Biomedical subjects

P Anton

Publications and source records attributed to P Anton.

At least 19 recordsLinked to original sources

PubMed2001

Diethyl phthalate, a chemotactic factor secreted by Helicobacter pylori.

The structure of a small-molecule, non-peptide chemotactic factor has been determined from activity purified to apparent homogeneity from Helicobacter pylori supernatants. H. pylori was grown in brucella broth media until one liter of solution had 0.9 absorbance units. The culture was centrifuged, and the bacteria re-suspended in physiological saline and incubated at 37 degrees C for 4 h. A monocyte migration bioassay revealed the presence of a single active chemotactic factor in the supernatant from this incubation. The chemotactic factor was concentrated by solid phase chromatography and purified by reverse phase high pressure liquid chromatography. The factor was shown to be indistinguishable from diethyl phthalate (DEP) on the basis of multiple criteria including nuclear magnetic resonance spectroscopy, electron impact mass spectroscopy, UV visible absorption spectrometry, GC and high pressure liquid chromatography retention times, and chemotactic activity toward monocytes. Control experiments with incubated culture media without detectable bacteria did not yield detectable DEP, suggesting it is bacterially derived. It is not known if the bacteria produce diethyl phthalate de novo or if it is a metabolic product of a precursor molecule present in culture media. DEP produced by H. pylori in addition to DEP present in man-made products may contribute to the high levels of DEP metabolites observed in human urine. DEP represents a new class of chemotactic factor.

Animals↗

PubMed2000

Human immunodeficiency virus type 1 infection is associated with significant mucosal inflammation characterized by increased expression of CCR5, CXCR4, and beta-chemokines.

Mucosal inflammation is characterized by increased expression of proinflammatory cytokines and chemoattractant chemokines, resulting in infiltration of immunocompetent cells. This study compared the degree of mucosal inflammation in human immunodeficiency virus type 1 (HIV-1)-infected gut mucosa with that in tissue samples from subjects with inflammatory bowel disease (IBD) and from healthy seronegative control subjects. Gut mucosal biopsy specimens were immunohistochemically stained and were evaluated by in situ imaging. There was significantly increased expression of HIV-1 coreceptors CCR5 and CXCR4, beta-chemokine RANTES, and macrophage inflammatory protein (MIP)-1alpha and MIP-1beta, as well as increased numbers of T cells in lamina propria of HIV-1-infected patients. The results were similar in patients with IBD and in HIV-1-infected patients, suggesting increased inflammation in the colon of HIV-1-infected patients. To further investigate the effect of inflammation in HIV-1-infected lamina propria, treatments that reduce immune activation in lamina propria must be evaluated.

Biopsy↗

PubMed2000

The accuracy of the Vivacare true pressure-sensitive periodontal probe system in terms of probing force.

BACKGROUND: The Hunter TPS Vivacare periodontal probe was invented to perform consistent, accurate and reliable periodontal examinations "with controlled pressures". AIMS: The aims of the present investigation are 3: (1) what is the accuracy of the probing force when various probe heads are used in a correct operation position; (2) what is the effect of over- and under-reading of the operation position on the probing force; (3) what is the accuracy of the probing force when different probe handles are used. The Hunter TPS probe consists of a tip connected to a special spring mechanism, which controls the pressure extended to the probe tip. According to the manufacturer, the force indicator lines coincide at approximately 20 g force. METHOD: The test apparatus consisted of an electronic balance, and an electronic caliper. 12 TPS probes tips and 3 handles were selected to test whether there were differences in force between probes. Each probe tip was adapted to the same handle and tested 10 times. In a 2nd test, the TPS handles and over-, accurate-, and under-readings were analyzed as to how they affect the probing force. The 12 TPS probe tips were connected to each of the 3 handles and tested 10x for each of 3 handles and levels. RESULTS: The range in force between TPS probe tips was 8.4 g (p<0.001). For the handles as well as for each of the readings of the operating positions, the differences were small and non-significant. However, between over- and under-readings, there were statistical significant differences. CONCLUSION: The conclusion is that the variation in force between probe tips is high and always above the manufacturer's "approximately" 20 g. As long as the same handle and the same probe tip are used together, and the examiner is reading the markings correct, the TPS probe is adequate.

