Inhibiting platelet-stimulated blood coagulation by inhibition of mitochondrial respiration.

Platelets are important mediators of blood coagulation that lack nuclei, but contain mitochondria. Although the presence of mitochondria in platelets has long been recognized, platelet mitochondrial function remains largely unaddressed. On the basis of a small amount of literature that suggests platelet mitochondria are functional, we hypothesized that the inhibition of platelet mitochondria disrupts platelet function and platelet-activated blood coagulation. To test this hypothesis, members of the tetrazole, thiazole, and 1,2,3-triazole families of small molecule heterocycles were screened for the ability to inhibit isolated mitochondrial respiration and coagulation of whole blood. The families of heterocycles screened were chosen on the basis of the ability of the heterocycle family to inhibit a biomimetic model of cytochrome c oxidase (CcO). The strength of mitochondrial inhibition correlates with each compound's ability to deter platelet stimulation and platelet-activated blood clotting. These results suggest that for this class of molecules, inhibition of blood coagulation may be occurring through a mechanism involving mitochondrial inhibition.

Association of plasma dilution with cardiopulmonary bypass-associated bleeding and morbidity.

OBJECTIVE: To investigate the relationship of cardiopulmonary bypass-associated plasma dilution with blood product transfusion and postoperative morbidity. DESIGN: Retrospective chart review. SETTING: Single academic medical center. PARTICIPANTS: Five hundred forty adults undergoing cardiac surgery between January 4, 2005 and September 19, 2007. INTERVENTIONS: Records were analyzed for demographics, blood volumes (BVs), and fluid balance. Plasma protein concentrations (% of baseline) at the end of bypass were calculated. The lowest and highest quartiles of plasma protein concentration were correlated with blood product administration and postoperative complications. MEASUREMENTS AND MAIN RESULTS: At the end of bypass, calculated plasma protein concentrations ranged from a low of 10% to a high of 111% of baseline. Concentrations below 45% of baseline were associated with increased blood product administration, longer ventilator support, and longer intensive care unit stay. CONCLUSIONS: Patient morbidity and likelihood of transfusion were associated with calculated plasma protein concentrations below 45% of baseline. Bleeding and administered fluids decrease both hematocrit and plasma proteins. Infusion of washed, salvaged blood or red blood cells raises hematocrit, but further dilutes clotting factors. If this dilution is excessive, coagulopathy may ensue. Patients with the smallest BVs are at greatest risk, but dilution can negatively impact patients with large BVs as well if the fluid used for cardiopulmonary bypass prime and anesthesia management represents a significant fraction of total BV.

Fibrin Strands Will Grow from Soluble Fibrin and Hang Up in an In Vitro Microcirculatory Viscoelastic Model: Is This a Major Cause of COVID-19 Associated Coagulopathy?

Viscoelastic testing (VET) by both TEG and ROTEM has demonstrated hypercoagulability early in corona virus disease 2019 (COVID-19) associated coagulopathy (CAC). Additional VET studies demonstrated fibrinolytic shutdown late in a majority of severely ill COVID-19 patients with an associated elevation of d-dimer. Elevated d-dimer confirms that coagulation, followed by fibrinolysis, has occurred. These findings imply that, during CAC, three enzymes-thrombin, Factor XIIIa and plasmin-must have acted in sequence. However, limitations in standard VET analyses preclude exploration of the earliest phases of clot induction, as well as clot formation and clot dissolution in flowing blood. Herein, we describe a novel method illuminating aspects of this unexplored area. In addition, we created an in vitro blood flow model in which the interactions of thrombin, Factor XIII and plasmin with fibrinogen can be studied, allowing the determination of soluble fibrin (SF), the highly unstable form of fibrin that precedes the appearance of a visible clot. This model allows the determination of the SF level at which fibrin microclots begin to form.

An algorithm for preventing bypass-associated dilutional (BAD) coagulopathy: Theory and example.

OBJECTIVES: Crystalloid administered during cardiopulmonary bypass may significantly dilute clotting factor concentrations, particularly in low blood volume patients. Should the administered fluid (pump prime plus IVs) drop the clotting factor concentrations below approximately 38%, almost all patients will bleed, heparin levels will be overestimated and excessive neutralizing protamine will be administered. This combination can render blood virtually unclottable. This paper describes algorithms that quantify dilution risk and the maximum fluid that can be safely administered. A confirmatory calculation to prevent excessive protamine administration is also described.

