PubMed Health⌕ Search

Biomedical subjects

H H Salem

Publications and source records attributed to H H Salem.

At least 19 recordsLinked to original sources
PubMed1997

Calpain cleavage of focal adhesion proteins regulates the cytoskeletal attachment of integrin alphaIIbbeta3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin clots.

The intracellular thiol protease calpain catalyzes the limited proteolysis of various focal adhesion structural proteins and signaling enzymes in adherent cells. In human platelets, calpain activation is dependent on fibrinogen binding to integrin alphaIIbbeta3 and subsequent platelet aggregation, suggesting a potential role for this protease in the regulation of postaggregation responses. In this study, we have examined the effects of calpain activation on several postaggregation events in human platelets, including the cytoskeletal attachment of integrin alphaIIbbeta3, the tyrosine phosphorylation of cytoskeletal proteins, and the cellular retraction of fibrin clots. We demonstrate that calpain activation in either washed platelets or platelet-rich plasma is associated with a marked reduction in platelet-mediated fibrin clot retraction. This relaxation of clot retraction was observed in both thrombin and ionophore A23187-stimulated platelets. Calcium dose-response studies (extracellular calcium concentrations between 0.1 microM and 1 M) revealed a strong correlation between calpain activation and relaxed clot retraction. Furthermore, pretreating platelets with the calpain inhibitors calpeptin and calpain inhibitor I prevented the calpain-mediated reduction in clot retraction. Relaxed fibrin clot retraction was associated with the cleavage of several platelet focal adhesion structural proteins and signaling enzymes, resulting in the dissociation of talin, pp60(c-)src, and integrin alphaIIbbeta3 from the contractile cytoskeleton and the tyrosine dephosphorylation of multiple cytoskeletal proteins. These studies suggest an important role for calpain in the regulation of multiple postaggregation events in human platelets. The ability of calpain to inhibit clot retraction is likely to be due to the cleavage of both structural and signaling proteins involved in modulating integrin-cytoskeletal interactions.

Blood Platelets↗
PubMed1997

Natural anticoagulants and the liver.

The regulation of blood coagulation is dependent on a complex interplay between procoagulant, anticoagulant and fibrinolytic proteins. Most of these proteins are synthesised in the liver and their levels are altered in patients with liver disease. The liver also plays an important role in the regulation of haemostasis throughout the clearance of activated clotting factors. It is therefore not surprising that the critically balanced coagulation system is dysregulated in patients with liver disease. In moderate liver failure bleeding disorders predominate, whereas in more advanced liver disease intravascular coagulation is commonly observed and contributes to the overall dysregulation of blood coagulation. In some patients, liver disease can be primarily caused by an abnormality of the coagulation system. These patients usually have a hypercoagulable state caused by a deficiency of a component of the natural anticoagulant system. These include protein C, protein S and antithrombin III. More recently, activated protein C resistance caused by a point mutation in the Factor V gene has been identified as an important risk factor for thrombosis. In these patients the abnormal Factor V is resistant to cleavage by activated protein C resulting in ongoing uncontrolled procoagulant drive. Both hepatic and portal vein thrombosis have been reported in these patients. Appropriate management of these patients should include a thorough assessment of their natural anticoagulant proteins and exclusion of activated protein C resistance as the cause of their thrombotic disorder.

Antithrombin III↗
PubMed1997

Efferent neurons and vestibular cross talk in the frog.

