Hemostatic Disorders in Hormonally Active Pituitary Tumors.

Endocrinopathies encompass heterogeneous diseases that can lead to hemostasis disorders at various stages over their clinical course. Normal hemostasis requires an equilibrium between the processes of coagulation and fibrinolysis, which depend on multiple activators and inhibitors. To date, the influence of various hormonal disorders on the hemostatic system has been assessed many times. The aim of this review was to analyze hemostasis abnormalities that occur in patients with hormonally active pituitary tumors: corticotropinoma, somatotropinoma, prolactinoma, gonadotropinoma and thyrotropinoma. Authors discuss studies that examined coagulation and hemostasis parameters among patients with these tumors, as well as analyze antithrombotic prophylaxis approach for endogenous hypercortisolemia subjects in particular.

Development of new antiatherosclerotic and antithrombotic drugs utilizing F11 receptor (F11R/JAM-A) peptides.

Peptides with enhanced resistance to proteolysis, based on the amino acid sequence of the F11 receptor molecule (F11R, aka JAM-A/Junctional adhesion molecule-A), were designed, prepared, and examined as potential candidates for the development of anti-atherosclerotic and anti-thrombotic therapeutic drugs. A sequence at the N-terminal of F11R together with another sequence located in the first Ig-loop of this protein, were identified to form a steric active-site operating in the F11R-dependent adhesion between cells that express F11R molecules on their external surface. In silico modeling of the complex between two polypeptide chains with the sequences positioned in the active-site was used to generate peptide-candidates designed to inhibit homophilic interactions between surface-located F11R molecules. The two lead F11R peptides were modified with D-Arg and D-Lys at selective sites, for attaining higher stability to proteolysis in vivo. Using molecular docking experiments we tested different conformational states and the putative binding affinity between two selected D-Arg and D-Lys-modified F11R peptides and the proposed binding pocket. The inhibitory effects of the F11R peptide 2HN-(dK)-SVT-(dR)-EDTGTYTC-CONH2 on antibody-induced platelet aggregation and on the adhesion of platelets to cytokine-inflammed endothelial cells are reported in detail, and the results point out the significant potential utilization of F11R peptides for the prevention and treatment of atherosclerotic plaques and associated thrombotic events.

Homocysteine and alpha-1 antitrypsin concentration in patients with subclinical hypercortisolemia.

PURPOSE: Glucocorticoids have particularly strong impact on the thromboembolic complications. A factor which increases the risk of thrombosis is hyperhomocysteinemia, observed in patients with hypercortisolemia. Proinflammatory factors also affect the haemostatic balance. There has been an extensive research which estimates hemostatic system in patients with Cushing's syndrome. Undoubtedly, much fewer publications are available on thromboembolic complications in patients with Subclinical Cushing's Syndrome (SCS). The purpose of this study was to estimate of homocysteine (HCY) and alpha-1 antitrypsin (α1ATp) concentrations in patients with SCS. MATERIALS AND METHODS: We studied 35 patients (56.0 ± 15.0 years) with SCS and 33 healthy volunteers (53.3 ± 17.7 years). In all subjects the analysis of HCY and α1ATp concentration in serum was determined with an immunonephelometric method. P-values below 0.05 were considered statistically significant. RESULTS: A comparison of HCY and α1ATp mean concentrations in patients with SCS and healthy representatives indicated statistically higher values of both analysed parameters in the sera of patients than in the healthy controls (p values were 0.018 and 0.008, respectively). In the patients with SCS a negative correlation between α1ATp and cortisol concentration in overnight dexamethasone test was found (p=0.017, R=-0.40). We did not reveal any statistically significant correlation between the concentrations of HCY and α1ATp, and coagulation parameters such as INR, APTT, fibrinogen concentration in patients with SCS. CONCLUSIONS: On the basis of the obtained results, a slight increase in the concentration of homocysteine and α1ATp is observed in patients with SCS, which may influence vascular complications.

The role of immunohistochemistry in histopathological diagnostics of clinically "silent" incidentally detected adrenal masses.