Postoperative bypass bleeding: a bypass-associated dilutional (BAD) coagulopathy?

A number of associations with post-bypass bleeding have been described in the accompanying paper. Herein we hypothesize that dilution is an underlying cause through a malign series of bypass-associated events. Heparinized blood behaves anomalously when diluted. Clotting times first shorten somewhat, then--as the dilution of whole blood approaches 50%--rapidly lengthen to unclottability. During cardiopulmonary bypass, low blood volume patients are at a significant risk of clotting factor dilution which will always be more severe than the level of whole blood dilution. If severe enough, this dilution may lower plasma clotting factors to a critical level and may result in excess protamine administration, secondary to overestimation of heparin. The presence of un-neutralized protamine combined with critically lowered clotting factors leads to marked coagulopathy.

Extreme Plasma Dilution Decreases Heparin and Protamine Cardiopulmonary Bypass Requirements: A Case Report on a Jehovah's Witness Patient.

The primary focus of cardiopulmonary bypass management in Jehovah's Witness patients is the need to conserve blood. A consequence of these strategies inevitably results in hemodilution that is frequently extreme enough to dilute clotting factors and potentially impair coagulation. The purpose of this case report is to demonstrate that a hemodiluted patient requires less heparin to sustain anticoagulation and less protamine to reverse heparin at cardiopulmonary bypass termination. Patient harm may ensue unless the effects of extreme hemodilution are recognized.

Non-Hodgkin's lymphoma in Job's syndrome: a case report and literature review.

Job's or hyper immunoglobulin E recurrent infection syndrome (Hyper-IgE syndrome) is a rare, often inherited multisystem disorder, characterized by cutaneous abscesses, pneumonia, elevated IgE levels and skeletal defects. We report a case of a 22-year-old man with Job's syndrome who presented with back pain. He was found to have diffuse large B-cell lymphoma involving his second lumbar vertebrae and spleen. Treatment with dose-adjusted EPOCH-rituximab (DA-EPOCH-R) chemotherapy achieved a complete remission after 4 cycles. A review of reported cases of lymphoma in Job's syndrome indicates an increase in relative risk of 259 (95% confidence interval 102, 416). The cause of the increased risk has yet to be defined but has similarities to a pathogenetic model of AIDS related lymphoma. In previous reports of lymphoma in Job's syndrome, patients presented with extranodal disease and had poor outcomes. With appropriate chemotherapy and hematological support, lymphoma associated with Job's syndrome can achieve complete remission.

ProteinChip Array analysis of microdissected colorectal carcinoma and associated tumor stroma shows specific protein bands in the 3.4 to 3.6 kDa range.

Multiple pathways of carcinogenesis have been associated with colorectal carcinomas, including the adenoma-carcinoma sequence. The non polyposis coli gene has also been implicated in the pathogenesis of these tumors. Identification of the epithelial-mesenchymal interaction may help in understanding the pathways of invasion and may lead to the development of new, non-invasive tools for the diagnosis and prognosis of colon carcinomas. A ProteinChip Array technology (SELDI=Surface Enhanced Laser Desorption Ionization) has been developed enabling analysis and profiling of complex protein mixtures from a few cells. This study describes the protein analysis of approximately 500-1000 freshly obtained cells from normal and malignant colonic epithelium and its associated stroma by SELDI-TOF-MS (Surface Enhanced Laser Desorption Ionization Time-of-Flight Mass Spectrometry). Pure cell populations of normal and malignant epithelium as well as stroma (without tumor cells) were selected by microdissection from 9 patients. A pattern of 3 peptides of 3.48, 3.55 and 3.6 kDa, which were increased in the colon tumor epithelium and stroma compared to associated normal colon and stroma in all 9 patients, was observed. Coupling microdissection with SELDI represents a powerful tool to identify cell and tumor specific proteins and to understand molecular events underlying the invasive event in colorectal carcinomas. The presence of certain proteins in invasive carcinomas may lead to the development of non invasive biomarkers for the identification or detection of recurrence of colorectal malignancies.

Trapping radioactive carbon dioxide during cellular metabolic assays under standard culture conditions: description of a unique gas-capturing device.

