ADAM10/17-dependent release of soluble c-Met correlates with hepatocellular damage.

The signalling pathway elicited by hepatocyte growth factor (HGF) and its receptor c-Met is indispensable for liver development and regeneration. It has been described that c-Met is released from the cell surface by a disintegrin and metalloprotease 10 (ADAM10) resulting in a soluble c-Met form known as sMet. Using the human hepatocellular HepG2 and hepatic stellate cell LX2 lines we show that sMet is released from the cell surface of liver cells by both ADAM17 and ADAM10, with ADAM17 appearing to be the major proteinase. Moreover, using a mouse model of 3,5-diethoxycarbonyl- 1,4-dihydroxycollidine (DDC)-induced hepatobiliary obstruction we show that serum levels of sMet correlate well with the liver damage state and consecutive regeneration as well as with established markers of liver damage such as alanine aminotransferase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and total bilirubin. However, sMet exhibited remarkably better correlation with liver damage and inflammation than did serum tumour necrosis factor α (TNF-α), whose shedding is also mediated by ADAM proteolytic activity. Our results indicate that the proteolytic activity of ADAM10/17 is essential for regulating HGF/c-Met signalling during acute liver damage and following regeneration and that the differential serum levels of sMet together with expression of c-Met/HGF might be a useful indicator not only for damage, but also for ongoing liver regeneration.

A guide to murine platelet structure, function, assays, and genetic alterations.

Platelets play an important role in hemostasis, thrombosis and several other biological processes. The adaptability of mice to genetic manipulation and their genetic similarity to humans has resulted in a plethora of murine models to study platelet function. Although murine platelets differ from human platelets with regard to size, number and structure, functionally they are very similar. Thus, studies which employed these model systems have greatly improved our current understanding of the contribution of platelets to hemostasis and thrombosis. This review presents general recommendations with respect to collection, isolation and processing of murine platelets. It also describes the assays currently available to study platelet function and critically assesses their utility. The extensive literature on the effects of genetic alterations on murine platelet function is considered in detail. This review is intended to provide a convenient source of reference for platelet investigators.

A guide to murine coagulation factor structure, function, assays, and genetic alterations.

Murine blood coagulation factors and function are quite similar to those of humans. Because of this similarity and the adaptability of mice to genetic manipulation, murine coagulation factors and inhibitors have been extensively studied. These studies have provided significant insights into human hemostasis. They have also provided useful experimental models for evaluation of the pathophysiology and treatment of thrombosis. This review contains recommendations for obtaining, processing and assaying mouse blood hemostatic components, and it summarizes the extensive literature on murine coagulation factor structure and function, assays and genetic alteration. It is intended to be a convenient reference source for investigators of hemostasis and thrombosis.

105th birth anniversary of professor Frantisek Pór, M.D.

Professor Frantisek Pór, M.D., was one of the most remarkable physicians in Czechoslovakia. He graduated at the German Medical Faculty of Charles University in Prague, in 1926. He was a founder of the First Internal Clinic of the Medical Faculty of P J. Safárik University and of the Faculty Hospital in Kosice. Prof. F. Pór, M.D., was the Head of the First Internal Clinic from 1948 until 1971. During his active professional life he educated 11 associate professors and 3 full professors. He was also a founder of Eastern Slovakian Medical Meetings in Nový Smokovec, the High Tatras, in 1961. The "Memorial Meeting of Professor F. Pór, M.D." has been organized by the Medical Society in Kosice since 1994. The last one was held in the Faculty Hospital of L. Pasteur in Kosice, on April 28, 2003.

A monoclonal antibody to the fibrinogen gamma-chain alters fibrin clot structure and its properties by producing short, thin fibers arranged in bundles.

BACKGROUND: We previously reported that hamster monoclonal antibody 7E9, which reacts with the C-terminus of the gamma-chain of mouse fibrinogen, inhibits factor (F)XIIIa-mediated cross-linking, platelet adhesion to fibrinogen, and platelet-mediated clot retraction; in addition, it facilitates thrombolysis. OBJECTIVES: To understand the mechanism(s) by which 7E9 acts, we have now studied the effect of 7E9 IgG, 7E9 F(ab')2, and 7E9 Fab on fibrin clot structure using electron microscopy and measurements of clot physical properties. RESULTS: By transmission electron microscopy, 7E9 IgG was found to bind primarily to the ends of the fibrinogen molecule. 7E9 IgG and 7E9 F(ab')2, both of which are bivalent, were capable of binding to two fibrinogen molecules simultaneously. Scanning electron microscopy of clots formed in the presence of equimolar concentrations of fibrinogen and 7E9 IgG demonstrated the presence of very short and thin fibers (63% reduction in fiber diameter) arranged in unusual bundles, surrounding large pores. Clots formed in the presence of 7E9 demonstrated a marked increase in permeation (approximately 25-fold increase in perfusion rate at constant pressure), an approximately 50% reduction in dynamic storage modulus (G'; a reflection of decreased clot stiffness), and an approximately 38% increase in loss tangent (tan delta; a reflection of the clot's ability to undergo irreversible deformation). These clots also showed decreased absorbance at 350 nm, reflecting the clot structure produced by 7E9 IgG. The effects of 7E9 IgG were not observed with control hamster IgG, 7E9 F(ab')2, or 7E9 Fab fragments, indicating requirements for both the binding properties and mass of 7E9 IgG. CONCLUSIONS: These data indicate that 7E9 antibody affects fibrin clot structure in a way that is consistent with the enhanced fibrinolysis we reported previously. Together with our previous observations, we conclude that 7E9 is directed at a strategically important region of fibrinogen with regard to platelet function, FXIIIa-mediated cross-linking, clot retraction, fibrin structure, and fibrinolysis. Thus targeting this region of fibrinogen may have antithrombotic therapeutic potential.

