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Biomedical subjects

E F Grabowski

Publications and source records attributed to E F Grabowski.

At least 19 recordsLinked to original sources
PubMed2005

Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome.

BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNF-alpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human umbilical vein (macrovascular) endothelial cells (HUVECs). RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/- 0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx-1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS.

Cell Membrane↗
PubMed2001

Shear stress decreases endothelial cell tissue factor activity by augmenting secretion of tissue factor pathway inhibitor.

Monolayers of human umbilical vein endothelial cells were activated with 50 U/mL interleukin-1alpha (IL-1alpha) for 3 hours and simultaneously conditioned with shear stresses of 0, 0.68, or 13.2 dyne/cm(2) in a parallel-plate flow chamber. In the presence of an inflow buffer containing 100 nmol/L factor X and 10 nmol/L factor VII, production of factor Xa, a measure of functional tissue factor (TF), was determined as the product of outflow concentration of factor Xa (chromogenic assay performed under quasi-static flow conditions after the shear period) and flow rate. Similarly, production of TF pathway inhibitor (TFPI) was estimated as the product of antigenic TFPI (by enzyme-linked immunosorbent assay) in the supernatant and flow rate. In parallel experiments, total RNA was isolated for determination of amplification products of TF mRNA by reverse transcription-polymerase chain reaction. We found that shear stress reduced factor Xa production (mean+/-SE; n=number of experiments) from 13.33+/-1.14 (n=16) fmol/minxcm(2) at 0 shear stress to 5.70+/-2.51 (n=5) and 0.54+/-0.54 (n=4) fmol/minxcm(2) at shear stresses of 0.68 and 13.2 dyne/cm(2), respectively. At the same time, immunogold labeling showed that TF antigen on the endothelial surface increased >5-fold with shear stress, whereas TFPI antigen on the surface increased 2-fold. The secretion of TFPI (appearance of new supernatant TFPI) rose from 7.4+/-2.4 (n=12) x10(-)(3) fmol/minxcm(2) at 0 shear stress to 23.7+/-7.3 (n=9) and 50.2+/-14.3 (n=4) x10(-)(3) fmol/minxcm(2) at 0.68 and 13.2 dyne/cm(2), respectively. TF mRNA amplification products were not markedly changed by shear stress. We conclude that acute application of shear stress reduces functional, but not antigenic, expression of TF by intact, activated endothelial cell monolayers in a manner associated with shear stress-augmented endothelial cell secretion of TFPI.

Antigens↗
PubMed1999

Comparison of tissue factor pathway in human umbilical vein and adult saphenous vein endothelial cells: implications for newborn hemostasis and for laboratory models of endothelial cell function.

In this work we have undertaken a comparative study of human umbilical vein endothelial cells (HUVECs) and human saphenous vein endothelial cells (HSVECs) with respect to functional and antigenic tissue factor (TF), tissue factor pathway inhibitor (TFPI), and TF mRNA. Monolayers of each cell type (passage 2, except where specified) were grown to confluence and then activated for 4 h with either 50 U/mL IL-1-alpha or 10 microg/mL tumor necrosis factor-alpha. Activated factor X appearing in supernatant was measured using a chromogenic assay, and both Northern blots and quantitative RT-PCR were performed to assess concentrations of TF mRNA accompanying activation. The role of TFPI was separately determined by ELISA for supernatant TFPI antigen, and by measurements of production of activated factor X in the presence of 0, 5, 15, or 50 microg/mL of an antibody directed against TFPI. To address a non-TF pathway endothelial cell function, antigenic concentrations of tissue plasminogen activator for both cell types was also determined by ELISA. HUVECs were found to produce 2.4- to 3.5-fold more functional TF. No significant HUVEC-HSVEC differences were detected in TF antigen, supernatant TFPI, anti-TFPI affinity for endothelial cell-associated TFPI, TF mRNA or its amplification products, and tissue plasminogen activator. Immunostaining for TF antigen, however, may have failed to detect a modest HUVEC-HSVEC difference. Our finding with respect to functional TF indicates that HUVECs and HSVECs are not equivalent in terms of models for endothelial cell function in small children versus adults.

Adult↗
PubMed1998

Vascular hemostasis in flowing blood in children.

This review considers differences in hemostasis among newborns, children, and adults from the standpoint of the vascular endothelium and, where appropriate, in the presence of flowing blood. Special procoagulant features of newborn hemostasis include unusually large von Willebrand factor multimers, augmented platelet transport under flow conditions, and greater ability of newborn endothelium to generate tissue factor. Special anticoagulant features in the newborn include increased vessel wall glycosaminoglycan activity, elevated alpha2-macroglobulin, and increased percentage of free protein S. The net effect of the differences is that hemostasis is generally achieved in all age groups but is developmental in nature. In addition to congenital hypercoagulable states and catheter placement, developmental vascular anomalies appear to constitute a thrombotic risk, at least in some children (and possibly adults).

Adult↗
PubMed1996

Variability of platelet degranulation by different contrast media.