Electronics, Medical↗

PubMed2000

Neurokinin-1 receptor expression in inflammatory bowel disease: molecular quantitation and localisation.

BACKGROUND: Substantial evidence implicates the neuropeptide substance P (SP) in mucosal immunoinflammatory responses. Autoradiographic studies have suggested a disturbance in SP receptor expression in inflammatory bowel disease (IBD). AIMS: Because of technical limitations such as poor cellular resolution with autoradiography, we used molecular methods to specifically localise the cellular expression of the neurokinin-1 receptor (NK-1R) in IBD colon, and to quantitate NK-1R mRNA expression levels therein. METHODS: In situ hybridisation and immunohistochemistry were used to localise NK-1R mRNA and protein, respectively, in normal, ulcerative colitis (UC), and Crohn's disease (CD) colonic resections. NK-1R mRNA expression levels of normal, UC, and CD mucosal biopsies were quantitated by competitive reverse transcription-polymerase chain reaction. RESULTS: NK-1R expression was localised to lamina propria mononuclear cells, epithelium, submucosal vasculature, smooth muscle, and myenteric plexus of normal and IBD colon. No ectopic NK-1R expression was observed in IBD. However, we found increased numbers of NK-1R expressing lymphoid cells in IBD tissue, aberrant negative epithelial expression of NK-1R in UC, and increased expression of NK-1R in CD myenteric plexus. Quantitation of NK-1R mRNA expression in IBD colonic mucosal biopsies revealed marked upregulation of NK-1R mRNA levels compared with non-inflamed mucosal expression levels (p<0.01). CONCLUSIONS: This report demonstrates the strategic localisation and upregulation of NK-1R expression in IBD colon, and thereby suggests the involvement of substance P in the pathophysiological symptoms of IBD.

Adult↗

PubMed1999

Reduction of the nociceptive response to gastric distension by nitrate ingestion in rats.

BACKGROUND: Dietary nitrates are known to produce nitric oxide in the stomach, which may influence gastric function. AIM: To investigate whether nitrate ingestion modifies gastric sensitivity to distension through a mechanism involving nitric oxide production. METHODS: Nociception, associated with gastric distension ranging from 10 to 40 mmHg, was assessed in anaesthetized rats by the amplitude of cardiovascular depressor responses. Gastric volume corresponding to each distension was recorded. The following intragastric administrations (1 mL) were performed before distension: water (control), KNO3, NaNO3, KCl, NaCl (all at 0.1 mmol/kg), standard food (0.5 g), sodium nitroprusside, a nitric oxide donor (5 mg/kg), and haemoglobin, a nitric oxide scavenger (150 mg/kg) given either with water or KNO3. RESULTS: In controls, the fall in blood pressure increased from 7.8 +/- 2.0 to 31.6 +/- 2. 7 mmHg at distending pressures from 10 to 40 mmHg, respectively. KNO3 significantly reduced the amplitude of blood pressure response for the highest distending pressures (35 and 40 mmHg), while KCl induced a reduction in blood pressure response at all gastric pressures. NaNO3 and NaCl did not induce significant changes in distension-induced depressor responses. Administration of 0.5 g of standard food or sodium nitroprusside reproduced the effect of KNO3, which was reversed by haemoglobin. None of the compounds modified the gastric pressure-volume relationship, except KNO3, which increased gastric volume for the lowest distending pressures, and haemoglobin, which reduced the volume for the highest pressure. CONCLUSIONS: Ingestion of potassium nitrate reduces the sensitivity to gastric distension, through a mechanism involving nitric oxide.

Animals↗

PubMed1999

Susceptibility of Lewis and Fischer rats to stress-induced worsening of TNB-colitis: protective role of brain CRF.

