Intravascular Compartment Research Articles

Mass kinetics of apolipoprotein A-I in interstitial fluid after administration of intravenous apolipoprotein A-I/lecithin discs in humans

Apolipoprotein kinetics are customarily determined by modeling time curves of specific radioactivity or isotopic enrichment in plasma after intravenous infusion of radiolabeled lipoproteins or stable isotope-enriched amino acids. However, this provides no information on the fractional rate of transfer of the apolipoprotein from plasma to interstitial fluid (k(p-if)) or its mean residence time in interstitial fluid (MRT(if)). To determine these parameters for a pharmacologic dose of exogenous apolipoprotein A-I (apoA-I) given intravenously as apoA-I/lecithin discs, we measured apoA-I in plasma and prenodal leg lymph in five healthy men before, during, and after a 4 h infusion at 10 mg/kg/h. ApoA-I concentrations in plasma and lymph were modeled by linear compartmental models (SAAM II version 1.1), using lymph albumin to adjust for the effects of variations in lymph flow rate. k(p-if) averaged 0.75%/h (range, 0.33-1.32), and MRT(if) averaged 29.1 h (14.1-40.0). Neither parameter was correlated with the distribution volume (57-105 ml/kg) or the fractional elimination rate (1.44-2.91%/h) of apoA-I, determined by modeling plasma apoA-I concentration alone. Although used here to study the mass kinetics of apoA-I, if combined with infusion of a tracer, analysis of lymph could also expand the modeling of endogenous apolipoprotein kinetics.

Effects of adenosine on systemic glycocalyx volume in goats

It has been suggested that adenosine increases perfused capillary volume by reducing the effective dimension of a blood excluding intravascular glycocalyx compartment (Klitzman 1979; VanTeeffelen 2005). In the present study, the effect of adenosine on systemic glycocalyx volume was assessed in goats by comparing the intravascular distribution volumes (Vd) of labeled red blood cells (RBCs, glycocalyx impermeable tracer) and neutral Dextran 40 (MW 40 kDa, glycocalyx permeable tracer). In open-chest anesthetized goats (n=6), Dextran 40 and RBCs were simultaneously infused via the vena femoralis. Plasma Vd was calculated from the steady-state dilution of RBCs and systemic hematocrit, and was 29.5 ± 2.1 (SEM) and 36.8 ± 2.2 ml/kg BW in control and during adenosine, respectively (P=0.05). Vd of dextran 40 was derived from its estimated plasma concentration at t = 30 s after injection and was 55.1 ± 5.6 and 38.7 ± 3.7 ml/kg in control and during adenosine (P<0.001). Systemic glycocalyx volume was 25.6 ± 1.9 and 1.9 ± 3.5 ml/kg in control and during adenosine (P<0.01). It is concluded that adenosine reduces systemic glycocalyx volume. This finding supports the concept that dynamic modulation of glycocalyx volume by adenosine may contribute to local control of organ blood flow by lowering microvascular resistance and enhancing capillary exchange capacity. Support by NHF (2003B181 to JB), NWO (902-16-192 to JT), and KNAW (HV).

Vasculoprotective properties of the endothelial glycocalyx: effects of fluid shear stress

The endothelial glycocalyx exerts a wide array of vasculoprotective effects via inhibition of coagulation and leucocyte adhesion, by contributing to the vascular permeability barrier and by mediating shear stress-induced NO release. In this review, we will focus on the relationship between fluid shear stress and the endothelial glycocalyx. We will address the hypothesis that modulation of glycocalyx synthesis by fluid shear stress may contribute to thinner glycocalyces, and therefore more vulnerable endothelium, at lesion-prone sites of arterial bifurcations. Finally, we will discuss the effects of known atherogenic stimuli such as hyperglycaemia on whole body glycocalyx volume in humans and its effect on endothelial function.

