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and its potent synthetic analogs downregulate TF and upregulate thrombomodulin expression on monocytic cells, counteracting the effects of TNF at the level of transcription. The human TF gene has characteristic binding sequences for activator protein-1 (AP-1) (c-Jun/c-Fos), nuclear factor-kappaB (NF-kappaB), Sp-1, and early growth response factor-1 (Egr-1). In this study, we investigated the regulatory mechanisms by which 1,25(OH)(2)D(3) inhibits TNF-induced TF expression in human monocytic cells. 1,25(OH)(2)D(3) reduced basal and TNF-induced TF activities. Gel-shift assay and luciferase assay with the respective reporter vectors showed that 1,25(OH)(2)D(3) reduced basal and TNF-induced activities of the nuclear proteins AP-1 and NF-kappaB, but not Egr-1. 1,25(OH)(2)D(3) inhibited TNF-induced phosphorylation of c-Jun without affecting phosphorylation of the other pathways. On the other hand, 1,25(OH)(2)D(3) directly inhibited nuclear binding and activities of NF-kappaB in the nucleus without affecting phosphorylation of the NF-kappaB activation pathway. These results indicate that 1,25(OH)(2)D(3) suppresses basal and TNF-induced TF expression in monocytic cells by inhibition of AP-1 and NF-kappaB activation pathways, but not of Egr-1. Our results may help to elucidate the regulatory mechanisms of 1,25(OH)(2)D(3) in TF induction, and may have physiological significance in the clinical challenge to use potential 1,25(OH)(2)D(3) analogs in antithrombotic therapy as well as immunomodulation and antineoplastic therapy of leukemia.

FSAP (Factor VII-activating protease) can cleave and inactivate PDGF-BB (platelet-derived growth factor-BB) and thereby inhibits VSMC (vascular smooth-muscle cell) proliferation. The auto-activation of FSAP is facilitated by negatively charged polyanions such as heparin, dextransulfate or extracellular ribonucleic acids. Since auto-activation is essential for the anti-proliferative function of FSAP, the influence of nucleic acids as cofactors for the FSAP-mediated inhibition of PDGF-BB was investigated. Natural or artificial RNA was an effective cofactor for FSAP mediated PDGF-BB degradation, whereas the effect of DNA was weak. RNA-induced cleavage of PDGF-BB was inhibited by serine protease inhibitors. The pattern of PDGF-BB cleavage was identical with either heparin or RNA as a cofactor. One of the cleavage sites in PDGF-BB was at the positions 160-162 (R160KK162), which is an important region for receptor binding and activation. In VSMCs, PDGF-BB-stimulated DNA synthesis was inhibited by FSAP in the presence of RNA. RNA was more effective than DNA and the cofactor activity of RNA was neutralized after pretreatment with RNase. FSAP binding to RNA protected the nucleic acid from degradation by RNase. These data are relevant to situations where extracellular nucleic acids released from necrotic or apoptotic cells could activate local FSAP, leading to inhibition of PDGF-BB.

Upon vascular injury, locally controlled haemostasis prevents life-threatening blood loss and ensures wound healing. Intracellular material derived from damaged cells at these sites will become exposed to blood components and could contribute to blood coagulation and pathological thrombus formation. So far, the functional and mechanistic consequences of this concept are not understood. Here, we present in vivo and in vitro evidence that different forms of eukaryotic and prokaryotic RNA serve as promoters of blood coagulation. Extracellular RNA was found to augment (auto-)activation of proteases of the contact phase pathway of blood coagulation such as factors XII and XI, both exhibiting strong RNA binding. Moreover, administration of exogenous RNA provoked a significant procoagulant response in rabbits. In mice that underwent an arterial thrombosis model, extracellular RNA was found associated with fibrin-rich thrombi, and pretreatment with RNase (but not DNase) significantly delayed occlusive thrombus formation. Thus, extracellular RNA derived from damaged or necrotic cells particularly under pathological conditions or severe tissue damage represents the long sought natural "foreign surface" and provides a procoagulant cofactor template for the factors XII/XI-induced contact activation/amplification of blood coagulation. Extracellular RNA thereby reveals a yet unrecognized target for antithrombotic intervention, using RNase or related therapeutic strategies.

