Inhibition of tissue factor limits the growth of venous thrombus in the rabbit.

Antibody mediated inhibition of tissue factor (TF) function reduces thrombus size in ex vivo perfusion of human blood over a TF-free surface at venous shear rates suggesting that TF might be involved in the mechanism of deep vein thrombosis. Moreover, TF-bearing monocytes and polymorphonuclear (PMN) leukocytes were identified in human ex vivo formed thrombi and in circulating blood. To understand the role of TF in thrombus growth, we applied a rabbit venous thrombosis model in which a collagen-coated thread was installed within the jugular vein or within a silicon vein shunt. The effect of an inhibitory monoclonal antirabbit TF antibody (AP-1) or Napsagatran, a specific inhibitor of thrombin, was quantified by continuously monitoring 125I-fibrinogen incorporation into the growing thrombi. The antithrombotic effect obtained with the anti-TF antibody was comparable to the effect observed with the thrombin inhibitor napsagatran suggesting that in this animal model the thrombus propagation is highly TF dependent. Immunostaining revealed that TF was mostly associated with leukocytes within the thrombi formed in the jugular vein or in the silicon vein shunt. Ex vivo perfusion experiments over collagen-coated coverslips demonstrated the presence of TF-bearing PMN leukocytes in circulating blood. The results suggest that in rabbits venous thrombus growth is mediated by clot-bound TF and that blocking the TF activity can inhibit thrombus propagation.

Vascular endothelial growth factor KDR receptor signaling potentiates tumor necrosis factor-induced tissue factor expression in endothelial cells.

Vascular endothelial growth factor (VEGF) and tumor necrosis factor-alpha (TNF-alpha) have been shown to synergistically increase tissue factor (TF) expression in endothelial cells; however, the role of the VEGF receptors (KDR, Flt-1, and neuropilin) in this process is unclear. Here we report that VEGF binding to the KDR receptor is necessary and sufficient for the potentiation of TNF-induced TF expression in human umbilical vein endothelial cells. TF expression was evaluated by Western blot analysis and fluorescence-activated cell sorting. In the absence of TNF-alpha, wild-type VEGF- or KDR receptor-selective variants induced an approximate 7-fold increase in total TF expression. Treatment with TNF alone produced an approximate 110-fold increase in total TF expression, whereas coincubation of TNF-alpha with wild-type VEGF- or KDR-selective variants resulted in an approximate 250-fold increase in TF expression. VEGF lacking the heparin binding domain was also able to potentiate TF expression, indicating that heparin-sulfate proteoglycan or neuropilin binding is not required for TF up-regulation. Neither placental growth factor nor an Flt-1-selective variant was capable of inducing TF expression in the presence or absence of TNF. Inhibition of protein-tyrosine kinase or protein kinase C activity significantly blocked the TNF/VEGF potentiation of TF up-regulation, whereas phorbol 12-myristate 13-acetate, a protein kinase C activator, increased TF expression. These data demonstrate that KDR receptor signaling governs both VEGF-induced TF expression and the potentiation of TNF-induced up-regulation of TF.

Chemical versus isotopic equilibrium and the metabolic fate of glycolytic end products in the heart.

Recent studies of isotope exchange across lactate dehydrogenase (LDH) and alanine aminotransferase (AAT) in hearts call into question whether both reactions are in equilibrium. To compare the oxidative and non-oxidative fates of glycolytic end products, isolated rabbit hearts were perfused with 5 mM [2-13C] glucose and 2.5 mM [3-13C] pyruvate: with (n = 6) and without (n = 7) stimulation of pyruvate oxidation using dichloroacetate (DCA), and during normal perfusion or hypoxia (n = 7/n = 6, +/- DCA). 13C NMR spectroscopy of intact hearts confirmed a steady-state enrichment level in both alanine and lactate. 1H- and 13C-NMR spectroscopy of tissue extracts identified the fractions of lactate, alanine and glutamate pools formed from each exogenous substrate. Glycolysis from glucose accounted for 22 +/- 7% of lactate formed and 10 +/- 2% of alanine formed in control hearts, and 16 +/- 2% lactate and 15 +/- 2% alanine in hypoxic hearts (mean +/- S.E.M.). In contrast, exogenous pyruvate formed 36 +/- 5% of the lactate pool, and 86 +/- 3% of the alanine pool in controls and 47 +/- 3% of lactate and of 67 +/- 3% alanine during hypoxia. [2(-13)C] glucose did not contribute to oxidative energy production via the TCA cycle as determined from low 13C enrichment of glutamate C5 from glucose (< 2%), while [3-13C] pyruvate accounted for 84 +/- 7% of labeled glutamate C4. Thus, exogenous pyruvate out-competed the metabolism of glucose, indicating low glycolytic activity. At 40 min, 96 +/- 2% of the total alanine was labeled from either glucose or pyruvate, confirming equilibrium at AAT. However, only 55 +/- 10% of total lactate was labeled, suggesting that the LDH reaction is not in rapid equilibrium within the myocardium.