Photothrombic activity of m-THPC-loaded liposomal formulations: Pre-clinical assessment on chick chorioallantoic membrane model

The objective of this study was to evaluate the ability of meso-tetra(hydroxyphenyl)chlorin (m-THPC) encapsulated into liposomal formulations to occlude neovascularization. Two m-THPC formulations including conventional or plain liposomes (Foslip) based on dipalmitoylphosphatidylcholine (DPPC) and the corresponding long-circulating poly(ethylene glycol) (PEG)-modified liposomes (PEGylated liposomes: Fospeg) were evaluated as delivery systems. Using the chick chorioallantoic membrane (CAM) as in vivo model, the fluorescence pharmacokinetic behaviour of encapsulated m-THPC reflecting the rate of the extravasation of the dye from the CAM vasculature and its photothrombic effectiveness were determined. This study was focused on the influence of the drug and/or light doses on the mean retention time of m-THPC within the CAM blood vessels after intravenous injection, and its photothrombic efficacy. Irrespective of the formulations tested and the drug doses injected, similar fluorescence pharmacokinetic profiles were obtained. The fluorescence contrast reached a steady state 30 s after injection. Constant positive values of the fluorescence contrast suggest that m-THPC is confined into the intravascular compartment during the experimental time (500 s). However, the photodynamic therapy assays showed that Foslip appears to be less potent than Fospeg in terms of photothrombic activities on the CAM model. For instance, the light dose necessary to induce the desired vascular damage with Foslip was twice (100 J/cm 2) higher than with Fospeg (50 J/cm 2). It can be inferred that this pre-clinical study showed that the formulation based on PEGylated liposomes technology offers a suitable delivery system for the treatment of choroidal neovascularization associated with age-related macular degeneration.

The in vivo cardiac kinetics of Tc-99m N-NOET are accelerated in obese, hyperlipidemic Zucker rats

Previous studies demonstrated that the intrinsic myocardial washout of bis(N-ethoxy,N-ethyldithiocarbamato)nitrido technetium(V) (technetium 99m N-NOET) was affected by changes occurring in the intravascular compartment such as variations in circulating lipid levels. We sought to determine whether the myocardial kinetics of Tc-99m N-NOET were affected by hyperlipidemia in a clinically relevant experimental model. Tc-99m N-NOET (50 MBq/kg) and thallium 201 (12 MBq/kg) were injected intravenously into hyperlipidemic fa/fa Zucker rats (n=6) and their lean, normolipidemic littermates (n=8), and dual-isotope in vivo planar imaging was performed for 60 minutes. In vivo image quantification indicated significantly faster cardiac washout of Tc-99m N-NOET in fa/fa animals versus lean animals (time constant, 411+/-64 minutes vs 1094+/-226 minutes, respectively; P<.05), whereas Tl-201 cardiac washout was not affected (356+/-85 minutes vs 337+/-53 minutes, respectively; P=not significant). The cardiac kinetics of Tc-99m N-NOET, but not those of Tl-201, were accelerated after intravenous injection of the tracer in fa/fa Zucker rats with circulating lipid levels similar to those encountered clinically in hyperlipidemic patients. The relationship between lipidemia and the rate of Tc-99m N-NOET myocardial washout warrants further clinical investigation.

A Multiparametric Assessment of Oxygen Efflux from the Brain

A quantitative understanding of unidirectional versus net extraction of oxygen in the brain is required because an important factor in calculating oxidative metabolism by calibrated functional magnetic resonance imaging (fMRI) as well as oxygen inhalation methods of positron emission tomography (15O2-PET) and nuclear magnetic resonance (17O2-NMR)) is the degree of oxygen efflux from the brain back into the blood. Because mechanisms of oxygen transport from blood to brain are dependent on cerebral metabolic rate of oxygen consumption (CMRO2), cerebral blood flow (CBF), and oxygen partial pressure (pO2) values in intravascular (Piv) and extravascular (Pev) compartments, we implemented multimodal measurements of these parameters into a compartmental model of oxygen transport and metabolism (i.e., hemoglobin-bound oxygen, oxygen dissolved in plasma and tissue spaces, oxygen metabolized in the mitochondria). In the alpha-chloralose anesthetized rat brain, we used magnetic resonance (7.0 T) and fluorescence quenching methods to measure CMRO2 (2.5+/-1.0 micromol/g min), CBF (0.7+/-0.2 mL/g min), Piv (74+/-10 mm Hg), and Pev (16+/-5 mm Hg) to estimate the degree of oxygen efflux from the brain. In the axially distributed compartmental model, oxygen molecules in blood had two possible fates: enter the tissue space or remain in the same compartment; while in tissue there were three possible fates: enter the blood or the mitochondrial space, or remain in the same compartment. The multiparametric results indicate that the probability of unmetabolized (i.e., dissolved) oxygen molecules reentering the blood from the tissue is negligible and thus its inclusion may unnecessarily complicate calculations of CMRO2 for 15O-PET, 17O-NMR, and calibrated fMRI methods.

