Biomedical subjects

M Mirshahi

Publications and source records attributed to M Mirshahi.

At least 19 recordsLinked to original sources

PubMed2001

Mineralocorticoid receptor-mediated signaling regulates the ion gated sodium channel in vascular endothelial cells and requires an intact cytoskeleton.

The PCR analysis followed by sequence alignment showed that both the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes were expressed in the human vascular endothelial cell line (ECV). The growth and multiplication of the ECV in culture were influenced by both aldosterone and the MCR-specific antagonist ZK 91587. Following double labelled immunofluorescence recorded by confocal microscopy, both the MCR and the ENaC were found to colocalize with the tubulin filaments in ECV cells in situ; no association was observed with cellular actin. ZK 91587 not only eliminated the basal expression, but it also impaired the transactivation of the ENaC gene by aldosterone. The disruption of actin and tubulin by cytochalasin D and colchicine, respectively, resulted in the total elimination of ENaC induction by aldosterone. These studies suggest that (i) the transcriptional regulation of the ENaC gene by the MCR-mediated signalling is not restricted to epithelial cells and requires cytoskeleton integrity in ECV cells in situ, (ii) tubulin may form a new and novel mediator in cell regulation, and (iii) the vascular tone may actually be regulated via transactivation of the ion gated sodium channel in the endothelial cell of the blood vessels under direct, receptor-mediated action of aldosterone.

Actins↗

PubMed2001

Epithelial sodium channel and the mineralocorticoid receptor in cultured rat Müller glial cells.

Müller glial cells are the major non-neuronal cells of the retina. They are involved in retinal function and exert a profound influence on the function of retinal neurons. We present an in vitro study of the localization of the mineralocorticoid receptor (MCR) and of the epithelial sodium channel (ENaC) in rat Müller glial cells isolated from rat retina, using respectively, a polyclonal antiserum raised against the rat purified MCR, and a rabbit polyclonal antibody against the 14-amino acid (aa) peptide QGLGKGDKREEQGL, which corresponds to the N-terminal region (44-58aa) of the alpha-subunit of the ENaC. In an immunocytochemical study using anti-MCR and anti-ENaC antibodies, the MCR was detected as a protein present in the cytoplasm and in the nucleus, whereas ENaC was detected as a membrane-bound protein. Reverse transcription-polymerase chain reaction (RT-PCR) analysis using specific primers, 5'-CTGCCTTTATGGATGATGGT-3' (sense), 5'-GTTCAGCTCGAAGAAGA-3' (antisense) for ENaC and 5'-AGGCTACCACAGTCTCCCTG-3' (sense) and 5'-GCAGTGTAAAATCTCCAGTC-3' (antisense) for MCR, showed expression of the ENaC and MCR genes in Müller cells. The presence of ENaC and MCR was detected as the predicted bands of 520 bp and 843 bp, respectively. In both cases, 100% identity was observed between the sequences of rat Müller cell (RMC) PCR products and rat kidney. Interestingly, the basal levels of ENaC were increased in vitro by the MCR-specific hormone, aldosterone. Thus, our results strongly suggest that the Müller glial cells may play a role in the regulation of extracellular Na+ concentration, which could be regulated by steroid-mediated sodium uptake.

Aldosterone↗

PubMed2000

Fibrinogen Alès: a homozygous case of dysfibrinogenemia (gamma-Asp(330)-->Val) characterized by a defective fibrin polymerization site "a".

Congenital homozygous dysfibrinogenemia was diagnosed in a man with a history of 2 thrombotic strokes before age 30. His hemostatic profile was characterized by a dramatically prolonged plasma thrombin clotting time, and no clotting was observed with reptilase. Complete clotting of the abnormal fibrinogen occurred after a prolonged incubation of plasma with thrombin. The release of fibrinopeptides A and B by thrombin and of fibrinopeptide A by reptilase were both normal. Thrombin-induced fibrin polymerization was impaired, and no polymerization occurred with reptilase. The polymerization defect was characterized by a defective site "a," resulting in an absence of interaction between sites A and a, indicated by the lack of fragment D(1) (or fibrinogen) binding to normal fibrin monomers depleted in fibrinopeptide A only (Des-AA fm). By SDS-PAGE, the defect was detected on the gamma-chain and in its fragment D(1). The molecular defect determined by analysis of genomic DNA showed a single base change (A-->T) in exon VIII of the gamma-chain. The resulting change in the amino acid structure is gamma 330 aspartic acid (GAT) --> valine (GTT). It is concluded that the residue gamma-Asp(330) is essential for the normal functioning of the polymerization site a on the fibrinogen gamma-chain.