A galvanic stimulus (30- to 120-s, 0.3-mA constant current pulse) was used to depolarize the spike-generating region of horizontal and anterior canal afferent neurons. The galvanically induced spike activity from these neurons served as a driving input to the efferent vestibular system in the bullfrog. Efferent-mediated effects were assessed by intracellular recordings of posterior canal afferent spike activity, either ipsilateral or contralateral to the driving stimulus. Ipsilateral to the driving stimulus, efferent-mediated spike rate changes occurred in 62 (39%) of 158 posterior canal afferent neurons. Ipsilateral efferent-mediated effects were overwhelmingly excitatory (92%). Of responding units, 3% were inhibited during stimulus application and 5% showed mixed responses involving 3-20 s of inhibition followed by facilitation. Contralateral to the driving stimulus, efferent-mediated spike rate changes occurred in 18 (23%) of 77 posterior canal afferent neurons. Contralateral efferent-mediated effects were overwhelmingly inhibitory (95%). Only one unit was facilitated during stimulation and no mixed responses to contralateral stimulation were observed. Analysis of the coefficient of variation in interspike intervals (CV) before and during stimulation showed no significant efferent-mediated effects on spike train noise. Comparisons of resting spike rates between units showing efferent-mediated effects and those that did not were in general agreement with previous studies. Responding units had a lower mean spike rate (6.8 +/- 0.70 spikes/s, mean +/- SE) than did nonresponding units (10.7 +/- 0.42 spikes/s, mean +/- SE; P < 0.001; 2-tailed t-test of log-normalized data). Comparison between groups in the regularity of their resting spike rates, as quantified by CV, showed considerable overlap. When responding and nonresponding units with similar resting spike rates were compared, responding units had more irregular resting spike rates than did nonresponding units (P < 0.004; 2-tailed, paired t-test). In most cases (77%) the temporal pattern and general shapes of efferent-mediated responses mirrored the driving input of the galvanically activated afferent neurons. The other 23% of efferent-mediated responses exhibited a marked adaptation of the response. Adapting and nonadapting units were not significantly different in their mean resting spike rates or in the regularity of their resting spike rates.

Animals↗
PubMed1996

The bioactive phospholipid, lysophosphatidylcholine, induces cellular effects via G-protein-dependent activation of adenylyl cyclase.

The naturally occurring phospholipid, lysophosphatidylcholine (lyso-PC), regulates a broad range of cell processes, including gene transcription, mitogenesis, monocyte chemotaxis, smooth muscle relaxation, and platelet activation. Despite the growing list of cellular effects attributable to lyso-PC, the mechanism(s) by which it alters cell function have not been elucidated. In this report, we have examined the effects of exogenous lyso-PC on signal transduction processes within a variety of lyso-PC-responsive cells, including human platelets, monocyte-like THP-1 cells, and the megakaryoblastic cell line, MEG-01. Pretreatment of each of these cells with increasing concentrations of lyso-PC (25-150 microg/ml) was associated with a progressive increase in the cytosolic concentration of cAMP. The accumulation of cAMP in platelets correlated closely with the ability of lyso-PC to inhibit multiple platelet processes, including platelet aggregation, agonist-induced protein kinase C activation, thromboxane A2 generation, and the tyrosine phosphorylation of platelet proteins. In each of the cell types examined, the ability of lyso-PC to increase the cellular levels of cAMP was synergistically enhanced by pretreating the cells with the cAMP phosphodiesterase inhibitor, theophylline (5 mM), and was specifically inhibited by the P-site inhibitor of adenylyl cyclase, 2,5-dideoxyadenosine. A role for the stimulatory G-protein, Gs, in the lyso-PC-induced activation of adenylyl cyclase was suggested by the ability of the GTPase inhibitor, guanylyl 5'-thiophosphate (0.2 mM), to inhibit the lyso-PC-stimulated increase in cAMP, and also by the ability of cholera toxin to inhibit increases in membrane GTPase activity in response to lyso-PC. The functional significance of lyso-PC-induced activation of adenylyl cyclase was investigated in MEG-01 cells. Treatment of these cells with either lyso-PC or dibutyryl cAMP for 36-40 h resulted in a 3-5-fold increase in the surface expression of the natural anticoagulant protein, thrombomodulin (TM). The ability of lyso-PC to increase TM expression was abolished by pretreating these cells with the adenylyl cyclase inhibitor, 2,5-dideoxyadenosine, whereas the dibutyryl cAMP-induced increase in TM remained insensitive to adenylyl cyclase inhibition. These studies define an important role for the adenylyl cyclase signaling system in mediating cellular effects induced by lyso-PC.

Adenylyl Cyclases↗
PubMed1996

Focal adhesion kinase (pp125FAK) cleavage and regulation by calpain.

Focal adhesion kinase (125 kDa form; pp125FAK) is a widely expressed non-receptor tyrosine kinase that is implicated in integrin-mediated signal transduction. We have identified a novel means of pp 125FAK regulation in human platelets, in which this kinase undergoes sequential proteolytic modification from the native 125 kDa form to 90, 45 and 40 kDa fragments in thrombin-, collagen- and ionophore A23187-stimulated platelets. The proteolysis of pp125FAK was prevented by pretreating platelets with the calpain inhibitors calpeptin or calpain inhibitor-1, and was reproduced in vitro by incubating immunoprecipitated pp125FAK with purified calpain. Proteolysis of pp125FAK resulted in a dramatic reduction in its autokinase activity and led to its dissociation from the cytoskeletal fraction of platelets. These studies define a novel signal-terminating role for calpain, wherein proteolytic modification of pp125FAK attenuates its autokinase activity and induces its subcellular relocation within the cell.