Over-Expression of Prolylcarboxypeptidase Enhances Plasma Prekallikrein Activation on Chinese Hamster Ovary Cell Membranes.

Recent investigations indicate that there is a physiologic plasma prekallikrein (PK) activator on the membrane of endothelial cells (HUVEC). PK complexes with high molecular weight kininogen (HK) and HK is PK's receptor on HUVEC. When assembled on HUVEC, PK is activated to plasma kallikrein independent of FXIIa by the serine protease prolylcarboxypeptidase (PRCP) (Km=9 nM). PRCP has been shown to be a PK activator when isolated from HUVEC (JBC 277:17962, 2002) and produced as a recombinant protein (Blood 103:4554, 2004). To additionally confirm that human PRCP is a physiologic PK activator, PRCP was over-expressed in Chinese hamster ovary cells (CHO). CHO were transfected with full-length PRCP under the control of a CMV promoter and CHO rPRCP was expressed as a fusion protein with C-terminal enhanced green fluorescent protein (EGFP). The presence of rPRCP in transfected CHO was detected by real time RT-PCR, immunoblot and immunoprecipitation. In CHO cells, PRCP mRNA and PK activation are 2–3-fold higher than controls. The increase in PRCP-induced PK activation on the transfected CHO cells parallels the increase in PRCP antigen expression on the transfected cells, as determined by anti-PRCP and anti-GFP antibodies. PK activation of the transfected cells is blocked by siRNA to the translation initiation site of PRCP, anti-PRCP antibody, and Z-Pro-Pro-aldehyde dimethyl acetate, with IC50 of the last 2 inhibitors of 0.01 and 7.0 mM respectively. Cellular localization of PRCP in intact cells using confocal microscopy and flow cytometry indicate that its over-expression results in its placement on the CHO cell plasma membrane. These investigations independently confirm that prolylcarboxypeptidase is expressed on cell membranes and its expression increases PK activation. The endothelial cell membrane expression of PRCP resulting in PK activation contributes to the constitutive level of bradykinin in the intravascular compartment to regulate blood pressure homeostasis.

Development and Brain Delivery of Chitosan−PEG Nanoparticles Functionalized with the Monoclonal Antibody OX26

The inhibition of the caspase-3 enzyme is reported to increase neuronal cell survival following cerebral ischemia. The peptide Z-DEVD-FMK is a specific caspase inhibitor, which significantly reduces vulnerability to the neuronal cell death. However, this molecule is unable to cross the blood-brain barrier (BBB) and to diffuse into the brain tissue. Thus, the development of an effective delivery system is needed to provide sufficient drug concentration into the brain to prevent cell death. Using the avidin (SA)-biotin (BIO) technology, we describe here the design of chitosan (CS) nanospheres conjugated with poly(ethylene glycol) (PEG) bearing the OX26 monoclonal antibody whose affinity for the transferrin receptor (TfR) may trigger receptor-mediated transport across the BBB. These functionalized CS-PEG-BIO-SA/OX26 nanoparticles (NPs) were characterized for their particle size, zeta potential, drug loading capacity, and release properties. Fluorescently labeled CS-PEG-BIO-SA/OX26 nanoparticles were administered systemically to mice in order to evaluate their efficacy for brain translocation. The results showed that an important amount of nanoparticles were located in the brain, outside of the intravascular compartment. These findings, which were also confirmed by electron microscopic examination of the brain tissue indicate that this novel targeted nanoparticulate drug delivery system was able to translocate into the brain tissue after iv administration. Consequently, these novel nanoparticles are promising carriers for the transport of the anticaspase peptide Z-DEVD-FMK into the brain.