FSAP (Factor VII-activating protease) is a novel plasma-derived serine protease that regulates haemostasis as well as vascular cell proliferation. FSAP undergoes autoactivation in the presence of polyanionic macromolecules such as heparin and RNA. Competition experiments suggest that RNA and heparin bind to the same or overlapping interaction sites. A proteolysis approach, where FSAP was hydrolysed into smaller fragments, was used to identify the polyanion-binding site. The EGF (epidermal growth factor)-like domains EGF2 and EGF3 of FSAP are the major interaction domains for RNA. The amino acids Arg170, Arg171, Ser172 and Lys173 within the EGF3 domain were essential for this binding. This is also the region with the highest positive net charge in the protein and is most probably located in an exposed loop. It is also highly conserved across five species. Disruption of disulphide bridges led to the loss of RNA and heparin binding, indicating that the three-dimensional structure of the EGF3 domain is essential for binding to negatively charged heparin or RNA. The identification of polyanion-binding sites will help to define the role of FSAP in the vasculature.

FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K(D) estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.

Proteins that fail to attain their correct three-dimensional structure are retained in the endoplasmic reticulum (ER) and eventually degraded within the cells. We investigated the degradation of mutant proteins, using naturally occurring protein C (PC) mutants (Arg178Gln and Cys331Arg) which lead to congenital deficiencies. Chinese hamster ovary (CHO) cells were transfected with normal or mutant expression vectors. The introduction of mutation at Asn329 of an unusual sequence Asn-X-Cys for N-linked glycosylation revealed that the mutation at Cys331, which may preclude a formation of disulfide bond with Cys345, resulted in no addition of N-linked oligosaccharides at Asn329. PC mutants with 4 glycosylation sites were gradually glycosylated in the ER, and the fourth glycosylation site is less accessible for glycosylation as reported for PC in plasma. The half lives of PC178 and PC331 mutants were about 5 and 4 h, respectively. PC mutants were degraded, but the degradation was inhibited by inhibitors for proteasome. Mannose trimming of N-linked oligosaccharides after glucose removal targeted PC mutants for degradation by proteasomes. And also the inhibition of glucose trimming immediately led to mannose trimming, resulting in the accelerated degradation of PC mutants. These degradations were inhibited by mannosidase I inhibitor, kifunensine. These results indicate that the initiation of mannose trimming by mannosidase I leads to the proteasomemediated degradation of glucose-trimmed or untrimmed PC mutants.

Objective: Immediate loss of thrombomodulin activity in the endothelium of vein grafts has been demonstrated during 90 min exposure to arterial circulation; this loss of activity is ascribed as an important cause of early thrombosis. Conventional ex vivo gene transfection after vein harvest cannot cover this acute period immediately after implantation. We have established a highly efficient non-viral gene therapy protocol utilizing modified transferrin receptor-facilitated gene transfer. Using this technique, we examined whether in vivo thrombomodulin gene therapy, directed to the endothelium of rat veins 2 days prior to grafting, may prevent thromboresistance impairment of vein grafts under simulated arterial circulation., Methods: Abdomen of SD rat was opened and cationic liposome:transferrin:thrombomodulin gene complexes or the vector without DNAs were applied to the inferior vena cava of rats while blood flow was reduced by proximal and distal clamping. After 2 days, the transfected veins were harvested and thrombomodulin expression and thromboresistance properties determined before and after exposure to an artificial circuit., Results: The trial of gene transfection using variable doses of DNAs confirmed that 7.5 microg of total DNAs was the most efficient quantity for thrombomodulin gene transfection to IVCs, although accompanying an increase of gene expression in other downstream organs. By transfection of the thrombomodulin gene in IVCs, the generation capacity of activated protein C in venous endothelium increased three-fold compared with veins treated with vector alone (P<0.01). Under simulated arterial circulation, perfusion of veins treated with vector alone decreased thrombomodulin activity to 36% of preperfused levels (P<0.01), whereas transfected grafts preserved the activity at normal vein endothelium levels even after perfusion. Consequently, the increase in endothelial thrombin activity induced by simulated arterial circulation was markedly attenuated in transfected veins (P<0.01), while immunohistochemistry confirmed the preservation of endothelial lining., Conclusions: Transferrin receptor-facilitated in vivo gene transfer to the inferior vena cava resulted in sufficient thrombomodulin gene expression immediately after graft implantation and subsequent maintenance of thromboresistance even after exposure to arterial pressure. Although further studies are needed, the present results suggest the possibility of gene therapy targeting acute phases of vein graft disease.