Measurement of carbon dioxide levels has been employed to follow cellular metabolic reactions for quite some time. By radio-labeling substrate molecules and evaluating the radioactivity levels of the carbon dioxide released, insight into metabolic pathways can be gleaned. Currently, no carbon dioxide capturing device is available that can be used with large volume cell monolayers growing under standard conditions within a regular commercially available culture flask. In this note we describe a simple device for collecting radio-labeled carbon dioxide from a standard culture flask. The device is independent of the culture flask, but can be attached for metabolic measurements allowing cells to be grown under standard conditions prior to study. The presented design permits convenient transfer of the device between flasks without contaminating or disturbing cells growing within the flasks. Data are presented demonstrating the reproducibility of measurements made with multiple devices with different substrate concentrations and varying periods of time, ranging up to 3 h.

A Functional Model of Cytochrome c Oxidase: Thermodynamic Implications.

The environment of the CuI ion in the distal ligand group decides the fate of the reduction of O2 by the two analogues 1 and 2 of the heme a3 CuB center in cytochrome c oxidase. The fourfold coordination by N in 1 favors the CuII oxidation state and leads to a 4 e- -4 H+ reduction and the formation of H2 O under physiological conditions, while with 2 a 2 e- -2 H+ reduction occurs to form the cytotoxic H2 O2 .

Mitochondrial proteome: toward the detection and profiling of disease associated alterations.

Existing at the heart of cellular energy metabolism, the mitochondrion is uniquely positioned to have a major impact on human disease processes. Examples of mitochondrial impact on human pathology abound and include etiologies ranging from inborn errors of metabolism to the site of activity of a variety of toxic compounds. In this review, the unique aspects of the mechanisms related to the mitochondrial proteome are discussed along with an overview of the literature related to mitochondrial proteomic exploration. The review includes discussion of potential areas for exploration and advantages of applying proteomic techniques to the study of mitochondria.

The complexity of the count: considerations regarding lymph node evaluation in colorectal carcinoma.

In patients with colorectal carcinoma, studies have reported improved survival with increasing numbers of retrieved lymph nodes. These findings are puzzling, as increased node sampling was not correlated with significant change in disease staging. Although the physiologic processes underlying this correlation between number of lymph nodes sampled and survival remain unknown, the reported correlation has caused modifications to clinical and non-clinical practices. Herein, we review the literature and discuss potential etiologies responsible for the observed increased survival statistics. Literature regarding colorectal lymph node anatomy, molecular aspects of colorectal cancer, changes in tumor characteristics and utilization of lymph node sample numbers are evaluated. In addition, we present the mathematical concepts available for probabilistic prediction of diagnostic confidence based upon sample size. From evaluation of the aggregate literature, certain facts emerge which are not easily identified within the individual studies. Colorectal carcinoma appears to encompass a number of individual disease entities with different physiologic characteristics and likelihoods of metastasis. In addition, it appears the improved survival is likely multifactorial including effects from intrinsic tumor biology and tumor-host interactions along with ever changing clinical practices. Finally, because lymph node count is dependent on a number of variables and is correlated, but unlikely to be causally associated with survival, use of this number as a quality indicator is unwarranted. Based on statistical considerations, the current recommended goal of 12-15 recovered lymph nodes without evidence of metastatic disease provides approximately 80% negative predictive value for colorectal carcinoma metastasis.

Oxygen metabolism and a potential role for cytochrome c oxidase in the Warburg effect.

By manipulating the physical properties of oxygen, cells are able to harvest the large thermodynamic potential of oxidation to provide a substantial fraction of the energy necessary for cellular processes. The enzyme largely responsible for this oxygen manipulation is cytochrome c oxidase, which resides at the inner mitochondrial membrane. For unknown reasons, cancer cells do not maximally utilize this process, but instead rely more on an anaerobic-like metabolism demonstrating the so-called Warburg effect. As the enzyme at the crossroads of oxidative metabolism, cytochrome c oxidase might be expected to play a role in this so-called Warburg effect. Through protein assay methods and metabolic studies with radiolabeled glucose, alterations associated with cancer and cytochrome c oxidase subunit levels are explored. The implications of these findings for cancer research are discussed briefly.