Measurement of relative blood volume changes during haemodialysis: merits and limitations

Dialysis hypotension is estimated to occur in 20% ofhaemodialysis (HD) sessions [1] and can lead to seriousvascular complications such as cerebral infarctionand cardiac and mesenteric ischaemia [2,3]. It maycontribute to chronic overhydration due to an inabilityto reach dry weight and may lead to under-dialysis[1,2,4]. Prevention of dialysis hypotension, therefore,is an important challenge to the dialysis staff. Theinitiating factor in the pathogenesis of dialysis hypo-tension is a decrease in blood volume which resultsfrom the imbalance between the ultrafiltration rate andthe plasma refilling rate [5]. Devices that continuouslyand non-invasively monitor relative blood volume(RBV) changes during HD are being advocated as atool to maintain an adequate volume of the intravas-cular compartment in order to avoid dialysis hypo-tension [6–8]. Nowadays, most manufacturers haveincorporated an RBV monitor in their dialysis appa-ratus, but evidence-based knowledge on how to use theRBV data in order to optimize the dialysis prescriptionof the individual patient is lacking. Moreover, there areconflicting data in the literature on the predictive valueof RBV changes for the occurrence of dialysis hypoten-sion. In this review, we will outline the pathophysio-logical response to ultrafiltration-induced reductionsinbloodvolume,evaluatetheRBVmeasuringmethods,discuss the relationship between RBV changes andbloodpressureanddiscussseveralfactorsthatinfluencethe validity of RBV measurements.

Modelling the bold response, a numerical approach

A comprehensive model of the BOLD response is presented, in which differential equations for cerebral O2 and hemoglobin (Hgb) transport are solved numerically. Several features of cerebral O2 transport can be investigated, and the consistency of current conceptions of BOLD responses tested. This study applies the model to the effects of arterial hematocrit, PaCO2 and PaO2, in responses elicited by functional activation (fBOLD) and physiological pertubation (pBOLD). The model uses 4 major compartments: arterial, capillary and venous as well as an extravascular compartment. Within the intravascular compartment the distribution of O2 between Hgb-bound and physically dissolved phases is updated for every time step, t, as a function of local pH, PCO2 and DPG. Total content of deoxy-Hgb is calculated as the sum of arterial, capillary and venous concentrations weighted by their respective volume fractions. The corresponding R2*-contribution is calculated as proposed by Ogawa et al. (1993). Relationships between PaO2, PaCO2 and CBF, as well as between CBF and CBV were taken from previous studies. The model predicted BOLD effects of the expected magnitude (1–3% at 1.5 T), and confirmed an inverse relation between fBOLD and baseline CBF. A slight decrease in fBOLD response was seen with high baseline hematocrit and constant CBF, but an increase was seen when concomitant flow decreases were included (in agreement with empirical results). During hypercapnia the predicted pBOLD response was high when CBF changes alone were included (figure 1: green curve), but attenuated by the accompanying CBV change (blue curve). The response was elevated when all effects were included (red curve), mainly due to the effect of arterial hyperoxia (hyperventilation), less to pH changes. Several physiological effects thus were predicted in accordance with established results. The model suggests that known effects of hematocrit may be due to accompanying flow changes. During hypercapnia, arterial hyperoxia is an important factor which should be taken into account when used in calibration experiments for CMRO2 determination. Numerical simulation seems helpful in interpreting BOLD effects caused by complex physiological responses.

Dynamic contrast‐enhanced MRI for assessing the disease activity of multiple myeloma: A comparative study with histology and clinical markers

To examine whether parameters of dynamic, contrast-enhanced MRI (DCE MRI) reflect the degree of infiltration and vessel density in corresponding bone marrow biopsy specimens. The pelvis of 24 patients with multiple myeloma (MM) was examined using contrast-enhanced DCE MRI. Biopsy was obtained from the spina iliaca posterior superior. Using a two-compartment model (assuming one intravascular and one interstitial compartment), the parameters amplitude (A, the maximum, relative signal increase over baseline) and the exchange rate constant (k(ep), describing the redistribution of contrast agent from the interstitial into the intravascular compartment) in the biopsied region were calculated and compared with the histological and clinical data. DCE MRI parameters were significantly higher in lesions with marked infiltration than with mild or no infiltration (P < 0.05). The amplitude normalized to that in the iliac artery (A(n)), but not k(ep), was higher in lesions with high vessel-density at histology (P = 0.01). Higher k(ep) levels were found in presence of increased serum immunoglobulins. Increased contrast uptake in the bone marrow of MM patients indicates at least moderate tumor involvement. Furthermore, the contrast enhancement correlates with vessel-density and serum markers of disease activity.

