Fibrinolysis biomarker, thrombin, and activated protein C level alterations after coagulation activation depend on type of thrombophilia and clinical phenotype.

Background: Recently, we have shown alterations in the anticoagulant response to recombinant activated factor VII (rFVIIa)-induced coagulation activation in patients with thrombophilia. Objectives: This study aimed to extend this in vivo model to fibrinolysis biomarkers. Methods: This interventional in vivo study included 56 patients with thrombophilia and previous venous thromboembolism (VTE+), 38 without VTE (VTE-), and 35 healthy controls. Plasma levels of D-dimer, plasmin-α2-antiplasmin (PAP) complex, and plasminogen activator inhibitor-1 (PAI-1) were monitored for over 8 hours after rFVIIa infusion (15 µg/kg) along with thrombin markers and activated protein C (APC). Results: Throughout cohorts, median PAP increased by 40% to 52% (P < 3.9 × 10-10) and PAI-1 decreased by 59% to 79% (P < 3.5 × 10-8). In contrast to thrombin-antithrombin (TAT) complex, which also increased temporarily (44% to 115%, P < 3.6 × 10-6), changes in PAP and PAI-1 did not reverse during the observation period. The area under the measurement-time curves (AUCs) of PAP and TAT, which are measures of plasmin and thrombin formation, respectively, were each greater in the VTE+ cohort than in healthy controls (median PAP-AUC = 0.48 vs 0.27 ng·h/L [P = .003], TAT-AUC = 0.12 vs 0.03 nmol·h/L [P = 2.5 × 10-4]) and were correlated with one another (r = 0.554). As evidenced by the respective AUCs, asymptomatic factor (F)V Leiden carriers showed less PAP formation (0.22 vs 0.41 ng·h/L, P = 9 × 10-4), more pronounced PAI-1 decline (0.10 vs 0.18 ng·h/L, P = .01), and increased APC formation (28.7 vs 15.4 pmol·h/L, P = .02) than those within the VTE+ group (n = 19 each). Conclusion: rFVIIa-induced thrombin formation is associated with fibrinolysis parameter changes outlasting the concomitant anticoagulant response. Both correlate with thrombosis history in FV Leiden and might help explain its variable clinical expressivity.

Potentials of Endothelial Colony-Forming Cells: Applications in Hemostasis and Thrombosis Disorders, from Unveiling Disease Pathophysiology to Cell Therapy.

Endothelial colony-forming cells (ECFCs) are endothelial progenitor cells circulating in a limited number in peripheral blood. They can give rise to mature endothelial cells (ECs) and, with intrinsically high proliferative potency, contribute to forming new blood vessels and restoring the damaged endothelium in vivo. ECFCs can be isolated from peripheral blood or umbilical cord and cultured to generate large amounts of autologous ECs in vitro. Upon differentiation in culture, ECFCs are excellent surrogates for mature ECs showing the same phenotypic, genotypic, and functional features. In the last two decades, the ECFCs from various vascular disease patients have been widely used to study the diseases' pathophysiology ex vivo and develop cell-based therapeutic approaches, including vascular regenerative therapy, tissue engineering, and gene therapy. In the current review, we will provide an updated overview of past studies, which have used ECFCs to elucidate the molecular mechanisms underlying the pathogenesis of hemostatic disorders in basic research. Additionally, we summarize preceding studies demonstrating the utility of ECFCs as cellular tools for diagnostic or therapeutic clinical applications in thrombosis and hemostasis.