Multiplet structure of 13C NMR signal from glutamate and direct detection of tricarboxylic acid (TCA) cycle intermediates.

For the first time, 13C NMR signals are shown from 13C-enriched, low-level tricarboxylic acid (TCA) cycle intermediates from extracts of normal cardiac tissue. As the low tissue content of the key intermediates alpha-ketoglutarate (alpha-KG) and succinate (SUC) in normal, well perfused tissues has until now precluded direct NMR detection from intact tissues and tissue extracts, 13C NMR signal from glutamate has generally been used to infer the isotopomer patterns of intermediates that are in chemical exchange with glutamate. However, the required assumptions regarding intracellular compartmentation for such indirect analysis have not been previously tested, as glutamate is largely cytosolic while the TCA cycle enzymes are located in the mitochondria. Chromatographic isolation of alpha-KG and SUC from heart tissue extracts allowed isotopomer analysis to be performed for comparison with that of glutamate. At steady state, a direct relationship between glutamate and alpha-ketoglutarate isotopomers was found, but succinate isotopomers matched those of glutamate only in hearts that displayed negligible contributions from the oxidation of unlabeled endogenous carbon sources.

Kinetic analysis of dynamic 13C NMR spectra: metabolic flux, regulation, and compartmentation in hearts.

Control of oxidative metabolism was studied using 13C NMR spectroscopy to detect rate-limiting steps in 13C labeling of glutamate. 13C NMR spectra were acquired every 1 or 2 min from isolated rabbit hearts perfused with either 2.5 mM [2-13C]acetate or 2.5 mM [2-13C]butyrate with or without KCl arrest. Tricarboxylic acid cycle flux (VTCA) and the exchange rate between alpha-ketoglutarate and glutamate (F1) were determined by least-square fitting of a kinetic model to NMR data. Rates were compared to measured kinetics of the cardiac glutamate-oxaloacetate transaminase (GOT). Despite similar oxygen use, hearts oxidizing butyrate instead of acetate showed delayed incorporation of 13C label into glutamate and lower VTCA, because of the influence of beta-oxidation: butyrate = 7.1 +/- 0.2 mumol/min/g dry wt; acetate = 10.1 +/- 0.2; butyrate + KCl = 1.8 +/- 0.1; acetate + KCl = 3.1 +/- 0.1 (mean +/- SD). F1 ranged from a low of 4.4 +/- 1.0 mumol/min/g (butyrate + KCl) to 9.3 +/- 0.6 (acetate), at least 20-fold slower than GOT flux, and proved to be rate limiting for isotope turnover in the glutamate pool. Therefore, dynamic 13C NMR observations were sensitive not only to TCA cycle flux but also to the interconversion between TCA cycle intermediates and glutamate.

Cardiac responses to induced lactate oxidation: NMR analysis of metabolic equilibria.

The role of lactate as a source of pyruvate oxidation in supporting cardiac work, energetics, and formation of oxidative metabolites was examined in normal myocardium. 13C- and 31P-nuclear magnetic resonance (NMR) spectra were acquired from isolated rabbit hearts supplied 2.5 mM [3-13C]lactate or [3-13C]pyruvate with or without stimulation of pyruvate dehydrogenase (PDH) by dichloroacetate (DCA). Similar workloads determined by rate-pressure products were noted with pyruvate (21,700 +/- 2,400; mean +/- SE) and lactate (18,970 +/- 1,510). Oxygen consumption was similar in all four groups with means between 19.0 and 22.2 mumol.min-1.g dry weight-1 (SE = 1.6-2.0) as was the ratio of phosphocreatine to ATP with means between 1.8 and 2.1 (SE = 0.1-0.6). Intracellular pH, determined from 31P-NMR spectra, was essentially the same with pyruvate (7.06 +/- 0.02) and lactate (7.05 +/- 0.04). 13C enrichment of glutamate was higher with lactate (92%) than with pyruvate (70%). Pyruvate plus DCA induced no change in glutamate content at 9-10 mumol/g, but 13C enrichment increased to 83%, while lactate plus DCA maintained enrichment at 90%. Levels of alpha-ketoglutarate were lower with lactate (1.81 mumol/g) than with pyruvate (2.36 mumol/g). Lactate plus DCA elevated glutamate by 60% with a proportional increase in alpha-ketoglutarate. Thus the balance between glutamate and alpha-ketoglutarate was affected by substrate supply only and not by PDH activation. The results suggest that the equilibrium between alpha-ketoglutarate and glutamate is sensitive to cytosolic redox state, an important consideration for 13C-NMR analyses that rely on glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)