BACKGROUND: The detectability of adrenal incidentalomas (incidentally found adrenal tumours) in the whole population is estimated at 0.1%; 0.42% in non-endocrine patients and at 4.3% in oncologically diagnosed ones. Even up to 16% of incidentalomas of adrenal glands can be malignant lesions. The issue of crucial importance is the histopathological differentiation between benign lesions and malignant tumours of the adrenal cortex and medulla. OBJECTIVES: To evaluate whether the immunohistochemical analysis of the expression of p53, p21, PCNA and Ki67 in the tumour's tissue can be useful in the histopathological diagnostics of adrenal incidentalomas and whether it is important for prognosis. MATERIAL AND METHODS: Our series consisted of 74 tumour samples from 164 patients operated for incidentalomas. There were 43 cortical adenomas, 11 cortical adrenocarcinomas and 20 PHEOs (including 5 malignant lesions). Using monoclonal antibodies, the expression of p53, p21, PCNA and Ki67 was evaluated. RESULTS: We found a statistically significant correlation between the expression of p53, p21, Ki67 and the differential diagnosis of adrenal cortical adenoma and adrenocortical carcinoma (for proteins: p53 p=0.010, for p21 p=0.010, for Ki67 p<0.001). The statistical significant correlation between PCNA protein and diagnosis of adrenal cortical adenoma and adrenocortical carcinoma was not found. The statistically significant correlation between p21, PCNA proteins and the diagnosis of benign and malignant PHEOs was not estimated. There was no expression of Ki67 or p53 protein above the assumed level in benign and malignant pheochromocytomas. The statistically significant correlation between p53, p21, PCNA or Ki67 and the occurrence of metastases in adrenocarcinoma and malignant PHEOs was not found.

Genomic structure, organization and promoter analysis of the human F11R/F11 receptor/junctional adhesion molecule-1/JAM-A.

The F11-receptor (F11R) (a.k.a. JAM-1, JAM-A, CD321) is a cell adhesion molecule of the immunoglobulin superfamily involved in platelet adhesion, secretion and aggregation. In addition, the F11R plays a critical role in the function of endothelial cells and in platelet adhesion to inflamed endothelium. In the present study, we used partial sequences of the human F11R gene, F11R cDNAs, and information in unannotated human genome databases, to delineate the F11R gene. We found that the F11R gene is composed of 13 exons (E1a, 1b, 1c, E1-E10) encoding two groups of mRNAs differing in length and sequence at their 5' UTRs, referred to as type 1 and type 2 messages. Type 1 cDNAs are shorter at the 5' end and contain a region not found within type 2 messages. Type 1 mRNAs are present in endothelial cells (EC), platelets, white blood cells and in the cell lines CMK, HeLa, K562, HOG and A549, while type 2 messages are limited to EC. Type 1 messages contain exons E1-E10 whereas type 2 messages usually contain exons E1a, 1c, part of E1 and E2-E10. The translation start site is localized in the 3' end of E1, common for both type 1 and type 2 messages. Expression of these messages is regulated by two alternative promoters, P1 and P2. P1 is a TATA-less promoter containing an initiator element, multiple transcription start sites, several GC and CCAAT boxes, and GATA, NF-kappaB and ets consensus sequences. The cloned P1 drives efficient expression of the luciferase reporter gene. A high level of similarity between human P1 and its rat and mouse counterparts was observed. Promoter P2, located upstream of P1, contains a TATA box, GC boxes, a CCAAT box and GATA and ets consensus sequences. 3' RACE provided evidence for variability in the 3' UTR due to the presence of two polyadenylation signals. The finding of multiple regulatory sites in the promoters supplements the biochemical evidence that the F11R has several different roles in the functional repertoire of endothelial cells, platelets and other cells. In particular, the presence of NF-kappaB provides additional evidence to the significance of the F11R function in the initiation of inflammatory thrombosis.

Nesidioblastosis--a rare cause of hypoglycaemia in adults.

A case of suspected clinically hormonally active insulinoma in a 48-year-old woman is presented. Despite the lack of features, which might correspond to the insulinoma in radiological examinations, the patient was qualified for a distal subtotal pancreatectomy and then, due to persistent hyperinsulinism, for total pancreatectomy. The insulinoma was found neither in a palpable examination of the pancreas nor in the intraoperative ultrasonic examination. In a histopathological examination supplemented with immunohistochemical tests, nesidioblastosis - a rare cause of hypoglycaemia in adults - was diagnosed.

Identification of ecto-PKC on surface of human platelets: role in maintenance of latent fibrinogen receptors.