Endotheliale koloniebildene Zellen (endothelial colony forming cells, ECFCs) sind endotheliale Vorläuferzellen, die in begrenzter Zahl im peripheren Blut zirkulieren. Sie unterstützen das Wachstum adulter Endothelzellen (ECs), haben ein hohes intrinsisches Proliferationspotential und tragen zur Neubildung von Blutgefäßen und Reparatur von Geweben in vivo bei. ECFCs können aus peripherem oder Nabelschnurblut isoliert werden und können in vitro in großen Mengen als autologe ECs kultiviert werden. Nach Differenzierung in Kultur liefern ECFCs einen exzellenten Ersatz für adulte Endothelzellen, da sie dieselben phenotypischen, genotypischen, und funktionellen Eigenschaften besitzen. In den letzten zwanzig Jahren wurden ECFCs von Patienten mit verschiedenen vaskulären Erkrankungen ex vivo verwendet, um die Pathophysiologie von Krankheiten zu studieren und Zell-basierte therapeutische Ansätze wie vaskuläre regenerative Therapie, tissue engineering, oder Gentherapie zu entwickeln. In diesem Review geben wir eine Übersicht über vergangene Studien, in denen ECFCs zur Aufklärung molekularer Mechanismen, die der Pathogenese hämostaseologischer Erkrankungen unterliegen, in der Grundlagenforschung verwendet wurden. Außerdem zeigen wir eine Zusammenfassung vorangehender Studien, die den Nutzen von ECFCs als zelluläres Werkzeug für Diagnose oder Therapie im klinischen Feld von Thrombose und Hämostase demonstrieren.

Antidrug antibodies against the polyethylene glycol moiety inhibit the procoagulant activity of therapeutic polyethylene glycolated factor VIII.

BACKGROUND: The standard therapy for patients with hemophilia A (HA) is the replacement with factor VIII (FVIII) therapeutics. To overcome the limitation of short half-life of wild-type FVIII protein, polyethylene glycol (PEG) can be coupled to therapeutic FVIII to improve pharmacokinetics. OBJECTIVES: We aimed to characterize antibodies developed against a FVIII therapeutic PEGylated with a 40-kDa PEG (40PEG-BDDFVIII) in 2 patients with mild HA. METHODS: An inhouse bead-based immunoassay was developed to characterize and confirm the specificity of the detected antibodies. The neutralizing nature of the antibodies toward PEGylated therapeutics was determined by a modified Nijmegen-Bethesda assay. RESULTS: Two out of 46 patients treated with 40PEG-BDDFVIII developed inhibitory antibodies toward the drug. Switching to a non-PEGylated FVIII successfully increased the FVIII activity in both patients. In patient 1, antibodies were raised against FVIII and PEG. Anti-FVIII antibodies were of the immunoglobulin (Ig)G isotype, whereas anti-PEG antibodies were of IgG, IgM, and IgA isotypes. In patient 2, antibodies of IgG and IgA isotypes were directed only against the PEG moiety. Competitive assays confirmed the specificity of the antibodies against PEG. The applied Nijmegen-Bethesda assay revealed that patients' anti-PEG antibodies and AGP3, an antibody against the backbone of PEG, can inhibit all currently available PEGylated therapeutics but to different degrees. No inhibitory FVIII antibodies were detected. CONCLUSION: Antibodies against the PEG moiety of 40PEG-BDDFVIII abolished the efficacy of the drug. This is the first report on real-world experiences with the development of neutralizing anti-PEG antibodies after treatment with PEGylated FVIII therapeutics in mild HA.

Ex Vivo Modeling of the PC (Protein C) Pathway Using Endothelial Cells and Plasma: A Personalized Approach.

BACKGROUND: The endothelial cell-dependent PC (protein C) pathway is critically involved in the regulation of coagulation, anti-inflammatory, and cytoprotective signaling. Its reactivity shows high interindividual variability, and it contributes to prothrombotic disorders, such as the FVL (factor V Leiden) mutation. METHODS: Endothelial colony-forming cells (ECFCs) were isolated from heparinized peripheral blood from healthy individuals and FVL carriers. Confluent monolayers of ECFCs were overlaid with plasma, and thrombin formation was initiated by addition of tissue factor (1 pmol/L). Subsequently, thrombin and APC (activated PC) formation rates were measured over time using oligonucleotide-based enzyme capture assays. To induce downregulation of TM (thrombomodulin) expression, ECFCs were stimulated with IL-1ß (interleukin 1ß). In vivo APC response rates were monitored in study participants after infusion of low-dose rFVIIa (recombinant activated factor VII). RESULTS: The median peak APC concentration was 1.12 nmol/L in experiments with IL-1ß stimulated ECFCs and 3.66 nmol/L without IL-1ß. Although thrombin formation rates were comparable, APC formation rates were significantly higher in FVL carriers (n=6) compared to noncarriers (n=5) as evidenced by a higher ratio between the area under the curve of APC generation to the area under the curve of thrombin generation (median 0.090 versus 0.031, P=0.017). These ex vivo results were correlated with an increased APC response to rFVIIa-induced thrombin formation in FVL carriers in vivo. CONCLUSIONS: Patient-specific ex vivo modeling of the PC pathway was achieved using blood-derived ECFCs. The correlation between in and ex vivo APC response rates confirms that the autologous PC model accurately depicts the in vivo situation.