Blood Platelets↗
PubMed1996

Altered thrombomodulin staining in blistering dermatoses.

The immunocytochemical distribution of thrombomodulin (TM) was examined in sections of skin from patients with blistering dermatoses occurring in the presence and absence of acantholysis. Skin sections were stained using polyclonal and monoclonal anti-human TM antibodies and were correlated with the staining pattern that resulted when using the monoclonal antibody 32-2B, which recognises the chief desmosomal adhesion molecule desmoglein I (DG I). Our study demonstrates a loss of TM staining in acantholytic dermatoses, occurring only in the region of actual disruption of the intercellular bridging between keratinocytes in the stratum spinosum. The thrombomodulin antigen expression paralleled the DG I expression. The strong correlation between the DG I and TM immunostaining pattern in both normal skin and acantholytic dermatoses supports the concept that TM has a role in mediating adhesion processes between keratinocytes.

Acantholysis↗
PubMed1996

Non-receptor protein tyrosine kinases and phosphatases in human platelets.

There is now a large and rapidly growing body of information on the different types of non-receptor tyrosine kinases and phosphatases present within platelets. These enzymes appear to play a critical role in co-ordinating, integrating and amplifying signals from multiple cell surface receptors. Despite considerable progress in this area of research over the last decade, a coherent understanding of how these enzymes fit into the complex communication networks of platelets remains elusive. The challenge ahead will be to define the molecular interactions and hierarchies between tyrosine kinases, phosphatases and other platelet signalling enzymes, and to pinpoint the key phosphorylation reactions required for the induction of specific platelet responses.

Animals↗
PubMed1995

Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses.

Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.

Calcium↗
PubMed1995

An essential role for lysophosphatidylcholine in the inhibition of platelet aggregation by secretory phospholipase A2.

The release of secretory phospholipase A2 (sPLA2) into the mammalian circulation may contribute to the development of hemorrhagic and inflammatory diseases. sPLA2 has previously been shown to alter the behavior of platelets, leukocytes, and endothelial cells, although the molecular basis for these cellular effects has not been established. Our studies indicate that the inhibition of platelet aggregation by snake, bee venom, and pancreatic sPLA2 is dependent on a plasma cofactor. This cofactor resides within the lipoprotein fraction of plasma, with 54%, 31%, and 11% of the activity present in the high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low density lipoprotein (VLDL) fractions, respectively. Delipidation of HDL and LDL was associated with the complete loss of platelet-inhibitory activity. Incubation of purified sPLA2 with the HDL fraction of plasma resulted in the time-dependent generation of lysophosphatidylcholine (lysoPC). The formation of lysoPC correlated with the inhibition of platelet aggregation. Purified lysoPC (10 to 100 micrograms/mL) inhibited platelet aggregation and dense granule release induced by thrombin (0.05 U/mL), collagen (1 micrograms/mL), ionophore A23187 (2 mumol/L), ADP (12.5 mumol/L), and adrenaline (3.2 mumol/L). The inhibition of platelet aggregation by lysoPC was dose-dependent and correlated with decreased fibrinogen binding to glycoprotein IIb-IIIa. Our studies indicate that the enzymatic generation of lysoPC from plasma lipoproteins is essential for the sPLA2-mediated inhibition of platelet activation in the presence of albumin. These results raise the possibility that the toxic effects of circulating sPLA2 may be due in part to the generation of the bioactive lysophospholipid, lysoPC.

Animals↗
PubMed1995

Production and characterization of recombinant human proteinase inhibitor 6 expressed in Pichia pastoris.