We assessed the role of central corticotropin-releasing factor (CRF) in stress-induced worsening of colitis in inbred rat strains with hypo (Lewis/N) and hyper (Fischer344/N) CRF responses to stress. Intracolonic administration of 2,4,6-trinitrobenzenesulfonic acid (TNB) induced colitis of similar severity in both strains as assessed on day 7 by macroscopic scoring, histological evaluation, tissue myeloperoxidase (MPO) activity, and decrease in food intake and body weight. Colitis was inhibited by daily intracerebroventricular injections of CRF in both strains. Chronic stress (3 h/day, water avoidance or wrap restraint on alternate days for 6 days) aggravated colitis more in Lewis than Fischer rats (71 and 22% further increase in MPO activity, respectively). The CRF antagonist astressin injected intracerebroventricularly enhanced the colitis response to stress and caused mortality in both strains. Fischer rats had higher plasma corticosterone levels 20 min after stress alone on day 1 and after TNB plus stress on days 1 and 3 compared with Lewis. These data show that central CRF restrains the proinflammatory action of stress in experimental colitis.

Animals↗

PubMed1998

Substance P (neurokinin-1) receptor is a marker of human mucosal but not peripheral mononuclear cells: molecular quantitation and localization.

Reciprocal communication between the immune system and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.

Biomarkers↗

PubMed1998

Low-level exposure to diquat induces a neurally mediated intestinal hypersecretion in rats: involvement of nitric oxide and mast cells.

Diquat, a nonselective desiccant herbicide, induces a significant secretion of fluid into the lumen of the gastrointestinal tract of rats at sublethal doses (from 0.5 to 50 mg/kg). This study investigated the effect of an acute low-level exposure to diquat (0. 1, 0.5, and 1 mg/kg) on intestinal net water flux and the mechanisms involved. In anesthetized rats, an intestinal loop (7 cm) was infused with Ringer's buffer containing [14C]-polyethylene glycol 4000. After equilibration, diquat (0.1, 0.5, and 1 mg/kg) was added to Ringer's buffer during 60 min. Net water flux was calculated according to [14C] activity determined in the effluent collected at 15-min intervals. Infused in the intestinal loop for 60 min at doses of 0.5 and 1 mg/kg but not at 0.1 mg/kg, diquat induced an intestinal net water secretion during 180 min with a maximal effect at the highest dose used and during the first hour following the end of diquat infusion. Diquat-induced (1 mg/kg) intestinal net water secretion was blocked by a neurotoxin, tetrodotoxin (5 micrograms/kg iv), doxantrazole (5 mg/kg ip), a mast cell stabilizer, and two inhibitors of NO synthases: l-NAME (25 mg/kg ip) and aminoguanidine (2 mg/kg ip). It is concluded that a single low-level (0.5 and 1 mg/kg) intrajejunal administration of diquat induces a net water intestinal secretion and that this secretory effect is nerve-mediated, implying mast cell degranulation and NO release.

Animals↗

PubMed1998

Mucosal subepithelial binding sites for the bacterial chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP).

BACKGROUND: The bacterial chemotactic peptide N-formyl-methionine-leucine-phenylalanine (FMLP) is produced by enteric flora and is one of the factors implicated in contributing to inflammatory bowel disease. Expression of receptors for FMLP on human phagocytes (polymorphs and monocytes) is well established, but there is conflicting evidence regarding the potential expression of FMLP receptors on other cells within the mucosa, particularly the epithelial cells. AIMS: To map FMLP receptors within intestinal mucosa using several different experimental approaches. METHODS AND RESULTS: Radioligand binding assays with 'H-FMLP' revealed no specific binding to primary cultured colonic enterocytes or to the cell line HT29, whereas neutrophils, as expected, exhibited specific binding with a Kd of 19 nM and approximately 2 x 10(4) receptors per cell. FITC labelled FMLP exhibited specific, displaceable binding on flow cytometry to neutrophils and monocytes but not to 10 gastrointestinal epithelial cell lines. Isolated lamina propria lymphocytes and peripheral blood lymphocytes exhibited no binding. To confirm the absence of receptors on epithelia, reverse transcription polymerase chain reaction for mRNA for the classic FMLP receptor was performed. While the presence of message was detected in activated peripheral blood phagocytes, it was not detected in epithelial cell lines. To exclude the possibility of FMLP binding to other receptors such as tachykinin receptors on epithelia, FITC labelled FMLP binding in tissue sections confirmed that the binding is subepithelial--that is, in the lamina propria. CONCLUSION: Receptors for FMLP are subepithelial and map to the lamina propria of the gastrointestinal mucosa.