Adult↗

PubMed2000

Thrombotic dysfibrinogenemia. Fibrinogen "Caracas V" relation between very tight fibrin network and defective clot degradability.

Fibrinogen Caracas V is a thrombotic dysfibrinogenemia with an Aalpha 532 Ser-->Cys mutation characterized by a tight fibrin network formed of thin fibers responsible for a less porous clot than a normal one. In the present work, fibrinogen Caracas V is further characterized in order to understand the relationship between the structural defect and thrombophilia. This thrombotic disorder has been attributed to a tight fibrin network responsible for a decreased permeation of flow through the clot, leading to defective thrombus lysis due to a diminished availability of fibrinolytic enzymes to the inner fibrin surface. Correction of clot structure anomaly, by addition of dextran 40 to fibrinogen before clotting, induces an improvement in fibrin degradation that was attributed to an increase in porosity. The pulmonary embolism observed in this family has been related to an hyper rigidity of the clot, an anomaly that is also corrected by dextran. Furthermore, this abnormal fibrinogen binds more albumin than does normal fibrinogen, a phenomenon attributed to the mutation of serine in Aalpha-532 by cysteine. Therefore, this fibrinogen shows a striking similarity to the fibrinogen Dusart, allowing us to confirm that the alphaC-terminal part of fibrinogen plays an important role in fibrin structure, and to conclude that the anomaly of fibrin network observed in fibrinogen Caracas V is responsible for a deficient thrombus lysis.

Albumins↗

PubMed2000

Paradoxical effects of mineralocorticoids on the ion gated sodium channel in embryologically diverse cells.

PCR analysis and Western blotting revealed the expression of the mineralocorticoid receptor (MCR) and the epithelial sodium channel (ENaC) genes at the level of RNA, DNA, and protein in several leukemic cell lines, fibroblasts from human cornea, and epithelial cells from ocular tissues. Following immunofluorescence, the MCR appeared to be primarily nuclear whereas the ENaC was almost exclusively membrane-bound. Paradoxically, the MCR-specific antagonist ZK 91587 actually stimulated the multiplication of human erythroblastic leukemia cells, contrary to the inhibitory effect of the antagonist RU 26752 on the multiplication of corneal fibroblasts; both effects were opposed by aldosterone. In quantitative PCR, both basal and aldosterone-induced levels of ENaC were diminished by ZK 91587 in the corneal fibroblast, in contrast to the stimulation observed in the retinal pigmentary epithelium. Thus, contrary to the existing notions, (a) antimineralocorticoids can act both as agonists and antagonists, and (b) the receptor-mediated action of mineralocorticoids on the sodium channel is not restricted to the epithelial cell.

Aldosterone↗

PubMed2000

Development of a sensitive method for the determination of ganciclovir by reversed-phase high-performance liquid chromatography.

Ganciclovir is a nucleoside analogue widely used in the treatment of cytomegalovirus infections, which affects mainly immunocompromised patients. Recently, new pharmaceutical dosage forms based on the use of albumin nanoparticles have been developed for improving the efficacy of this drug. The aim of this study was to develop an analytical HPLC method for the determination of ganciclovir in both pharmaceuticals (i.e. albumin nanoparticles) and biological medium samples. The chromatography was performed on a reversed-phase encapped column (LiChrospher Select B C8) with a mobile phase consisting of acetonitrile in 0.05 M ammonium acetate (pH 6.5; 2: 98, v/v). Acyclovir was used as internal standard and the detection wavelength was 254 nm. The limit of quantitation of ganciclovir was 50 ng/ml and the average recoveries over a concentration range of 0.05-10 microg/ml ranged from 98 to 102%. Precision did not exceed 5%. In summary, this assay is a selective, sensitive and reproducible method for the determination of the ganciclovir in albumin nanoparticles. It can be successfully applied to the estimation of the ganciclovir uptake by cultured human corneal fibroblasts.

Antiviral Agents↗

PubMed2000

Demonstration of the mineralocorticoid hormone receptor and action in human leukemic cell lines.