Sequential recruitment of neutrophils into lung and bronchoalveolar lavage fluid in LPS-induced acute lung injury

Infiltration of activated neutrophils [polymorphonuclear leukocytes (PMN)] into the lung is an important component of the inflammatory response in acute lung injury. The signals required to direct PMN into the different compartments of the lung have not been fully elucidated. In a murine model of LPS-induced lung injury, we investigated the sequential recruitment of PMN into the pulmonary vasculature, lung interstitium, and alveolar space. Mice were exposed to aerosolized LPS and bronchoalveolar lavage fluid (BAL), and lungs were harvested at different time points. We developed a flow cytometry-based technique to assess in vivo trafficking of PMN in the intravascular and extravascular lung compartments. Aerosolized LPS induced consistent PMN migration into all lung compartments. We found that sequestration in the pulmonary vasculature occurred within the first hour. Transendothelial migration into the interstitial space started 1 h after LPS exposure and increased continuously until a plateau was reached between 12 and 24 h. Transepithelial migration into the alveolar air space was delayed, as the first PMN did not appear until 2 h after LPS, reaching a peak at 24 h. Transendothelial migration and transepithelial migration were inhibited by pertussis toxin, indicating involvement of Galphai-coupled receptors. These findings confirm LPS-induced migration of PMN into the lung. For the first time, distinct transmigration steps into the different lung compartments are characterized in vivo.

Macroprolactin; high molecular mass forms of circulating prolactin

Two high molecular mass forms of prolactin (PRL) in serum have been identified by gel filtration chromatography (GFC): macroprolactin (big-big PRL, > 100 kDa) and big PRL (40-60 kDa). Macroprolactin has a variable composition and structure, but is most frequently a complex of PRL and IgG, with a molecular mass of 150-170 kDa. It is formed in the circulation following pituitary secretion of monomeric PRL but has a longer half-life, and the PRL in the complex remains reactive to a variable extent in immunoassays. In the majority of subjects little or no macroprolactin can be detected in serum, but in some individuals it may be the predominant immunoreactive component of circulating PRL and the cause of apparent hyperprolactinaemia. Owing to its high molecular mass, macroprolactin appears to be confined to the intravascular compartment and much evidence indicates that it has minimal bioactivity in vivo and is not of pathological significance. Nevertheless, hyperprolactinaemia due to macroprolactin can lead to diagnostic confusion and unnecessary further investigation and treatment if it is not recognized as such. Macroprolactin is a common cause of apparent hyperprolactinaemia with some assays and it is essential that laboratories introduce screening programmes to examine samples with elevated total immunoreactive PRL for the presence of macroprolactin and determine the monomeric PRL component which is known to be bioactive in vivo. A number of screening tests have been described; that based on the precipitation of macroprolactin with polyethylene glycol has been the most widely validated and applied. The reference technique of GFC should be available for confirmation and further investigation of samples, giving equivocal results in screening tests. In comparison with macroprolactin, little is known about big PRL. It is a more consistent component of total serum PRL but rarely, if ever, the cause of hyperprolactinaemia. Further research is required into the nature of macroprolactin and big PRL, the relationships between high molecular mass forms of PRL, and their clinical significance.

Involvement of CD147 in overexpression of MMP-2 and MMP-9 and enhancement of invasive potential of PMA-differentiated THP-1