Activated Factor XI is Increased in Plasma in Response to Surgical Trauma but not to Recombinant Activated FVII-Induced Thrombin Formation.

AIM: Feedback activation of factor XI (FXI) by thrombin is believed to play a critical role in the amplification phase of thrombin generation and to contribute to thrombosis development and hemostasis. However, the activation of FXI by thrombin has been shown in vitro to require a cofactor. In this study, the role of thrombin in activated FXI (FXIa) formation in vivo is investigated. METHODS: The study population comprised probands in whom coagulation activation was triggered by low-dose (15 µg/kg) recombinant activated factor VII (rFVIIa, n=89), of whom 34 with (VTE+) and 45 without a history of venous thromboembolism (VTE-), and patients undergoing major orthopedic surgeries (n=45). FXIa was quantified via an enzyme capture assay using a monoclonal FXI-specific antibody. Thrombin formation was monitored using an oligonucleotide-based enzyme capture assay and the thrombin activation markers prothrombin fragment 1＋2 (F1＋2) and thrombin-antithrombin complex (TAT). RESULTS: In the rFVIIa cohort, FXIa and thrombin remained below their lower limit of quantification of 3.48 and 1.06 pmol/L, respectively. By contrast, during the surgeries, median FXIa levels increased from 3.69 pmol/L pre-operatively to 9.41 pmol/L mid-operatively (P=4·10-4) and remained significantly elevated 24 h thereafter, with 9.38 pmol/L (P=0.001). Peak levels of F1+2 were comparable in the VTE+, VTE-, and surgery cohort (235, 268, and 253 pmol/L), whereas peak TAT levels were higher in the surgery cohort (53.1, 33.9, and 147.6 pmol/L). CONCLUSIONS: Under in vivo conditions, the activation of FXI requires specific local features that are present at the wounded site including potential cofactors of thrombin.

The zeaxanthin epoxidase is degraded along with the D1 protein during photoinhibition of photosystem II.

The xanthophyll zeaxanthin is synthesized in chloroplasts upon high light exposure of plants and serves central photoprotective functions. The reconversion of zeaxanthin to violaxanthin is catalyzed by the zeaxanthin epoxidase (ZEP). ZEP shows highest activity after short and moderate high light periods, but becomes gradually down-regulated in response to increasing high light stress along with down-regulation of photosystem II (PSII) activity. ZEP activity and ZEP protein levels were studied in response to high light stress in four plant species: Arabidopsis thaliana, Pisum sativum, Nicotiana benthamiana and Spinacia oleracea. In all species, ZEP protein was degraded during photoinhibition of PSII in parallel with the D1 protein of PSII. In the presence of streptomycin, an inhibitor of chloroplast protein synthesis, photoinhibition of PSII and ZEP activity as well as degradation of D1 and ZEP protein was strongly increased, indicating a close correlation of ZEP regulation with PSII photoinhibition and repair. The concomitant high light-induced inactivation/degradation of ZEP and D1 prevents the reconversion of zeaxanthin during photoinhibition and repair of PSII. This regulation of ZEP activity supports a coordinated degradation of D1 and ZEP during photoinhibition/repair of PSII and an essential photoprotective function of zeaxanthin during the PSII repair cycle.

The Verbal Fluency Decline After Deep Brain Stimulation in Parkinson's Disease: Is There an Influence of Age?