We studied the expression of the mineralocorticoid receptor (MCR), and of the amiloride-sensitive sodium channel (ASSC) regulated by the MCR, in human leukemic cell lines. Cell extracts from TF1 (proerythroblastic), HEL (human erythroblastic leukemia) and U937 (myeloblastic) cell line were positive for the ASSC, as a 82 kDa band in Western blots developed with the aid of a polyclonal antibody raised against the peptide QGLGKGDKREEQGL, corresponding to the region 44-58 of the alpha subunit of the epithelial sodium channel (ENaC) cloned from rat colon, linked to KLH. The polyclonal antibody against the MCR revealed a single band of about 102 kDa in extracts from HEL and TF1 cells. The immunofluorescent labelling of the MCR in all cell lines showed a nucleocytoplasmic localization of the receptor but the ASSC was exclusively membrane-bound and these results were confirmed by confocal microscopy. The expression of the MCR in the HEL cells was evident as a predicted band of 843 bp (234 amino acids) in electrophoresis of the PCR product obtained after total RNA had been reverse transcribed and then amplified using the primers 5'-AGGCTACCACAGTCTCCCTG-3' and 5'-GCAGTGTAAAATCTCCAGTC-3' (sense and antisense, respectively). The ENaC was similarly evident with the aid of the primers 5'-CTGCCmATG GATGATGGT-3' (sense) and 5'-GTTCAGCTCGAAGAAGA-3' (antisense) as a predicted band of 520 bp. In both cases, 100% identity was observed between the sequences of the PCR products compared to those from known human sources. The multiplication of the HEL cells was influenced by antagonists (RU 26752, ZK 91587) targeted for specificity to the MCR and this was selectively reversed by the natural hormone aldosterone. These steroids also provoked chromatin condensation in the HEL population. These permit new and novel possibilities to understand the pathobiology of human leukemia and to delineate sodium-water homeostasis in nonepithelial cells.

Amino Acid Sequence↗

PubMed2000

Distribution of albumin nanoparticles in animals induced with the experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease characterised by a disruption of the blood-brain barrier (BBB), demyelination and a relevant inflammatory reaction with an intense infiltration of macrophages. These neurological disorders are similar to those observed in the multiple sclerosis (MS) disease. The use of different liposomes and adeno-associated virus has been proposed for improving the treatment of this pathogenesis. The aim of this work was to evaluate the potential and capacity of albumin nanoparticles to reach the central nervous system (CNS) in EAE-induced rats. For this purpose, the distribution of biotinylated nanoparticles within the CNS was studied. Albumin carriers were mainly found in the lumbar portion of the spinal cord, overlying the meningeal and perivascular areas. The optic chiasma, iris and the area of the Purkinje cells of the cerebellum revealed also an intense presence of these carriers. Finally, immunohistochemical studies also revealed that circulating macrophages (ED1), which migrate to damaged sites, and resident activated microglial cells (OX42) were involved in the distribution of albumin nanoparticles. In summary, the use of nanoparticles may be useful for the design of new pharmaceutical dosage forms able to target the lesions associated with alterations of the BBB.

Animals↗

PubMed2000

Identification of visual arrestin (S-antigen) in retinal pigmented epithelial cells.

PURPOSE: Inactivation of photolyzed rhodopsin requires phosphorylation of this receptor and binding of the 48 kDa regulatory protein arrestin (S-antigen). Arrestin is also to cause an autoimmune disease, uveoretinits, that resembles uveitis in humans. In this study we demonstrate the presence of visual arrestin in retinal pigment epithelial cells (RPE) in culture. METHODS: Bovine RPE were isolated. Mouse and rat monoclonal and rabbit polyclonal antibodies against visual arrestin, and a synthetic peptide "GFLGELTSSEVATEVPFRLM" (a pathogenic sequence corresponding to residues 340 to 359 of human visual arrestin), and rabbit polyclonal antibody against the specific peptide "EDPDTAKESFQ" for bovine visual arrestin were used to detect arrestin in RPE cells. Using visual arrestin specific primer, RT-PCR of RNA from RPE was performed. RESULTS: By western blots analysis a 48 kDa protein, corresponding to visual arrestin was detected with both mAb and polyclonal antibodies in extracts of RPE cells. RT-PCR analysis of RNA from RPE cells confirmed the presence of arrestin mRNA of predicted 377 bp and exhibited 100% homology with visual arrestin 48 kDa. CONCLUSION: Visual arrestin proteins present in RPE may be involved in the desensitization of G-protein-coupled receptors in RPE cells and in arrestin uveopathogenesis.