[Tenth memorial of professor Frantisek Pór, MD]. / X. memoriál profesora MUDr. Frantiska Póra.

Professor Frantisek Pór, MD, was one of the most important physicians in Czechoslovakia. He graduated in German Medical Faculty of Charles University in Prague in 1926. He was a founder of the first Internal Clinic of Medical Faculty of P. J. Safárik University and of Faculty Hospital in Kosice. Professor F. Pór, MD, was the head of the 1st Internal Clinic from 1948 until 1971. During his active professional life he educated eleven assistant professors and three full professors. He was also a founder of Eastern Slovakian Medical Meetings in Nový Smokovec, High Tatras in 1961. Medical Society in Kosice organized "Memorial of Professor F. Pór, MD" from 1994 every year and the last was held in April 28, 2003 in Faculty Hospital of L. Pasteur in Kosice.

A hamster antibody to the mouse fibrinogen gamma chain inhibits platelet-fibrinogen interactions and FXIIIa-mediated fibrin cross-linking, and facilitates thrombolysis.

Murine models employing genetically altered mice have the potential to provide important new information about the hemostatic system, but before such data can be extrapolated to humans it is necessary to define the similarities and differences between murine and human hemostasis. After establishing the similarities of murine fibrinogen to human fibrinogen in its pattern of proteolysis in response to plasmin and its cross-linking by factor XIIIa, we studied a new hamster monoclonal antibody (mAb) 7E9 that reacts with the gamma chain of mouse fibrinogen. This antibody inhibits platelet adhesion to fibrinogen, platelet-mediated clot retraction, platelet aggregation, and FXIIIa-mediated cross-linking of fibrin; it also facilitates tissue plasminogen activator (tPA)-mediated lysis of fibrin formed either in the absence or presence of platelets. These data provide evidence that the C-terminus of mouse fibrinogen gamma chain, like that of human fibrinogen, is involved in fibrinogen binding to platelets and FXIIIa-mediated cross-linking of fibrin. Our data raise the possibility that a therapeutic agent that targets the C-terminus of the gamma chain in human fibrinogen might have broad antithrombotic and profibrinolytic effects.

Platelet adhesion to fibrinogen coated at various densities.

Platelet adhesion to low-density coated fibrinogen induces greater protein tyrosine phosphorylation of SYK and FAK than adhesion to high-density coated fibrinogen, and leads to activation of integrin alpha IIb beta 3 on the luminal side of adherent platelets.

Albumin and heparin multilayer coatings for blood-contacting medical devices.

Three types of covalently crosslinked assemblies consisting of multiple (1) molecular layers of human serum albumin (HSA); (2) alternating layers of HSA and unfractionated heparin; and (3) alternating layers of HSA and partly depolymerized heparin fixed with one end to HSA were prepared on various surfaces. Adsorption of fibrinogen, IgG, and antithrombin (ATIII) from human citrated plasma on coated surfaces was evaluated by ELISA. Fibrinogen adsorption on coated ELISA plates was lower than that on bare polystyrene. There was no IgG adsorption on the HSA coating alone, but considerably high IgG adsorption was detected on the heparin-containing surface. The adsorption of ATIII increased with increasing heparin on the surface. The effect of multilayer coatings on platelets was tested by incubation of modified vascular prostheses with citrated blood. The most favorable interaction with platelets was observed on the HSA assembly. The interaction of platelets with the surface bearing unfractionated heparin was higher than that of the surface covered with partly depolymerized heparin. The long-term durability of the HSA-heparin coating was proven by a 21-day implantation of coated polyurethane plates in goat heart.

Preparation of monoclonal antibodies to murine platelet glycoprotein IIb/IIIa (alphaIIbbeta3) and other proteins from hamster-mouse interspecies hybridomas.