Surrogate tissue analysis: monitoring toxicant exposure and health status of inaccessible tissues through the analysis of accessible tissues and cells.

Genomics and proteomics have made it possible to define molecular physiology in exquisite detail, when tissues are accessible for sampling. However, many tissues are not accessible for human diagnostic evaluations or experimental studies, creating the need for surrogates that afford insight into exposures and effects in such tissues. Surrogate tissue analysis (STA) incorporating contemporary genomic and proteomic technologies may be useful in determining toxicant exposure and effect, or disease state, in target tissues at the pre- or early clinical stage. We present here a discussion of STA based on presentations given at the Society of Toxicology's 2003 annual meeting's "Innovations in Applied Toxicology" symposium. Speakers at the symposium (Box 1) discussed various potential applications of STA, including the use of peripheral blood lymphocytes (PBLs) as a source of genetic biomarkers to monitor radiation exposure; the use of gene expression analysis of PBLs and hair follicles as a means to monitor the impact of toxicants on inaccessible organs; the characterization of disease-associated gene signatures in peripheral blood mononuclear cells (PBMCs) of renal cell carcinoma (RCC) patients; the use of sperm RNA to determine genetic and environmental effects on sperm development in the testis; and the use of serum protein profiles to monitor the development and progression of various cancers. Also discussed are some of the challenges that must be overcome if the utility of STA is to be proven, and thus permit researchers to move this concept from the laboratory to the clinical environment.

Mitochondrial proteome: cancer-altered metabolism associated with cytochrome c oxidase subunit level variation.

Shifts in metabolism associated with tumorigenesis were first noted by Otto Warburg in the 1920s. In the ensuing decades many examples of the phenomenon have been elucidated while the underlying molecular mechanism has remained elusive. As the enzyme complex at the crux of oxidative phosphorylation, cytochrome c oxidase is uniquely positioned to have a very high impact on cellular metabolism. In this study, we test the hypothesis that there is a specific association between altered cytochrome c oxidase subunit levels and altered metabolism by combining the technique of reverse-phase protein microarray with radiolabeled glucose metabolic studies. Such a relationship is observed with five different cell lines, two of which (1542N and 1542T) are a matched set of normal and tumor-based lineages derived from the same prostate gland. By measuring the [(14)C]carbon dioxide production of a cell line metabolizing [1-(14)C]glucose and comparing those measurements to values obtained for the same cell line metabolizing [6-(14)C]glucose, we determined the relative utilization of the hexose monophosphate shunt and glycolysis progressing through the Krebs cycle metabolic pathway in each cell line. In all cases there is an increased utilization of hexose monophosphate shunt relative to glycolysis progressing through the Krebs cycle in tumor derived relative to normal derived cell lines. Additionally, there is an associated increase in the ratio of nuclear encoded cytochrome c oxidase subunits to mitochondrially encoded cytochrome c oxidase subunits in the tumor-derived cell lines. These results demonstrate an alteration in subunit levels of a single enzyme complex (cytochrome c oxidase) commensurate with tumor-altered metabolism.

Mitochondrial proteome: altered cytochrome c oxidase subunit levels in prostate cancer.

Laser capture microdissection was combined with reverse phase protein lysate arrays to quantitatively analyze the ratios of mitochondrial encoded cytochrome c oxidase subunits to nuclear encoded cytochrome c oxidase subunits, and to correlate the ratios with malignant progression in human prostate tissue specimens. Cytochrome c oxidase subunits I-III comprise the catalytic core of the enzyme and are all synthesized from mitochondrial DNA. The remaining subunits (IV-VIII) are synthesized from cellular nuclear DNA. A significant (P < 0.001, 30/30 prostate cases) shift in the relative concentrations of nuclear encoded cytochrome c oxidase subunits IV, Vb, and VIc compared to mitochondrial encoded cytochrome c oxidase subunits I and II was noted during the progression of prostate cancer from normal epithelium through premalignant lesions to invasive carcinoma. Significantly, this shift was discovered to begin even in the premalignant stage. Reverse phase protein lysate array-based observations were corroborated with immunohistochemistry, and extended to a few human carcinomas in addition to prostate. This analysis points to a role for nuclear DNA encoded mitochondrial proteins in carcinogenesis; underscoring their potential as targets for therapy while highlighting the need for full characterization of the mitochondrial proteome.