The human intracellular serine proteinase inhibitor, proteinase inhibitor 6 (PI-6), was expressed in the methylotropic yeast Pichia pastoris. The PI-6 cDNA was modified to encode six histidine residues immediately after the initiation codon, and was placed under the control of the P. pastoris alcohol oxidase promoter in the vector pHIL-D2. On the methanol induction, active recombinant PI-6 was produced within the yeast cells, and following cell lysis, was separated from yeast proteins by affinity chromatography using nickel nitrilo-tri-acetic acid (NTA) resin. The interaction of recombinant PI-6 with a range of serine proteinases was studied. Second order association rate constants (ka) were derived for the interaction with trypsin (1.8 x 10(6) M-1 s-1), thrombin (1.2 x 10(5) M-1 s-1), urokinase plasminogen activator (4.0 x 10(4) M-1 s-1), plasmin (1.3 x 10(6) M-1 s-1), and activated protein C (7.5 x 10(3) M-1 s-1). By monitoring complex formation, recombinant PI-6 was also shown to interact with factor Xa. No complex formation was observed with chymotrypsin, human leukocyte elastase, cathepsin G and tissue plasminogen activator, although PI-6 is apparently a substrate for chymotrypsin, leukocyte elastase and cathepsin G.

Amino Acid Sequence↗
PubMed1995

Immunohistochemical localization of thrombomodulin in normal human skin and skin tumours.

Thrombomodulin (TM) expression has been investigated in sections of normal human skin, in cultured normal human keratinocytes, and in a variety of skin tumours. TM was present in squamous epithelial cells in the spinous layer of normal epidermis and in the outer root sheath of hair follicles, but was absent in the cells of the basal layer. It appeared to be predominantly localized to the cell membrane and the intercellular bridges in these areas. Cultured normal human keratinocytes demonstrated functionally active constitutive TM expression on their cell surface. Immunoperoxidase staining of skin tumours using anti-human TM antibodies demonstrated a typical cell membrane positivity in tumours with squamous or hair follicle differentiation. Basal cell carcinomas showed TM expression only in areas where incomplete squamoid metaplasia occurred. Sweat gland tumours and lesions of the melanogenic system failed to express TM. The localization of TM by immunostaining in various benign and malignant skin tumours typically correlated with their normal skin element of origin. The physiological significance of TM expression in the epidermis is currently undefined.

Basal Cell Carcinoma↗
PubMed1994

Tyrosine kinases regulate the cytoskeletal attachment of integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) and the cellular retraction of fibrin polymers.

Integrins promote cell-substratum and cell-cell adhesion by acting as transmembrane linker molecules between extracellular adhesion proteins and the actin-rich cytoskeleton. The integrin alpha IIb beta 3 (platelet glycoprotein IIb/IIIa) is essential for platelet spreading, aggregation, fibrin clot retraction, and for the transduction of extracellular signals. We examined the effect of the specific tyrosine kinase inhibitor herbimycin A on integrin and cytoskeletal-mediated events in thrombin-stimulated platelets. Incubation of washed platelets for 24 h with herbimycin A (5 microM) abolished the thrombin-stimulated cytoskeletal enzyme activity of pp60c-src in parallel with a reduction in the tyrosine phosphorylation of multiple platelet proteins, as assessed with anti-phosphotyrosine immunoblots. However, thrombin-induced activation of protein kinase C and the production of thromboxane A2 were not altered by herbimycin A. Despite the absence of cytoskeletal pp60c-src enzyme activity, platelet shape change, aggregation, and serotonin release were unaltered following platelet stimulation with thrombin (0.05-1.0 unit/ml). Herbimycin A-treated platelets also demonstrated normal platelet aggregation in response to collagen (5 micrograms/ml), ionophore A23187 (2 microM), and ADP/adrenaline (10 microM each). However, the ability of herbimycin A-treated platelets to retract fibrin gels was significantly reduced. This defect in clot retraction was associated with reduced incorporation of integrin alpha IIb beta 3 into the cytoskeletal fraction of thrombin-aggregated platelets. Our studies suggest that tyrosine kinases in platelets regulate the cytoskeletal attachment of alpha IIb beta 3, as an essential process for the transmission of cellular contractile forces to fibrin polymers.

Benzoquinones↗
PubMed1994

Adhesion receptor activation of phosphatidylinositol 3-kinase. von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets.