BackgroundDuring infection and inflammation, circulating blood monocytes migrate from the intravascular compartments to the extravascular compartments, where they mature into tissue macrophages. The maturation process prepares the cells to actively participate in the inflammatory and immune responses, and many factors have been reported to be involved in the process. We found in our study that CD147 played a very important role in this process.ResultsBy using PMA-differentiated human monocyte cells line THP-1, we found that CD147 mediated matrix metalloproteinases (MMPs) expression of the leukemic THP-1 cells and thus enhanced the invasiveness of THP-1 cells. After 24 hours of PMA-induced monocyte differentiation, the mean fluorescence intensity of CD147 in differentiated THP-1 cells (289.61 ± 31.63) was higher than that of the undifferentiated THP-1 cells (205.1 ± 19.25). There was a significant increase of the levels of proMMP-2, proMMP-9 and their activated forms in the differentiated THP-1 cells. Invasion assays using reconstituted basement membrane showed a good correlation between the invasiveness of THP-1 cells and the production of MMP-2 and MMP-9. The difference in the MMPs expression and the invasive ability was significantly blocked by HAb18G/CD147 antagonistic peptide AP-9. The inhibitory rate of the secretion of proMMP-9 in the undifferentiated THP-1 cells was 45.07%. The inhibitory rate of the secretion of proMMP-9, the activated MMP-9 and proMMP-2 in the differentiated THP-1 cells was 52.90%, 53.79% and 47.80%, respectively. The inhibitory rate of invasive potential in the undifferentiated cells and the differentiated THP-1 cells was 41.82 % and 25.15%, respectively.ConclusionThe results suggest that the expression of CD147 is upregulated during the differentiation of monocyte THP-1 cells to macrophage cells, and CD147 induces the secretion and activation of MMP-2 and MMP-9 and enhances the invasive ability of THP-1 cells. The matured monocytes / macrophages, via their high expression of CD147, may play an important role in promoting the tissue repair or tissue damage during their inflammatory response.

Volume Status in Severe Malaria: No Evidence Provided for the Degree of Filling of the Intravascular Compartment

The study by Planche et al. [1] provides important new information addressing intracellular volume depletion in children with severe childhood malaria, but does not address the question of whether intravascular volume depletion (hypovolemic shock) is present. Using sophisticated methodology to determine total body water and extracellular water, they demonstrate a 6.7% deficit in total body water and an 11.7% deficit of intracellular water, providing an important indication of the volumes of fluid that may be required to optimize hydration. The data, however, do not address the degree of filling of the intravascular compartment, nor should they be used to answer the question about the state of tissue and organ perfusion. Indeed, we believe that these new data present no conflict with our previously reported findings. Using methods to study critical illness physiology that are widely employed within pediatric intensive care units for interpretation of circulatory status, we have demonstrated evidence for hypovolemia in 53 Kenyan children with severe malaria complicated by metabolic acidosis [2]. Our children were younger, had longer capillary refilling times (>3 s), lower central venous pressures (mean 2.9 cm H2O) and higher creatinines (>80 µmol/l): all features of compensated hypovolemic shock. Furthermore, hypotension (systolic BP 15). These findings also indicate important baseline differences in two cohorts of children studied. We agree that reconsideration of guidelines for acute fluid management is warranted, particularly when current recommendations await an adequate evidence base. Nevertheless, conflicting opinions on the question of volume status in children with severe malaria can be satisfactorily resolved only through prospective randomized trials that include both fluid resuscitation and control groups. While the design and conduct of such trials will involve considerable challenges, optimal fluid management will never be resolved on the basis of theoretical consideration alone.

Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Endothelial Tissue Factor Pathway Inhibitor.