BACKGROUND: DBS is commonly used to treat Parkinson's disease (PD). DBS is not considered to cause major cognitive side effects, but some research groups have reported that it can cause decreased verbal fluency. The influence of age on DBS cognitive outcome is unclear. We investigated the possible influence of patients' age, level of education, disease duration, disease progression, depression, and levodopa equivalent dose (LED) on verbal fluency performance in patients with PD who underwent DBS of the subthalamic nucleus (STN-DBS). In this article, we investigated the influence of demographic and clinical parameters, especially age, on cognitive performance post-DBS in PD patients. METHODS: Forty-three patients with PD and without major psychiatric illness (according to Diagnostic and Statistical Manual of Mental Disroders, Fourth Edition) were enrolled in the study. Median age was 64.0 years (range, 46-77). In 21 patients, the indication for DBS was established on clinical grounds in keeping with international guidelines; these patients underwent STN-DBS, and the remaining 22 did not. Cognitive performance in both groups was assessed by standard neuropsychological test batteries at baseline and after median follow-up of 7 months. RESULTS: A statistically significant decline in the semantic category of verbal fluency task was found in the STN-DBS group (P < 0.01). Linear regression model revealed an influence of age (P < 0.01) and disease duration (P < 0.01) in relation to this decline. CONCLUSIONS: This study confirms previous findings that verbal fluency declines after STN-DBS in PD patients in comparison to PD patients without DBS. This decline is related to age and disease duration.

Tissue-specific accumulation and regulation of zeaxanthin epoxidase in Arabidopsis reflect the multiple functions of the enzyme in plastids.

The enzyme zeaxanthin epoxidase (ZEP) catalyzes the conversion of zeaxanthin to violaxanthin, a key reaction for ABA biosynthesis and the xanthophyll cycle. Both processes are important for acclimation to environmental stress conditions, in particular drought (ABA biosynthesis) and light (xanthophyll cycle) stress. Hence, both ZEP functions may require differential regulation to optimize plant fitness. The key to understanding the function of ZEP in both stress responses might lie in its spatial and temporal distribution in plant tissues. Therefore, we analyzed the distribution of ZEP in plant tissues and plastids under drought and light stress by use of a ZEP-specific antibody. In addition, we determined the pigment composition of the plant tissues and chloroplast membrane subcompartments in response to these stresses. The ZEP protein was detected in all plant tissues (except flowers) concomitant with xanthophylls. The highest levels of ZEP were present in leaf chloroplasts and root plastids. Within chloroplasts, ZEP was localized predominantly in the thylakoid membrane and stroma, while only a small fraction was bound by the envelope membrane. Light stress affected neither the accumulation nor the relative distribution of ZEP in chloroplasts, while drought stress led to an increase of ZEP in roots and to a degradation of ZEP in leaves. However, drought stress-induced increases in ABA were similar in both tissues. These data support a tissue- and stress-specific accumulation of the ZEP protein in accordance with its different functions in ABA biosynthesis and the xanthophyll cycle.

Apathy in Parkinson's disease is related to executive function, gender and age but not to depression.

Deficits in executive functions occur in up to 93% of patients with Parkinson's disease (PD). Apathy, a reduction of motivation and goal-directed behavior is an important part of the syndrome; affecting both the patients as well as their social environment. Executive functions can be subdivided into three different processes: initiation, shifting and inhibition. We examined the hypotheses, (1) that apathy in patients with Parkinson's disease is only related to initiation and not to shifting and inhibition, and (2) that depression and severity of motor signs correlate with apathy. Fifty-one non-demented patients (19 = female) with PD were evaluated for apathy, depression and executive functions. Executive function variables were summarized with an index variable according to the defined executive processes. Linear regression with stepwise elimination procedure was used to select significant predictors. The significant model (R (2) = 0.41; p < 0.01) revealed influences of initiation (b = -0.79; p < 0.01), gender (b = -7.75; p < 0.01), age (b = -0.07; p < 0.05) and an age by gender interaction (b = 0.12; p < 0.01) on apathy in Parkinson's disease. Motor signs, depression and level of education did not influence the relation. These results support an association of apathy and deficits of executive function in PD. Initiation strongly correlates with apathy, whereas depression does not. We conclude, that initiation dysfunction in a patient with Parkinson's disease heralds apathy. Apathy and depression can be dissociated. Additionally, apathy is influenced by age and gender: older age correlates with apathy in men, whereas in women it seems to protect against it.