The cytoskeleton participates in the coordinated regulation of intracellular signaling molecules, following agonist stimulation of cells. We have demonstrated that von Willebrand factor (vWF) induced the cytoskeletal association and activation of phosphatidylinositol 3-kinase (PtdIns 3-kinase) in human platelets. The activation of PtdIns 3-kinase coincided with the tyrosine phosphorylation of multiple platelet proteins, as assessed by anti-phosphotyrosine immunoblotting. One of these tyrosine-phosphorylated proteins, pp60c-src, became specifically enriched in the cytoskeletal fraction of vWF-stimulated platelets. The vWF-stimulated cytoskeletal association of PtdIns 3-kinase and pp60c-src required platelet stirring and aggregation, was specifically blocked by an anti-GPIb monoclonal antibody, and was not observed in platelets lacking the glycoprotein Ib/IX complex (Bernard-Soulier syndrome). Pretreatment of normal platelets with 5 mM EDTA (37 degrees C for 90 min) or RGDS (2 mM), which disrupts the binding of various adhesive proteins to platelet integrins and inhibits fibrinogen-mediated platelet aggregation, did not alter the vWF-stimulated activation and cytoskeletal association of PtdIns 3-kinase and pp60c-src. Pretreatment of platelets with acetylsalicylic acid (1 mM) completely abolished vWF-stimulated production of thromboxane A2, dense granule release, and the activation of protein kinase C, without altering the activation and cytoskeletal translocation of PtdIns 3-kinase and pp60c-src. Our results suggest that vWF binding to the platelet adhesion receptor glycoprotein Ib/IX can mediate activation and translocation of both tyrosine and lipid kinase(s) independent of other agonists.

Aspirin↗
PubMed1994

Selective inhibitory effects of the anticoagulant activated protein C on the responses of human mononuclear phagocytes to LPS, IFN-gamma, or phorbol ester.

Recent studies have shown that infusion of the anticoagulant protein, activated protein C (APC), can ameliorate many of the systemic effects of endotoxemia in experimental animals, although the mechanisms in this action are unknown. We investigated the effects of APC on the responses of blood monocytes, alveolar macrophages, and cells of the monocyte line, THP-1, to stimulation in vitro by LPS, IFN-gamma, or PMA. Mononuclear phagocyte (MO) activation was associated with rapid production of TNF-alpha, down-regulation of the glycophosphatidylinositol-linked protein CD14 (the key MO receptor for complexes of LPS and LPS-binding protein responsible for intracellular signaling), and down-regulation of the related LPS-binding proteins CD11b and CD18. Addition of APC, but not the zymogen, PC, or active site-blocked APC, inhibited selected MO responses involving the CD14-dependent LPS-induced pathway of MO activation, or activation induced by IFN-gamma or PMA. Thus, APC inhibited the production of TNF-alpha and prevented down-regulation of membrane CD11b, CD14, and CD18, but had no effect on up-regulation of MHC class II, ICAM-1, or IL-2R, down-regulation of MO expression of another glycophosphatidylinositol-linked protein, CD59, or production of reactive oxygen intermediates. These data show that APC inhibits host cytokine production but maintains MO responses associated with adhesion, phagocytosis, and killing of Gram-negative bacteria, such that use of APC may be a logical and potent adjunctive therapy in select inflammatory diseases involving MO activation and damaging host cytokine overproduction.

Antigens, CD↗
PubMed1994

Purification and characterization of two forms of soluble thrombomodulin from human urine.

We have isolated and characterized two forms of soluble thrombomodulin from human urine. The purification procedure consisted of ultrafiltration, chromatography on DEAE-Sepharose, affinity chromatography on diisopropyl-phosphate-thrombin and/or monoclonal anti-thrombomodulin IgG affigel followed by reverse-phase HPLC. An active soluble form of thrombomodulin was purified 1600-fold from 34-l urine. The purified protein migrated as a doublet, with molecular mass 76/72 kDa under reducing conditions and 63/57 kDa under non-reducing conditions as determined by SDS/PAGE. Amino acid analysis of the 63/57-kDa soluble thrombomodulin confirmed sequence identity with human thrombomodulin and demonstrated N-terminal heterogeneity. Compared to membrane-type thrombomodulin, the purified 63/57-kDa soluble thrombomodulin was more active as a cofactor for protein-C activation. The second major thrombomodulin fragment urine is an inactive 35-kDa thrombomodulin polypeptide derived from the N-terminal extracellular region of thrombomodulin.

Chromatography, Affinity↗
PubMed1994

Protein C is responsible for the rapid inactivation of factor Va following blood clotting in vitro.

When whole blood is allowed to clot in vitro, factor V is rapidly activated to factor Va which is subsequently inactivated. We developed two monoclonal anti-protein C antibodies, one of which inhibits protein C activation and the other inhibits protein C activity. The addition of either antibody to blood before clotting in vitro significantly inhibited the inactivation of factor Va, confirming the essential role of protein C in mediating the rapid inactivation of factor Va.

Antibodies, Monoclonal↗