Activation of tissue factor (TF)-dependent coagulation is an early event in the pathogenesis of sepsis, responsible for microvascular thrombosis and consequent organ injury. We hypothesized that sepsis-induced TF procoagulant activity may be paralleled by a decreased expression or function of its natural inhibitor, tissue factor pathway inhibitor (TFPI). To test this hypothesis we used a non-human primate sepsis model in which adult baboons were infused with live E. coli at 109 CFU/kg (sublethal dose), 1010 CFU/kg (lethal dose) or saline (control group) and the animals were sacrified after 2, 8, or 24 hours. Lung tissue was snap frozen for protein and mRNA extraction or fixed and processed for light and electron microscopy. Tissue cryosections were immunostained using double/multiple fluorescence labeling approaches for TF, TFPI, factor VII/FVIIa and fibrin, in conjunction with cell markers for endothelial cells (CD31 or von Willebrand factor), leukocytes (PSGL-1), macrophages (CD68), PMN (myeloperoxidase) and platelets (gpIIb/IIIa). Large amounts of TF were detected in leukocytes, endothelial cells and platelet-rich microthrombi, starting from 2 hours and throughout the examined period. Concomitantly, confocal and electron microscopy analysis revealed increased leukocyte infiltration, platelet aggregates and fibrin deposition in the intravascular and interstitial compartments. In addition, TF induction was documented by semiquantitative RT-PCR, ELISA, western blot and factor Xa activation assays. Whereas TFPI mRNA showed only a modest increase, tissue-associated TFPI protein was found considerably decreased, especially during the first eight hours post E. coli infusion. Moreover, TFPI inhibitory activity of lung extracts from septic animals was 6–8 fold lower comparing to controls. TF activity measurements in the presence of inhibitory anti TFPI antibodies showed that only a very small fraction of endogenous TF was inhibited by tissue-associated TFPI, suggesting that most of the active TFPI available in the vascular compartment was depleted. The decrease of TFPI inhibitory potency cannot be exclusively explained by its proteolytic degradation, as we did not find significant amounts of truncated TFPI on western blots. In conclusion, our studies demonstrate that the exposure to septic and inflammatory stimuli lead to a decrease of TFPI-dependent endothelial anticoagulant potential, simultaneous with a strong TF-dependent procoagulant response. Activation of TF-dependent coagulation pathway not adequately countered by TFPI may have important roles in the pathogenesis of sepsis-associated disseminated intravascular coagulation. Strategies aimed to restore the physiological anticoagulant function of TFPI may help preventing sepsis-induced multiple organ dysfunction syndrome and death.

Mapping the Interaction of Peptide RPPGF with the Exodomain on Human Protease Activated Receptor 4 (PAR4).

Recent evidence from our laboratory indicates that the angiotensin converting enzyme breakdown product of bradykinin, Arg-Pro-Pro-Gly-Phe (RPPGF), binds to the active site of thrombin and binds to amino acids LDPR in the P4-P1 position on PAR1 (Am J Physiol 285:H183, 2003). Further, RPPGF and, an RPPGF analog, rOicPGF, individually inhibit the active site of both thrombin and factor VIIa (JPET In Press, 2004). Additional studies show that RPPGF at 2 mg/kg IP or rOicPGF at 0.44 mg/kg IP significantly delays carotid artery thrombosis in the Rose Bengal Model in the 129SF2 and C57BL mice (JPET, In Press, 2004). Both RPPGF and rOicPGF inhibit gamma thrombin-induced mouse platelet aggregation in PRP. Since mouse platelets express PAR4 and not PAR1, RPPGF or rOicPGF also interacts with mouse PAR4. Studies determined if RPPGF and rOicPGF bind human PAR4. Biotinylated-S41ILPAPRGYPGQ52 (biotin-SIL12), a peptide that encompasses the thrombin binding and cleavage site on human PAR4, specifically binds to peptide RPPGC linked to microtiter plates. Soluble RPPGF, rOicPGF, or unlabeled SIL12 inhibits biotin-SIL12 binding to RPPGC with an IC50 at about 1 mM. Scrambled RPPGF, FPRPG, does not block binding. CDNA of human PAR4 from Gly19-Arg78 was prepared from HEL cell mRNA by RT-PCR and cloned into Novagen pET31b vector. The recombinant PAR4 exodomain (rPAR4ec) was confirmed by N-terminal sequencing and its expected size was determined by mass spectrometry. Further, it is recognized by polyclonal antibody to PAR4, but not to antibodies to PAR1 or PAR3. RPPGFK-biotin specifically binds to rPAR4ec linked to microtiter plates. This binding is blocked by rOicPGF or RPPGF (IC50= 0.2 or 0.75 mM, respectively), but not peptide WPPGF. These data indicate that the N-terminal arginines are essential to interact with rPAR4ec. Further studies mapped the site where RPPGF binds to rPAR4ec. Alanine was substituted for proline at the P4 position (PAR4ec-P44A), the P2 position (rPAR4-P46A), or both the P4 and P2 position (rPAR4-p44A/P46A). RPPGFK-biotin binding to linked rPAR4ec is blocked by increasing concentrations (0.1–0.3 mM) soluble r PAR1ec-WT, rPAR4ec-WT, and rPAR4ec-P44A with an IC50 of 0.22 mM. In contrast, 20-fold excess (0.3 mM) rPAR4ec-P46A or rPAR4ec-P44A/P46A does not block RPPGFFK-biotin binding to rPARec linked to microtiter plates. These data indicate that the RPPGF binding site on rPAR4ec is Pro46. RPPGF, a naturally occurring biologic peptide, has the ability to bind to both PAR1 and PAR4 at the thrombin cleavage site. This information suggests that there are conformational identities among thrombin substrates. These data also suggest that the ACE breakdown product of bradykinin may be a thrombin inhibitor that contributes to the constitutive anticoagulant state of the intravascular compartment. Since RPPGF and rOicPGF are directed to 4 individual targets that contribute to thrombosis, these compounds could be used as model structures to develop single, small molecule inhibitors against multiple targets that contribute to thrombosis risk.