RATIONALE AND OBJECTIVES: It has been suggested that nonionic but not ionic contrast media degranulate blood platelets when mixtures of blood and contrast media are studied by flow cytometry. This phenomenon was further assessed in the current study not only by performing whole-blood platelet flow cytometry but also by performing flowing blood platelet aggregometry. The latter is a highly sensitive measure of platelet function. METHODS: Blood samples were collected from six normal donors and mixed with equal volumes of an ionic monomer (diatrizoate), a nonionic monomer (iohexol), an ionic dimer (ioxaglate), and a nonionic dimer (iodixanol). Samples were collected in the presence of no anticoagulant for 1 min prior to the addition of sodium citrate or in the presence of heparin (14.5 U/ml) or recombinant hirudin (60 micrograms/ml). All samples were fixed in formaldehyde within 30 min. RESULTS: Platelet degranulation was observed with one nonionic agent (iohexol) and one ionic agent (diatrizoate). Degranulation was not seen with iodixanol or ioxaglate. CONCLUSION: These findings indicate that degranulation is independent of the ionic or nonionic nature per se of contrast media. A possible explanation for this conclusion is suggested.

Anticoagulants↗
PubMed1995

Platelet degranulation induced by some contrast media is independent of their nonionic vs ionic nature.

We confirm that the phenomenon of platelet degranulation exists for both iohexol and diatrizoate, as reported earlier. In contrast to previous conclusions, however, we have determined that the degranulation is independent of the nonionic vs. ionic nature of the media per se, since degranulation was neither seen with nonionic iodixanol nor ionic ioxaglate. The degranulation, further, does not significantly augment platelet function, as measured by flowing whole blood platelet aggregometry.

Blood Platelets↗
PubMed1995

Endothelial cell function, including tissue factor expression, under flow conditions.

Vascular endothelium remains a dynamic interface between blood and the vessel wall. New developments make clear that many antithrombotic and prothrombotic responses of the endothelium depend on flow conditions in an adaptive manner: duration of a certain level of shear stress matters as well as level of shear. In general, over a time course of several hours, endothelium appears to be more actively antithrombotic under moderate shear conditions (e.g., 15 dynes/cm2), and more fibrinolytic under high (e.g., 30 dynes/cm2). Pulsatile flow and cyclic wall stress further modify these responses. Special consideration, moreover, must be given to branch points and regions of irregular geometry (i.e., stenoses, aneurysms) in the circulation. In such locations, predilection sites for thrombosis, lipid uptake, and atherosclerosis, low levels of fluid shear stress (e.g., 0.5 dynes/cm2), large gradients in fluid shear stress, and vessel wall bending stresses all become important. Preliminary work suggests that endothelial cells in such regions can become prothrombotic, leading to localized platelet adhesion/aggregation and fibrin formation on subendothelium and perhaps deeper structures following vessel injury.

Animals↗
PubMed1993

The functional expression of tissue factor by fibroblasts and endothelial cells under flow conditions.

The expression of tissue factor (TF) by a variety of vascular cell types under physiologic flow conditions is critical to factor X activation and in vivo clotting. Therefore, in a parallel-plate flow chamber (volume 40 microL) we mounted monolayers of human embryonic fibroblasts (FBs) or interleukin-1 alpha (IL-1 alpha) (5 U/mL x 4 hours)-stimulated human umbilical vein endothelial cells (ECs). Inflow buffer contained 10 nmol/L factor VIIa, 100 nmol/L factor X, and 2.0 mmol/L CaCl. With FBs, production of factor Xa (product of outflow concentration of factor Xa-and flow rate) increased 200-fold over the range of shear stress from 0 to 2.7 dynes/cm2. Production values (mean +/- SE (N)) were 7.93 +/- 0.024 (6), 312 +/- 7.3 (6), 688 +/- 33.1 (8), 1,033 +/- 119 (6), and 1,601 +/- 183 (7) fmol/cm2.minute at shear stresses of 0, 0.27, 0.68, 1.35, and 2.7 dynes/cm2, respectively. Further experiments at 0.68 dynes/cm2 indicated that factor Xa production increased with factor X concentration over the range from 3 to 100 nmol/L, but changed little from 300 to 1,000 nmol/L. With ECs, production was 0.13 +/- 0.86 (6), 8.17 +/- 1.65 (13), and 1.66 +/- 1.66 (5) fmol/cm2.minute at 0, 0.68, and 2.7 dynes/cm2, respectively. However, in the presence of an antibody directed against tissue factor pathway inhibitor (TFPI) production with ECs was augmented to 16.46 +/- 0.80 (8), 149.8 +/- 18.6 (8), and 48.9 +/- 10.3 (10), respectively, at these same shear stresses. Control experiments with factor VIIa, factor X, or both absent confirm for both cell types the specificity of the reaction for the TF pathway. Similarly, specificity for TF itself is shown by the virtual absence of factor Xa generation in the presence of the monoclonal antibody HTF1-7B8 directed against human TF. We conclude that ECs, even when activated, are normally unable to generate significant quantities of factor Xa in the presence of factors X and VIIa. However, significant quantities of factor Xa are possible in the presence of an inhibitor of TFPI. On the other hand, production of factor Xa by fibroblasts is markedly augmented by shear stress, yet independent of the availability of substrate factor X above an inflow concentration of 100 nmol/L. The latter suggests a direct effect of flow on the fibroblast monolayers, not substrate limitation by convective diffusion.