During cardiopulmonary bypass (CPB), the extrinsic coagulation system initiated by tissue factor (TF) is a major procoagulant stimulus. TF is present in surgical wounds and could be expressed on activated monocytes. However, recent studies have suggested that collagen stimulation rapidly induces TF by leukocyte-platelet complex formation. Therefore, the appearance of TF-bearing leukocytes and their effect on promoting coagulation were investigated in 5 patients undergoing coronary bypass surgery. Neutrophils and monocytes positive for CD41a and TF increased abruptly in circulating blood during CPB. Their increase was most prominent in blood pooled in the pericardial cavity. Monocytes, but not neutrophils positive for TF showed a second peak one day after operation, which accords with the increase in TF mRNA levels in leukocytes. Similarly, an increase in leukocytes positive for TF accords with the activated factor X generation assay using isolated leukocytes, and with an increase in thrombin-antithrombin complex in circulating blood. The second increase in TF-positive monocytes seems to be responsible for these coagulation parameters that remained high one day after operation. After 10 min of blood incubation stimulated by collagen in vitro, simulating activation in the pericardial cavity, significant increases in neutrophils and monocytes positive for TF and platelet were observed. Our present study suggested the involvement of two distinct mechanisms for the appearance of TF-bearing leukocytes responsible for promoting coagulation: the quick appearance being partly explained by the formation of leukocyte-platelet complex that occurs mainly in the pericardial cavity, and the slow appearance via transcriptional activation in monocytes.

Biocompatibility of extracorporeal circuits has mostly been investigated by sampling blood in circulation. However, a proportion of activated leukocytes leave circulation by sticking to the circuits, and might affect the circuit biocompatibility. To reveal these characteristics, we eluted the adsorbed leukocytes from circuits used for 6 patients by washing with ethylenediaminetetraacetic acid (EDTA)-HEPES buffer. 1.3 x 109 leukocytes, representing 5 to 10% of the circulating leukocytes, were collected. Most were neutrophils expressing CD11b antigens as high as those in circulation during cardiopulmonary bypass. Adsorbed monocytes expressed tissue factor higher than those in circulation (P < 0.05). An elution procedure by EDTA itself induced only a minimal activation of the leukocytes. These results indicate that extracorporeal circuits adsorb a large number of activated neutrophils and a small number of highly thrombogenic monocytes during their use. Although our data is limited, this elution procedure appears useful to investigate the biocompatibility of extracorporeal circuits.

Plasma plasmin inhibitor (PI) is a physiological inhibitor of plasmin-mediated fibrinolysis and constitutes a hemostatic component in blood plasma; hence its deficiency results in a severe hemorrhagic diathesis. We have carried out molecular analysis of American family members with congenital PI deficiency, and detected a single thymine deletion at nucleotide position 332 in exon 5. The deletion was found in both alleles of the homozygotes and in one allele of the heterozygotes, and the patterns of restriction fragment length polymorphism created by the mutation in the family members were compatible with their phenotypes. The deletion caused a frameshift leading to an alteration and shortening of the deduced amino acid sequence. The amino acid sequence consists of the first 83 amino acids of the N-terminal sequence of the normal PI and additional new amino acids, resulting in a mutant composed of 94 amino acids in contrast to 464 amino acids of the normal PI. In transient expression analysis, the mutant PI whose molecular size was compatible with the predicted amino acid sequence was detected in the lysates of the cells transfected with the mutated PI expression vector. The mutant PI was retained and underwent progressive degradation within the cells, and was minimally excreted into the media. These data indicate that this mutation is the cause of PI deficiency in this pedigree.