To obtain mouse-specific monoclonal antibodies (mAbs) against platelet proteins, an Armenian hamster was immunized with washed mouse platelets. Immune splenocytes were then fused with a nonsecreting murine myeloma cell line, and the resulting heterohybridomas were screened for antibody production utilizing an ELISA in which the target antigen was mouse platelets adsorbed onto microtiter plates in the presence of thrombin. Secondary screening assays included ELISA tests using murine fibrinogen or platelets from beta3-integrin knockout mice, flow cytometry, immunoblotting, immunoprecipitation, and a functional assay to identify antibodies that inhibit platelet-fibrinogen interactions. Hybridoma cells producing hamster mAbs against murine glycoprotein (GP) IIb/IIIa, fibrinogen, CD9, and other platelet integrins were identified. Two hybridomas (1B5 and 9C2) producing antibodies that react with the GPIIb/IIIa complex in immunoprecipitation analysis were subcloned twice. Functional analyses by means of aggregation and adhesion assays revealed that 1B5 completely inhibits platelet-fibrinogen interactions, whereas 9C2 does not affect platelet aggregation or platelet adhesion.

Platelet adhesion to fibrinogen, fibrin monomer, and fibrin protofibrils in flowing blood -- the effect of fibrinogen immobilization and fibrin formation.

Platelet fibrin(ogen) adhesive interactions were investigated in whole citrated blood using the rectangular perfusion chamber at wall shear rates of 300 and 1600 s(-1) with regard to the amount and structure of immobilized protein. Only single platelets adhered to adsorbed fibrinogen at both low and high surface fibrinogen concentrations and at 1600 s(-1) almost no adhesion was observed. When using spray-immobilized protein, platelet adhesion was significantly higher than to adsorbed protein. Conversion of adsorbed fibrinogen to fibrin monomer resulted in the formation of pronounced platelets aggregates and with the elevation of wall shear rate 50% decrease of adhesion took place. Degree of platelet adhesion to fibrin monomer was significantly influenced by immobilized protein concentration at both shear rates. However, the morphology (small and dense platelet aggregates) and extent of platelets adhered to fibrin pentamer was nearly the same at both shear rates. Starting with surface-bound fibrinogen and alternating addition of thrombin and fibrinogen fibrin pentamer was prepared using the stepwise synthesis. This methodology is based on the observation that at low concentration immobilized fibrin monomer binds fibrinogen in 1:1 molar ratio. The gradually formed fibrin of a defined size and composition can be a useful tool in the further understanding of the role of fibrin architecture in the pathophysiology of thrombosis.

Comparative study of human monocyte and platelet adhesion to hydrogels in vitro - effect of polymer structure.

Blood platelet and monocyte adhesion was studied in vitro with respect to the influence of hydrophilic polymer chemical functional groups and their charge. The results showed that the strongest adhesion of human monocytes was to coverslips covered with cationic polymer. Platelet adhesion to all tested polymers proved to be negligible; no differences related to the charge of the polymers used were observed. These results show the obvious difference between the biocompatibility and haemocompatibility in vitro which must be taken into consideration during polymer biological properties testing before clinical trials.

[Can epigastric pain and non-ulcerative dyspepsia in children and adolescents be Campylobacter pylori infection?]. / Mohou byt bolesti v epigastriu a neulcerózní dyspepsie u detí a dospívajících infekcním onemocnením Campylobacter pylori?

The authors examined 263 children and adolescents aged 5-20 years who suffered from so-called non-ulcerative dyspepsia or epigastric pain, because of the suspected presence of Campylobacter pylori. In 31.9% of the examined subjects direct microbiological methods revealed its presence in the gastroduodenal mucosa. The confidence limit of positive results of Campylobacter pylori in the population is between 27.58% and 38.84%, the probability being 95%. The authors proved a mutual correlation between endoscopic, histological, serological findings and detection of Campylobacter pylori by microbiological methods. The statistical significance of the correlation of campylobacter pylori and chronic gastritis B (mostly inactive) provides further support for the hypothesis of the aetiological role of Campylobacter pylori in the development of chronic gastritis B in children and adolescents. From the investigation it does not ensue, however, that colonization with Campylobacter pylori is associated with certain clinical symptoms in all instances. However, in the differential diagnosis of so-called non-ulcerative dyspepsia and epigastric pain in children and adolescents we must include infection with Campylobacter pylori among their possible causes.

[Aspects of social and health problems in the aged in the Kosice-Nové Mesto District]. / Niektoré stránky sociologickej a zdravotníckej problematiky starých l'udí v okrese Kosice-Nové mesto.

The author presents the results of repeated examinations of a group of 725 old people on the records of the geriatric welfare centre of the district Kosice-Nové mesto. Attention is paid to medico-social needs of old people, their demands, to the extent and quality of care and housing conditions. Some suggestions are presented for the health and social services.