Antibodies↗
PubMed1993

Platelet adhesion/aggregation in an in vitro model of coronary artery stenosis.

Platelet adhesion/aggregation (PAA) at a site of coronary artery stenosis is believed to be a process strongly modulated by local shear rates and the functional state of neighboring endothelium. One purpose of the present work, therefore, is to describe an in vitro model for the direct imaging of such PAA. Another is to apply the model to the question as to whether the use of nonionic vs. ionic contrast media (CM) in the presence of vascular endothelium contributes to PAA at the stenosis site. Toward these ends, we utilized a special flow chamber which incorporates a monolayer of endothelial cells (ECs), a step 66% flowpath constriction at a site preadsorbed with microfibrillar collagen, and arterial shear rates. By epifluorescence microscopy and digital image analysis of video recordings, PAA was found to be greater with dysfunctional ECs (pretreated with lysine acetylsalicyclate) than with normal ECs, thereby confirming a modulatory role in PAA of functionally intact ECs. When nonionic (iohexol) or ionic (ioxaglate, diatrizoate) CM was added to the flowing blood at a concentration of 20% by non-red cell volume, PAA was inhibited in the order diatrizoate > ioxaglate > iohexol > saline control. No inhibition by any CM was seen, however, when chamber prefill culture medium containing 20% by volume CM was displaced by CM-free blood, in simulation of bolus administration of CM. In terms of inhibition of PAA during percutaneous transluminal coronary angioplasty (PTCA), therefore, our model provides a conceptual basis by which one may anticipate in flowing blood no clear benefit of ionic over nonionic CM.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin↗
PubMed1993

Nonionic contrast media. Procoagulants or clotting innocents?

OBJECTIVES: Compared with ionic contrast media, nonionic contrast media cause fewer adverse reactions, including hemodynamic and electrophysiologic complications, and are better tolerated by patients. However, concern as to whether nonionic agents are associated with more thromboembolic complications has been raised in recent years. This article reviews in-vitro and in-vivo coagulation studies with nonionic contrast media, including the authors' current study using blood samples from percutaneous transluminal coronary angioplasty (PTCA) patients. METHODS: Articles for this review were selected on the basis of providing viewpoints representative of both sides of the clotting controversy involving nonionic contrast media. In addition, articles were selected that, in toto, include the several relevant aspects of thrombosis during PTCA: whole blood clotting, angiography catheters, angiography syringes, rheology, and vascular endothelium. The preliminary work described used platelet adhesion/aggregation to a collagen-coated surface under controlled conditions of heparinized, whole blood flow. Adhesion/aggregation was quantified by digital analysis of real-time images obtained by epifluorescence videomicroscopy. In parallel studies, changes in markers (membrane glycoproteins) of platelet activation were measured using whole blood flow cytometry. RESULTS: Although nonionic contrast media are weaker anticoagulants than ionic contrast media, no convincing evidence demonstrates that they are procoagulant. On the contrary, in-vitro studies suggest that ionic media may weaken the antithrombotic properties of vascular endothelium by direct endothelial injury. Certain clinical trials of PTCA patients substantially challenge the use of nonionics. Yet, Grabowski et al found no evidence of platelet activation by ioxaglate or iohexol in preliminary work. This result was obtained with two of the most sensitive platelet assays available: flowing whole blood aggregometry and whole blood flow cytometry. Attention should be given to the adequacy of heparinization in future clinical studies. CONCLUSIONS: Nonionic contrast media are not procoagulant in vitro in the hands of most research groups. The focus of current controversy, therefore, is whether there is a clinical and in-vivo difference in thrombotic events seen with nonionic versus ionic media during PTCA. Elements in this controversy include platelet activation, blood-foreign surface (catheter and syringe) interaction, rheology, vascular endothelium, and adequacy of heparinization.

Angioplasty, Balloon, Coronary↗
PubMed1993

Coagulation disorders in children.

Many new developments have taken place in the field of coagulation during the past year. We focus on the interesting new controversies surrounding vitamin K prophylaxis of the newborn infant, on the increasing information regarding the growth factors responsible for thrombopoiesis, and on the role of protein C in the genesis of thrombosis.

Bleeding Time↗
PubMed1992

Relationship of anticoagulation and radiographic contrast agents to thrombosis during coronary angiography and angioplasty: are there real concerns?

Radiographic contrast agents are essential for the performance of coronary angiography and angioplasty. Historical data show that thrombosis-related events have occurred since coronary angiography has been performed. Newer non-ionic agents have been shown to be safer than conventional high osmolar ionic agents especially in high risk patients, but concern has been raised about a potentially increased risk of thrombosis with the use of these agents. A review of basic and clinical evidence for this perception does not support the view that an increase in thrombosis-related events has occurred as a results of non-ionic contrast media use in coronary angiographic procedures.

Angioplasty, Balloon, Coronary↗