Amino Acid Sequence↗

PubMed2000

The epithelial sodium channel (ENaC) in rodent retina, ontogeny and molecular identity.

PURPOSE: To assess the appearance of the epithelial sodium channels (ENaC) during retinal development and establish its molecular identity. METHODS: Photoreceptors were isolated by horizontal sectioning of rat retina with a vibratome series 1000. Intact retina and isolated photoreceptors were analyzed for the developmental appearance of ENaC using the Polymerase Chain Reaction (PCR) technique. Immunofluorescence was conducted with the aid of an antibody raised against the 14 amino acids peptide QGLGKGDKREEQGL, corresponding to the N-terminal region (44-58 aa) of alpha ENaC. RESULTS: ENaC message was present in rat retina photoreceptors, isolated just one day after birth. The bipolar and ganglion cell layers, separated from whole retina by vibratome, also contained the ENaC message. The partial sequence of the photoreceptor ENaC (496 base) exhibited one hundred percent homology with the channel from rat known sources. Immunochemistry revealed that only the outer nuclear layer was positive for the ENaC in one-day-old rat; the inner segment became immunopositive at the age of 9 days. CONCLUSION: The ENaC is present in the retina and visible soon after birth. These observations suggest that ENaC may prove to have physiological importance in the retina but until now its function is unknown.

Amino Acid Sequence↗

PubMed1999

Immunochemical analysis of the sodium channel in rodent and human eye.

The presence of the amiloride-sensitive sodium channel (ASSC) in ocular tissues was studied with the aid of a polyclonal antiserum raised against the 14 amino acid peptide QGLGKGDKREEQGL. This sequence corresponds to the region 44-58 of the alpha subunit of the channel, termed ENaC, cloned from rat colon. The antibody titers, measured by the ELISA technique, rose to 1&ratio;2560 4 weeks after immunization, and this bleed was used in all subsequent experiments. Immunoblotting with the polyclonal anti-alphaENaC serum, revealed a major band of 82-86 kDa in extracts prepared from whole bovine or rat retina; a minor component of 92 kDa in the extract from bovine ciliary body may represent a glycosylated species. Immunohistochemistry, using the alphaENaC-specific antiserum, revealed strong fluorescence in specific areas of the rat and human eye. Pronounced labelling was observed in the epithelial cell layer of the retina, the lens, as well as both the pigmented and the nonpigmented epithelium of the ciliary body and the iris. All of the cell layers (epithelium, endothelium and fibroblasts) in the cornea, the blood vessels in the iris, and iris epithelium, were also strongly immunopositive. The somatic body of the photoreceptor cells (cones and rods) in the inner and outer segments could be traced to forming a synapse in both the internal and external portions of the internal nuclear layer. The bipolar cells and ganglia in the neuronal compartment also exhibited occasional immunofluorescence. The method of fixation and the source of the tissue were important parameters for the immunochemical localization of the ENaC. The resolution was very poor when rat eye was fixed in Bouin's solution but this method was satisfactory for human tissues. For rat eye, optimum resolution was obtained with AMeX fixation. This widespread distribution of the ENaC generally colocalizes with the previously observed immunopositivity for the mineralocorticoid receptor such that steroid hormone-mediated ion regulation would appear to add a new parameter to the functional expression of ocular tissues.

Amiloride↗

PubMed1999

General overview of mineralocorticoid hormone action.

The adrenal cortex elaborates two major groups of steroids that have been arbitrarily classified as glucocorticoids and mineralocorticoids, despite the fact that carbohydrate metabolism is intimately linked to mineral balance in mammals. In fact, glucocorticoids assured both of these functions in all living cells, animal and photosynthetic, prior to the appearance of aldosterone in teleosts at the dawn of terrestrial colonization. The evolutionary drive for a hormone specifically designed for hydromineral regulation led to zonation for the conversion of 18-hydroxycorticosterone into aldosterone through the catalytic action of a synthase in the secluded compartment of the adrenal zona glomerulosa. Corticoid hormones exert their physiological action by binding to receptors that belong to a transcription factor superfamily, which also includes some of the proteins regulating steroid synthesis. Steroids stimulate sodium absorption by the activation and/or de novo synthesis of the ion-gated, amiloride-sensitive sodium channel in the apical membrane and that of the Na+/K+-ATPase in the basolateral membrane. Receptors, channels, and pumps apparently are linked to the cytoskeleton and are further regulated variously by methylation, phosphorylation, ubiquination, and glycosylation, suggesting a complex system of control at multiple checkpoints. Mutations in genes for many of these different proteins have been described and are known to cause clinical disease.