Human platelets express a protein phosphorylation system on their surface. A specific protein kinase C (PKC) antibody, monoclonal antibody (MAb) 1.9, which binds to the catalytic domain of PKC and inhibits its activity, causes the aggregation of intact platelets while inhibiting the phosphorylation of platelet surface proteins. Photoaffinity labeling with 100 nM 8-azido-[alpha(32)P]ATP identified this ecto-PKC as a single surface protein of 43 kDa sensitive to proteolysis by extracellular 0.0005% trypsin. Inhibition of the binding of 8-azido-[alpha(32)P]ATP to the 43-kDa surface protein by MAb 1.9 identified this site as the active domain of ecto-PKC. Covalent binding of the azido-ATP molecule to the 43-kDa surface protein inhibited the phosphorylative activity of the platelet ecto-PKC. Furthermore, PKC pseudosubstrate inhibitory peptides directly induced the aggregation of platelets and inhibited azido-ATP binding to the 43-kDa protein. Platelet aggregation induced by MAb 1.9 and by PKC inhibitory peptides required the presence of fibrinogen and resulted in an increase in the level of intracellular free calcium concentration. This increase in intracellular free calcium concentration induced by MAb 1.9 was found to be dependent on the binding of fibrinogen to activated GPIIb/IIIa integrins, suggesting that MAb 1.9 causes Ca(2+) flux through the fibrinogen receptor complex. We conclude that a decrease in the state of phosphorylation of platelet surface proteins caused by inhibition of ecto-PKC results in membrane rearrangements that can induce the activation of latent fibrinogen receptors, leading to platelet aggregation. Accordingly, the maintenance of a physiological steady state of phosphorylation of proteins on the platelet surface by ecto-PKC activity appears to be one of the homeostatic mechanisms that maintain fibrinogen receptors of circulating platelets in a latent state that cannot bind fibrinogen.

Cloning of the human platelet F11 receptor: a cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation.

This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2-type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcgammaRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcgammaRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation. (Blood. 2000;95:2600-2609)

[Carcinoid: unusual clinical course]. / Rakowiak--nietypowe przebiegi kliniczne.

Carcinoid is a slowly developing neuroendocrine tumour. It appears with frequency of 1.5/100,000 persons. Usually it is localized in appendix, small intestine, rectum and bronchi. Clinical sings. of carcinoid syndrome develop in only 10% cases of tumour. We present three cases of carcinoid: the first one with evidences of heart insufficiency, the second one with evidences of colon cancer, the third one coexisting with Graves-Basedow disease.

Enhancement of human platelet aggregation and secretion induced by rapamycin.

BACKGROUND: Rapamycin is a new immunosuppressive drug of the macrolide type. Despite binding to one of the FK-binding proteins as the initial step in intracellular action, further effects differ from those of the other fungally derived macrolides, cyclosporine and tacrolimus. We have previously demonstrated an enhancement of agonist-mediated platelet activation by cyclosporine and tacrolimus which was associated with increased phosphorylation of two intracellular platelet proteins, p20 and p40. Because rapamycin utilizes the same class of binding proteins as tacrolimus, but its action is not associated with the inhibition of calcineurin, we postulated that if the stimulatory effect of cyclosporine or tacrolimus was due to calcineurin inhibition, rapamycin should not affect platelets in a similar fashion. METHODS: Normal, washed human platelets were treated with various concentrations of rapamycin (from ng to microg/ml), and pre-incubated at 37 degrees C with rapamycin for various periods (1-30 min). Several platelet functional parameters were measured in samples treated with rapamycin and these parameters were compared with control platelet samples treated with the vehicle for the same period. Platelet aggregations following exposure to ADP or to the thrombin equivalent, TRAP-6, were measured as changes in optical transmission in a Chronolog lumi-aggregometer. Each experiment was repeated at three or more times and the mean results were used for statistical comparison. RESULTS: Rapamycin-treated platelets demonstrated an increase in their dose- and time-dependent sensitivity to ADP, resulting in a significantly enhanced primary wave of ADP-induced platelet aggregation followed by a secondary wave of aggregation, indicative of granule secretion. Furthermore, rapamycin-treated platelets showed significantly enhanced sensitivity to TRAP-6 as demonstrated by an increase in the initial velocity of aggregation, an increase in their maximal extent of aggregation and an enhancement of granular ATP secretion. Concentrations of rapamycin in the ng range, as well as short pre-incubation times (within min), were sufficient to cause significant enhancement of agonist-induced platelet aggregation and secretion (P < 0.001) as compared with their vehicle controls. CONCLUSIONS: Rapamycin significantly potentiates agonist-induced platelet aggregation in a time- and dose-dependent manner. As these findings are similar to those observed with the other fungal macrolides, we hypothesize that inhibition of calcineurin may not be necessary for the increase in intracellular protein phosphorylation observed following exposure of platelets to cyclosporine or tacrolimus. Whether the rapamycin-induced enhancement of sensitivity to agonists and platelet hyperaggregability explains the thrombocytopenia observed in patients when high doses of rapamycin are administered in the clinical setting, and whether these effects are synergistic with cyclosporine, are questions which remain to be investigated.