Animals↗

PubMed1996

Cytokines and tumor vaccination.

Patients with locally advanced stages of renal cell carcinoma are at high risk of relapse or progress even after initial radical surgery. Based on the proven efficacy of adoptive and active immunotherapeutic approaches of metastatic renal cell carcinoma, a phase II trial was started in 1989 using autologous, Newcastle disease virus modified and lethally irradiated tumor cell vaccines in combination with low-dose recombinant interleukin-2 (1.8 million U) and recombinant interferon-alpha 2a (1.0 million U) for a surgical adjuvant treatment. Patients were vaccinated (subcutaneous injection) once a week for 8-10 weeks and the treatment was started about 4-10 weeks after surgery. Up to now more than 208 patients with locally advanced renal cell carcinoma (stages pT2-3a, N1-2, M0; pT3b-4, N0-2, M0) were vaccinated after initial radical surgery (tumor nephrectomy with lymph node dissection and ipsilateral adrenalectomy and if necessary in combination with en bloc removal of venous extensions). We overview a follow-up of 203 evaluable patients with a median disease-free survival of 21 months (range of 2-64 months). During this observation period 18 relapses (8.9%) were diagnosed with 3 local relapses (1.5%), 10 lymph node metastases (5%) and/or distant organ metastases in 9 cases (4.5%). These progressive patients' disease was treated by surgery and/or combined immunochemotherapy. Toxicity encountered on this tumor cell vaccination was mild (WHO grade 1) and was characterized by flu-like symptoms and fever up to 38.8 degree Celsius for some hours beginning at 4 hours after the vaccine/cytokine application. Occasionally a transient local inflammation at the site of injection was observed. The comparison of the risk-factor-adapted group of adjuvant treated renal cell carcinoma patients (locally advanced stages) with historical data gave evidence for an improvement in disease-free survival on vaccination treatment. Although this was not a prospective randomized trial, we can summarize that the surgical adjuvant treatment of autologous tumor vaccines in combination with low-dose cytokines may improve relapse-free and overall survival in patients with locally advanced renal cell cancer.

Adult↗

PubMed1995

Denovo urothelial carcinoma of the upper and lower urinary tract in kidney--transplant patients with end-stage analgesic nephropathy.

Patients with end-stage analgesic nephropathy bear a higher risk for urothelial cancer than do patients with other renal diseases. In a retrospective study in patients with analgesic nephropathy and kidney transplants we analyzed the prevalence and clinical course of de novo urothelial cancer. Diagnosis of analgesic nephropathy was based on the patients' history and clinical data. Only patients under cyclosporine treatment were included. Between 1968 and 1993, 2,371 kidney transplants were performed on 2,072 patients in the Department of Abdominal and Transplant Surgery. The prevalence of analgesic nephropathy was 3.1%. Of 65 patients with analgesic nephropathy and kidney transplants, 10 (15.4%) developed urothelial carcinoma; 10.8%, bladder cancer; and 9.1%, renal pelvic cancer. The mean age at diagnosis was 56.1 years. Urothelial cancer occurred on average at 33.6 months posttransplantation. On average, 6 of 10 patients with urothelial cancer died of the disease at 16.9 months after the diagnosis. All patients with urothelial bladder cancer had a muscle-infiltrating tumor of moderate or high grade. Since urothelial renal pelvic cancer occurred in 9.1% of our patients with analgesic nephropathy and urological screening is insufficient in patients on dialysis, we suggest that prophylactic nephroureterectomy be performed on one side before transplantation and on the contralateral side at 3-6 months after transplantation. An aggressive approach is indicated in patients with urothelial cancer of the bladder.

Analgesics↗

PubMed1995

Localized renal-cell carcinoma: detection of abnormal cells in peritumoral tissue. A cytophotometry and immunocytochemistry study.