Amiloride↗

PubMed1999

Copurification of selected glycolytic enzymes with retinal S-antigen (arrestin) by hydroxyapatite agarose chromatography of bovine retina.

PURPOSE: A variety of methods have been developed for the purification of S-antigen but a simple and rapid procedure based on hydroxyapatite-agarose (HA) adsorbtion is most widely used. In the present study, we investigated the nature of proteins purified with the aid of HA chromatography. METHODS: After elimination of retinal S-antigen by HA, the soluble extract of retinal tissue was readsorbed on HA. The proteins were thereafter desorbed by 10 to 500 mM phosphate buffer gradient. Two peaks obtained by SDS-PAGE were used for the generation of specific antisera for subsequent analysis by ELISA and Western blotting. RESULTS: Four proteins (two 48 kDa , one 50 kDa and one 46 kDa) were obtained in this manner. Partial amino acid sequencing permitted the identification of these proteins as alpha-enolase (48 kDa), gamma-enolase (48 kDa), Glucose-6-phosphate-Isomerase (50 kDa) and aspartate-amino-transferase (46 kDa). CONCLUSION: The selected glycolytic enzymes co-purified with retinal S-antigen by hydroxyapatite agarose chromatography of bovine retina.

Amino Acid Sequence↗

PubMed1999

Beta-arrestin-related proteins in ocular tissues.

PURPOSE: Proteins of the arrestin family contribute to the regulation of G-protein-mediated transduction. In this study, the presence of beta-arrestins in ocular tissues was investigated. METHODS: Mouse monoclonal and rabbit polyclonal antibodies were raised against the peptide Val-Asp-Thr-Asn-Ile-Leu-Glu-Leu-Asp-Thr-Asn-Asp-Asp-Asp-Ile, a sequence present in beta-arrestins 1 and 2 but absent from visual arrestin. These antibodies were used for the immunohistologic detection of beta-arrestins in parafin sections of rodent eyes fixed in Bouin's solution. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA from bovine retina, retinal pigmented epithelial (RPE) cells, lens epithelial cells, and human corneal fibroblasts was performed using beta-1 arrestin primers. RESULTS: In the eye, bet-arrestin staining predominated in RPE, inner segments of photoreceptors, synaptic spherules of rods, inner plexiform layer and ganglion cell fibers, epithelial cells from ciliary body, and vessels. RT-PCR amplified a 480 bp product, corresponding to the predicted length. The sequence of PCR products from bovine retina and RPE cells was identical with the bovine beta-arrestin mRNA. CONCLUSIONS: beta-arrestins were detected in several ocular tissues. In photoreceptor cells, their specific localization in the synaptic terminals and plexiform layer suggests a role of beta-arrestin in synaptic transmission. In other ocular tissues, the presence of beta-arrestin may be related either to adrenergic signal transduction or to signal transduction mediated by other G-protein-coupled receptors.

Amino Acid Sequence↗

PubMed1999

Abnormal fibrin clot architecture in nephrotic patients is related to hypofibrinolysis: influence of plasma biochemical modifications: a possible mechanism for the high thrombotic tendency?

Porosity, viscoelasticity and morphological properties of plasma fibrin from 16 nephrotic patients and 16 healthy volunteers were compared. Nephrotic patients were characterized by formation of tight and rigid plasma fibrin gels which resulted in a slower rate of fibrin lysis studied either under pressure-driven permeation or diffusional transport of fibrinolytic agents. These latter findings indicated that both abnormal fibrin network conformation and abnormal fibrin fiber structure were involved in hypofibrinolysis. Albumin supplementation up to 40 mg/ml partially restored normal fibrin architecture and increased the rate of fibrinolysis in these patients. Multiparametric analysis showed that nephrotic patients were mainly characterized by a low plasma albumin level (R = -0.85), a low albumin to fibrinogen ratio (R = -0.89) and a high resistance to lysis (R = -0.82). High triglycerides level was the only plasma modification related to the slower fibrin lysis rate (R = -0.54). High fibrin rigidity (G') was the only fibrin parameter simultaneously related to the nephrotic state (R = 0.75) and the lysis resistance (R = -0.71). After eliminating the effects of age, albumin and fibrinogen levels, low fibrin porosity (Ks) and low fiber mass-length ratio (mu) were the main features of the nephrotic state. These findings are discussed in relation to both the pathophysiology of thrombotic complications in nephrotic syndrome and their pharmacological prevention.