Activation of human platelets by protein kinase C antibody: role for surface phosphorylation in homeostasis.

A monoclonal antibody that inhibits protein kinase C (PKC) activity, as well as PKC pseudosubstrate inhibitory peptides, was found to cause aggregation of human platelets followed by granular secretion. Binding of this antibody to the platelet surface was demonstrated directly by flow cytometry and immunofluorescence microscopy. Assays of ecto-protein kinase activity revealed that this antibody inhibits the phosphorylation of five proteins on the platelet surface. The platelet aggregation induced by extracellular PKC inhibitors could be blocked by the addition of the membrane-impermeable phosphatase inhibitor, microcystin. Thus the inhibition of surface protein phosphorylation together with continuous dephosphorylation, namely, a decrease in the phosphorylation state of surface proteins, causes the activation of platelets. The aggregation caused by decreased surface phosphorylation appears to be initiated by the exposure of active fibrinogen-binding sites on the platelet surface, as demonstrated by the formation of fibrinogen-dependent microaggregates, as the first step in this process. We conclude that the phosphorylation of surface proteins by a platelet ecto-protein kinase C protects platelets from spontaneous aggregation and thus can play an important role in homeostatic mechanisms that maintain circulating platelets in a resting state.

Inhibition by modified oligodeoxynucleotides of the expression of type-1 plasminogen activator inhibitor in human endothelial cells.

Antisense phosphodiester and phosphorothioate oligodeoxynucleotides (23-residue or 24-residue oligodeoxynucleotides) were constructed for sequences of type-1 plasminogen-activator-inhibitor mRNA to assess their capability to modulate type-1 plasminogen-activator-inhibitor-mediated fibrinolysis. Antisense oligodeoxynucleotides were targeted at the mRNA sequence coding a signal peptide, at a part of the reactive center Ile342-Pro349, and at an internally translated segment Asn265-Leu272. The effect of antisense oligonucleotides on the concentration of type-1 plasminogen activator inhibitor in conditioned media and human endothelial cells was determined by the activity test with fibrin as a substrate, and by immunoprecipitation after metabolic labelling of cells with [35S]methionine. Three phosphorothioate oligodeoxynucleotides were specifically inhibitory while phosphodiester oligodeoxynucleotides with the same sequence did not show any activity. Phosphorothioate oligodeoxynucleotides 2, 4 and 6 inhibited the synthesis of type-1 plasminogen activator inhibitor in endothelial cells in a time-dependent and concentration-dependent manner. These data suggest that antisense oligodeoxynucleotides may be useful in the design of antithrombolytic therapeutics.

Antibodies to recombinant fragment 212-276 of protein C specifically recognize the intact human molecule.

The peptide fragment Pro212-Ile276 of human protein C was produced as a part of a fusion protein in Escherichia coli. The identity of the peptide was confirmed by immunoblotting experiments using specific antibodies to intact protein C. The peptide Pro212-Ile276 was isolated from the fusion protein after mild hydrolysis with formic acid by gel filtration and reverse-phase HPLC. This peptide fragment was used to produce antibodies specific for the heavy chain of protein C which recognized native protein C present in blood plasma. Antibodies to intact protein C reacted also with the Pro212-Ile276 peptide fragment, indicating that this region is immunogenic in intact protein C and may represent a native epitope.

Hypotensive activity of histidine-containing analogues of C-terminal hexapeptide of substance P.

Four new hexapeptide analogues of C-terminal Substance P fragment with increased solubility in aqueous solutions are described. The peptides contain histidine in positions 6, 8, 9 and 10, respectively. The effect of the structural changes on the hypotensive activity and antigenic properties of analogues was compared. It was found that substitution of amino acid residues in various positions in the C-terminal hexapeptide of Substance P resulted in different effects on the hypotensive and antigenic properties, respectively. Only the [His6] SP6-11 analogue had an unchanged antigenic structure when compared with the C-terminal region of Substance P, but it showed an almost total loss of hypotensive activity. The [His9] SP6-11 analogue retained 50% of the hypotensive activity of the C-terminal hexapeptide but showed a markedly reduced expression of the antigenic epitope localized in this region of Substance P.

Specificity of antisera obtained to substance P and its C-terminal hexapeptide.

Synthetic fragments and analogs were used to characterize specificity of antisera to SP and SP6-11. [Tyr8] SP and [Lys6] SP6-11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP7-11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP6-11 by His or Gly showed that all but Glu6 take part in the structure of the antigenic determinant.