To spare organ function, partial resection of early diagnosed renal-cell carcinoma (RCC) is applied for well-localized and small-volume RCC with increasing frequency, although recurrence of the tumor in the same kidney is occasionally observed. The aim of the present study was to establish objective prognostic parameters that would allow the selection of tumors suitable for an organsaving procedure. Of the 160 patients undergoing a radical nephrectomy, 67 were included in this study. In 7/45 patients with lymph-node dissection (15.6%), clinical staging revealed a false-negative lymph-node status. By means of conventional histopathology, multifocality could be demonstrated in 2/67 patients (3%); in 1/67 patients (1.5%), the ipsilateral adrenal gland was unexpectantly tumor-involved. Both tumor tissue and normal peritumoral tissue were examined for the presence of premalignant and tumor cells on the basis of DNA ploidy and of the expression of the tumor-associated G250 antigen, which is specifically expressed at the surface of renal cancer cells. In 40/67 (59.1%) peritumoral tissue specimens, cells with an abnormal DNA content could be observed using automated image analysis. In 12/67 cases (18%), cells obtained from peritumoral tissue also showed an aneuploid DNA histogram; 4/67 (6%) had a tumor-correlated DNA ploidy. Additionally, 38/67 (56.9%) of these tissues, histopathologically classified as normal, contained cells expressing the G250 antigen. These observations were independent of the stage or histological grade of the tumor. These data indicate that classic pathological parameters for tumor staging are insufficient for the detection of multifocality, occurring in more than 15% of cases.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Renal-cell carcinoma with intracaval neoplastic extension: stratification and surgical technique.

Surgical removal continues to be the mainstay in the treatment of renal-cell carcinoma with neoplastic venous extension. The steady improvement of surgical and anesthesiological techniques and the introduction of complete circulatory arrest has dramatically improved the morbidity even of patients with extensive thrombi. If ultrasound or computerized tomography (CT) scanning suggests the presence of a venous extension in a patient with renal-cell carcinoma, cavography, magnetic resonance imaging (MRI), transesophageal color-coded ultrasound, and echocardiography may be needed to resolve the questions of cranial extension and vascular wall infiltration. Surgical stratification and, thus, classification of the venous extension depend on the potential need for complete circulatory arrest. Surgical removal is done en bloc for smaller venous extensions and in a two-step procedure (radical nephrectomy followed by thrombectomy) for more extensive thrombi. In patients with infiltration of the suprahepatic inferior vena cava, the hepatic veins or atrium, pending thrombotic embolism, or large masses of suprahepatic thrombotic material, the use of cardiopulmonary bypass and complete circulatory arrest is recommended.

Aged↗

PubMed1995

Adjuvant treatment of locally advanced renal cancer with autologous virus-modified tumor vaccines.

We report on 208 patients with locally advanced renal-cell carcinoma who received a surgical adjuvant vaccination with autologous, Newcastle disease virus (NDV)-modified, and lethally irradiated tumor cells in combination with low-dose recombinant interleukin-2 and interferon-alpha. The pathological stage was defined as pT2-3a, N1-2, MO (n = 107); pT3b-4 NO, MO (n = 68); and pT3b-4, N1-2, MO (n = 23). The follow-up of 203 evaluable patients showed a median disease-free survival of 21+ months (range, 2-64+ months). In all, 18 relapses (9%) occurred in spite of initial vaccination therapy. Those patients presented with local relapse (n = 3), lymph node metastases (n = 10), and/or distant organ metastases (n = 9). All patients relapsing during the first 6 months after the onset of treatment had primary lymph node involvement of the disease. An analysis of the patient subgroup with a follow-up of more than 22 months showed 10 relapses among 56 patients (18%) along with a median follow-up of 39 months (range, 23-64 months). Toxicity was very mild, manifesting as flu-like symptoms and fevers of up to 38 degrees C. At 8 and 24 weeks after the start of vaccination, anti-NDV serum antibodies were detectable in 70% and 100% of the patients tested, respectively. In comparison with historical data based on the natural course of patients with locally advanced renal-cell cancer, our results demonstrate an improvement of the disease-free survival after surgical adjuvant treatment with autologous, NDV-modified tumor vaccines in combination with low-dose cytokines.