Adolescent↗

PubMed1998

Thrombus generation after adenovirus-mediated gene transfer into atherosclerotic arteries.

Thrombosis represents a major issue during arterial local delivery. We evaluated the occurrence of thrombosis after adenovirus (Ad)-mediated gene transfer into normal and atherosclerotic arteries. A replication-deficient Ad vector expressing the beta-galactosidase reporter gene (Ad.RSV betagal; 4 x 10(9) PFU) was injected into normal and atherosclerotic arteries (n = 11 in both groups). The contralateral artery received either an Ad vector carrying no transgene (Ad.MLPnull) (n = 7 in both groups, 4 x 10(9) PFU) or vehicle buffer (n = 4 in normal group, n = 8 in atherosclerotic group). Animals were sacrificed 3 days following gene transfer for thrombus detection and assessment of beta-galactosidase activity. Thrombus was absent in normal arteries and in atherosclerotic arteries injected with vehicle buffer only. In contrast, nonocclusive thrombus was present in atherosclerotic arteries injected with either Ad.RSV betagal (5 of 11) or Ad.MLPnull (3 of 7). Beta-galactosidase activity was predominantly found in the endothelial layer of the transfected arteries. Gene transfer and expression occurred despite the presence of the thrombus (4 of 5), and its efficiency did not significantly differ regardless of the thrombus. We conclude that thrombus frequently occurred in atherosclerotic arteries after Ad-mediated gene transfer. Further studies are warranted to identify the mechanisms of thrombus generation after Ad-mediated gene transfer into atherosclerotic arteries.

Adenoviridae↗

PubMed1998

Enhanced activation of the mineralocorticoid receptor in genetically hypertensive rats.

The relative abundance and availability of the mineralocorticoid receptor (MCR) appeared to be similar in the heart, kidney and ocular tissues of the genetically hypertensive SHR and normotensive WKY rats by a number of criteria including Western blotting, immunoprecipitation, dot blot analysis, and immunohistochemistry. On the other hand, the activation of the MCR, as judged by binding to DNA cellulose, was significantly enhanced in the hearts and kidneys of 14 week-old, hypertensive, SHR rats compared to the normotensive WKY animals. The activation of the renal MCR was elevated in the SHR strain even at the age of six weeks when the tail arterial pressure was statistically identical to that of the WKY strain. Thus, precocious receptor activation may represent a primary lesion leading to hypertension in the SHR strain, thereby providing a new model to elucidate the hypertensive state.

Animals↗

PubMed1998

Cellular source of human platelet secretory phospholipase A2.

Platelets are one source of the group II extracellular form of phospholipase A2 (sPLA2) which is involved in the amplification of local and systemic inflammation. Although sPLA2 protein has been described in human platelets, its presence in human megakaryocytes has not been yet established. We demonstrated in this study that the human erythroleukaemia (HEL) cell line, which has megakaryoblastic features, constitutively expresses sPLA2. Using an anti-rhsPLA2 monoclonal antibody (mAb BA11) and dot-blot detection, we showed that HEL cells and platelets release sPLA2 into incubation medium upon stimulation by thrombin. Similar results were obtained for sPLA2 activity detected by a spectrofluorescence assay. Enzymatic activity was abolished by mAb BA11 and by protamine. In both cell types, although released, the major part of sPLA2 remained in the cell pellet, and was probably adsorbed at non-specific membrane sites. Double labelling experiments using mAb BA11 and an anti-GPIIb antiserum revealed the presence of sPLA2 in human bone-marrow megakaryocytes. The use of reverse transcription-polymerase chain reaction conjugated with hybridization analysis demonstrated the presence of mRNA encoding for sPLA2 in platelets and HEL cells. Expression of sPLA2 in platelets and megakaryocytes at both transcriptional and post-translational levels strongly argues in favour of a megakaryocytic origin of platelet sPLA2 and rules out a role for endocytosis of the enzyme from plasma by circulating platelets.

Blood Platelets↗