Perioperative monitoring of circulating and central blood volume in cardiac surgery by pulse dye densitometry.

To determine perioperative changes in circulating (BVI) and central blood volume (CBVI) by a new dye dilution technique using pulse dye densitometry. Prospective observational study in the cardiac anesthesia and intensive care unit of a university hospital. Sixty-six patients undergoing coronary artery bypass surgery. Hemodynamic measurements by the dye dilution method using pulse dye densitometry were performed prior to skin incision and 3.3+/-1.4 h and 17+/-2.7 h after surgery. Based on conventional monitoring the therapeutic goals of hemodynamic therapy were achieved in all of the patients of this study. Despite a marked positive fluid balance which developed during surgery mean BVI decreased significantly after surgery while CBVI remained unchanged. Postoperative BVI deficits vs. preoperative values were observed in 78% of patients; these BVI deficits were profound in 29% of the cases. In contrast, 65% of the individual patients showed no or only minor postoperative changes in CBVI vs. preoperative values. Changes in the intravascular volume compartments affected BVI to a greater extent than CBVI. Therefore measuring circulating in addition to central blood volume may be useful to ensure a normal circulating blood volume that can compensate for any change in the central vascular compartment.

Intercompartmental fluid volume shifts during cardiopulmonary bypass measured by A-mode ultrasonography

To investigate the time course of fluid extravasation during cardiopulmonary bypass (CPB), we measured the peripheral tissue thickness (TT) by A-mode ultrasound in 34 patients undergoing elective cardiac surgery. TT of the forehead was determined by a handheld A-mode ultrasound device and 10 MHz Transducer at nine defined intervals, from the night before surgery until the first postoperative day. Mean calculated loss of 1700 +/- 40 mL (SEM) water during the fasting period resulted in a significant reduction of TT by 0.28 +/- 0.03 mm. From induction to start of CPB, rehydration with 1000 mL of fluid was performed and TT increased to baseline. After 60 min of extracorporal circulation, forehead TT increased significantly by 0.75 +/- 0.08 mm and remained unchanged until the end of surgery when the measured fluid gain was 1580 +/- 138 mL. At discharge from ICU, negative fluid regimen resulted in a balance of -127 +/- 146 mL whereas TT declined significantly to +0.16 +/- 0.09 mm compared to baseline. Dehydration due to fasting and the marked interstitial fluid extravasation during CPB could be detected by the changes of the peripheral TT. We conclude that parts of the fluid load during CPB are shifted from the intravascular compartment to the interstitial space in a time-dependent manner.

Effect of magnetic field gradients induced by microvasculature on NMR measurements of molecular self-diffusion in biological tissues

Presence of induced mesoscopic gradients of magnetic field in magnetically heterogeneous samples affects the measured value of apparent diffusion coefficient. This effect is investigated theoretically in the context of diffusion measurements in perfused biological tissues with blood as the paramagnetic compartment. It is shown that the apparent diffusion coefficient is sensitive to mutual correlations in vessel positions. Neglect of these correlations results in a failure of the commonly used model of microvasculature in which vessels are described as independently placed cylinders. The model is modified to account for intervessel correlations. The results indicate an underestimation of apparent diffusion coefficient in proportion to the magnetic susceptibility of intravascular compartment in agreement with published experimental data. The proportionality coefficient depends on the microvascular architecture. Comparison with experimental data yields a numerical value for a new model parameter that characterises the correlation in mutual positions of blood vessels.