Adjuvants, Immunologic↗

PubMed1995

Biochemotherapy of advanced metastatic renal-cell carcinoma: results of the combination of interleukin-2, alpha-interferon, 5-fluorouracil, vinblastine, and 13-cis-retinoic acid.

We conducted a phase I/II clinical trial evaluating the sequential outpatient combination of S.C. recombinant human interleukin-2 (rIL-2; given at 10 MIU/m2 b.i.d. on days 3-5 weeks 1 and 4 and at 5 MIU/m2 on days 1, 3, and 5 of weeks 2 and 3), s.c. recombinant human alpha-interferon (rIFN-alpha; given at 6 MIU/m2 on day 1 of weeks 1 and 4 and on days 1, 3, and 5 of weeks 2 and 3 and at 9 MIU/m2 on days 1, 3, and 5 of weeks 5-8), i.v. bolus 5-fluorouracil (5-FU; given at 1,000 mg/m2 once weekly during weeks 5-8), and i.v. bolus vinblastine (given at 6 mg/m2 once weekly during weeks 5 and 8) in conjunction with p.o. 13-cis-retinoic acid (13-C-RA; given at 35 mg/m2 daily during weeks 1-8). Therapy was always given in the outpatient setting. Grade 3 constitutional symptoms (malaise, chills, fevers, anorexia) were observed in 4%-8% of treatment cycles and required a 50% reduction in the doses of rIL-2 and rIFN-alpha. None of the patients experienced major 5-FU-related toxicities such as severe diarrhea and/or stomatitis; up to 20% of patients developed vinblastine-associated peripheral polyneuropathy, which was reversible after the cessation of therapy. 13-cis-Retinoic acid produced no significant side effect; no toxic death occurred. Among 24 patients with progressive metastatic disease, there were 4 complete remissions (lung, lymph nodes) and 6 partial remissions (lung, pleura, liver, lymph nodes, and peritoneal carcinosis), for an overall objective response rate of 42% (95% confidence interval, 22%-63%).(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗

PubMed1995

Uraemic medium causes endothelial cell dysfunction characterized by an alteration of the properties of its subendothelial matrix.

Uraemic patients suffer from haemorrhagic disorders and accelerated atherosclerosis. To evaluate the possible role of the vessel wall in these haemostatic alterations associated with uraemia, we investigated the effect of a uraemic milieu on human endothelial cell (EC) cultures and the reactivity of the extracellular matrices (ECM) generated by these cells towards platelets. EC cultures were exposed to a pool of sera (20% in the culture medium) obtained either from uraemic patients or from normal donors, and the following parameters were evaluated: (1) EC viability (trypan blue exclusion test); (2) von Willebrand factor (vWF) levels in supernatants and associated with ECM; (3) the reactivity of EC and EC-derived ECM towards platelets, measured 'ex vivo' under flow conditions (5 min, wall shear rate 800 s-1); and (4) ultrastructure of the ECM. The viability of EC cultured in the presence of uraemic sera was similar to controls. Platelet interaction with ECM generated by EC exposed to uraemic sera was significantly reduced (P < 0.05). This decrease was mainly related to a reduction in platelet adhesion (9.8 +/- 1.9% vs 16.7 +/- 1.8% in controls, P < 0.02). VWF levels in supernatants and associated with ECM were similar to controls. Ultrastructural analysis of the ECM generated by EC exposed to uraemic sera revealed a deficient matrix. An increased removal of EC was observed in experiments in which EC cultured in the presence of uraemic sera were perfused with citrated blood. These results indicate that a uraemic milieu induces quantitative and qualitative changes in the vascular subendothelium, characterized by a less intrincate network of fibrils, as well as a decreased attachment of EC and reduced thrombogenicity to the ECM. These changes may represent another mechanism which contributes to the haemostatic dysfunction observed in uraemic patients.