Increased mitochondrial content in remyelinated axons: implications for multiple sclerosis.

Mitochondrial content within axons increases following demyelination in the central nervous system, presumably as a response to the changes in energy needs of axons imposed by redistribution of sodium channels. Myelin sheaths can be restored in demyelinated axons and remyelination in some multiple sclerosis lesions is extensive, while in others it is incomplete or absent. The effects of remyelination on axonal mitochondrial content in multiple sclerosis, particularly whether remyelination completely reverses the mitochondrial changes that follow demyelination, are currently unknown. In this study, we analysed axonal mitochondria within demyelinated, remyelinated and myelinated axons in post-mortem tissue from patients with multiple sclerosis and controls, as well as in experimental models of demyelination and remyelination, in vivo and in vitro. Immunofluorescent labelling of mitochondria (porin, a voltage-dependent anion channel expressed on all mitochondria) and axons (neurofilament), and ultrastructural imaging showed that in both multiple sclerosis and experimental demyelination, mitochondrial content within remyelinated axons was significantly less than in acutely and chronically demyelinated axons but more numerous than in myelinated axons. The greater mitochondrial content within remyelinated, compared with myelinated, axons was due to an increase in density of porin elements whereas increase in size accounted for the change observed in demyelinated axons. The increase in mitochondrial content in remyelinated axons was associated with an increase in mitochondrial respiratory chain complex IV activity. In vitro studies showed a significant increase in the number of stationary mitochondria in remyelinated compared with myelinated and demyelinated axons. The number of mobile mitochondria in remyelinated axons did not significantly differ from myelinated axons, although significantly greater than in demyelinated axons. Our neuropathological data and findings in experimental demyelination and remyelination in vivo and in vitro are consistent with a partial amelioration of the supposed increase in energy demand of demyelinated axons by remyelination.

Postoperative analgesia and functional recovery after total-knee replacement: comparison of a continuous posterior lumbar plexus (psoas compartment) block, a continuous femoral nerve block, and the combination of a continuous femoral and sciatic nerve block.

BACKGROUND AND OBJECTIVES: Continuous femoral nerve block is a well-accepted technique for regional analgesia after total-knee replacement. However, many patients still experience considerable pain at the popliteal space and at the medial aspect of the knee. The goal of this study is to evaluate whether a psoas compartment catheter provides better postoperative analgesia than a femoral nerve catheter does and whether it is as effective as the combination of a femoral and a sciatic nerve catheter and, thus, improves functional outcome. METHODS: Ninety patients who underwent total-knee replacement under standardized general anesthesia participated in this prospective randomized study. Group FEM received a continuous femoral nerve block, group FEM/SCI received a combination of a femoral and a sciatic continuous nerve block, and group PSOAS received a continuous psoas compartment block. Patient-controlled analgesia with piritramide was available for 48 hours. Maximal bending and extending of the knee and walking distance was assessed during the first 7 days. A standardized telephone survey was conducted after 9 to 12 months to evaluate residual pain and functional outcome. RESULTS: Postoperative opioid consumption during 48 hours was significantly less in the FEM/SCI group (median: 18 mg; 25th/75th percentile: 6/40) compared with the FEM group (49 mg; 25/66) and the PSOAS group (44 mg; 30/62) (P =.002). Postoperative pain scores were not different, and no differences occurred with respect to short-term or long-term functional outcome. CONCLUSION: The FEM/SCI catheter is superior to FEM and PSOAS catheter with respect to reduced analgesic requirements after total-knee replacement, but functional outcome does not differ with those 3 continuous regional analgesia techniques.