Blood Platelets↗

PubMed1994

alpha-thrombin bound to extracellular endothelial matrix induces pronounced fibrin deposition and platelet thrombus growth in flowing non-anticoagulated human blood.

Previous studies have shown that thrombin binds to the extracellular endothelial matrix and remains biologically active. In the present study, the role of matrix-bound alpha-thrombin in thrombus formation was investigated by utilizing a model system of thrombogenesis. Plastic cover-slips coated with either matrix-bound alpha-thrombin or matrix-bound active site inactivated thrombin (DIP-alpha-thrombin) were positioned in parallel-plate perfusion chambers and subsequently exposed to non-anticoagulated human blood at a venous wall shear rate of 100/s. The blood was drawn directly from an antecubital vein by a roller pump placed distally to the perfusion chamber. The thrombotic deposits on the matrix, fibrin deposition and platelet thrombus volume, were morphologically evaluated. Matrix-bound alpha-thrombin enhanced the fibrin deposition and thrombus volume on matrices of non-stimulated endothelium with 91% (P < 0.001) and 94% (P < 0.05), respectively. In contrast, binding of DIP-alpha-thrombin to matrices of stimulated endothelium reduced the fibrin deposition by 33% (P < 0.05), but had no effect on the platelet thrombus volume. Translocation of thrombin molecules from upstream matrix areas to binding sites farther downstream on the matrix was indicated in experiments with matrices of stimulated endothelium, which showed enhanced fibrin deposition on downstream areas. Our findings are compatible with a prominent role for matrix-bound alpha-thrombin in thrombogenesis, and in particular on endothelial matrices without tissue factor. The role of matrix-bound alpha-thrombin on tissue factor containing matrices appears less prominent, although it is significant.

Blood Coagulation↗

PubMed1993

Modulation of procoagulant activity of extracellular endothelial matrix by anti-tissue factor antibody and the synthetic peptide Arg-Gly-Asp-Val. Experiments with flowing non-anticoagulated human blood.

Fibrin forms on and binds to the extracellular matrix of endotoxin-stimulated endothelium when exposed to flowing non-anticoagulated blood. These processes have been investigated by employing a human ex vivo perfusion model, a synthetic peptide Arg-Gly-Asp-Val and a monoclonal anti-tissue factor antibody which inhibits tissue factor/FVIIa-induced coagulation. Procoagulant extracellular matrix on plastic cover-slips was prepared from cultures of endotoxin-stimulated human endothelium following brief exposure to 0.1 M NH4OH. Non-anticoagulated blood was drawn directly from an antecubital vein by a pump at venous (100/s) and arterial (650/s) wall shear rates over the matrix-coated cover-slips positioned in parallel-plate perfusion chambers. Deposition of fibrin and platelets on the matrix was quantified by morphometry. Preincubation of the matrix with Arg-Gly-Asp-Val inhibited fibrin deposition by 80-90% at both venous (P < 0.001) and arterial shear (P < 0.05). However, the peptide had no effect on the clotting time in a modified one-stage clotting assay where coagulation was initiated by lysed endotoxin-stimulated endothelial cells, indicating that the peptide interfered with the binding of fibrin to the matrix in the perfusion model. Preincubation of the matrix with the anti-tissue factor antibody, which blocked the coagulant activity ( > 95%, P < 0.01) in the modified coagulation assay, also inhibited fibrin deposition on the matrix by 90-95% (P < 0.01) at both shear rates. In the absence of either inhibitor, platelets adhered preferentially to the fibrin meshwork, and more so at arterial shear. Platelet thrombus formation on the fibrin coat was in particular pronounced at arterial shear. Thus, it appears that the extracellular matrix of endotoxin-stimulated endothelium initiates coagulation predominantly through tissue factor/FVIIa and that the resulting fibrin meshwork forming on the surface induces rapid platelet thrombus formation. The inhibitory effect of Arg-Gly-Asp-Val on the binding of fibrin to the matrix may indicate the presence of specific matrix fibrinogen/fibrin binding site(s) with a recognition sequence of Arg-Gly-Asp.

Amino Acid Sequence↗