Expression Of GMP-140 Research Articles

Effects of xinfeng capsules on expression of platelet granule membrane protein 140 and platelet cluster of differentiation 40 ligand in peripheral blood of adjuvant arthritis rats.

Platelet GMP-140 and CD40L as specific markers of platelet activation play an important role in the morbidity and development of rheumatoid arthritis. The expression of GMP-140, CD40L increases in peripheral blood of AA rats. And they have correlation with voix pedis' swelling, AI. XFC could inhibit the inflammatory response through inhibiting platelet activation of AA rats, which means decreasing the expression of GMP-140, CD40L in peripheral blood. So, the voix pedis' swelling and AI were decreased as the result.

The role of protein kinases in glycoprotein GMP-140 expression on activated human platelets

The expression of glycoprotein GMP-140 (P-selectin, CD-62P) is stimulated in human platelets by vasopressin via G protein-coupled receptors. In order to clarify the subsequent intracellular signal pathway, we examined the role of protein kinases in vasopressin action on thrombocytic CD62P expression in normal subjects. Staurosporine, a protein kinase (PK) C inhibitor, inhibited platelet aggregation and CD62P expression, whereas phorbol ester (PMA), a PKC stimulator, provoked them. On the other hand, 8-bromo-adenosine-3′,5′-monophosphate-cyclic AMP (8-Br-cAMP), a membrane permeant cAMP analogue, had no effect on platelet activation. PMI, a PKA inhibitor, had a little effect on them. From these results, it is suggested that PKC plays a dominant role in AVP action on platelet aggregation and CD62P expression.

Expression of GMP-140 (P-selectin) correlates with graft viability in cold-preserved rat livers.

Ischemia/reperfusion injury is an inflammatory process involving cytokine release, Kupffer cell activation, and sinusoidal endothelial cell activation. GMP-140 is synthesized by endothelial cells. We analyzed by Western blotting the expression of GMP-140 in a syngeneic rat liver transplantation model using grafts preserved for different periods of time. Compared with prereperfusion samples, expression did not change significantly in freshly harvested and 4-hr preserved livers. In grafts preserved for 24 hr (100% survival), GMP-140 levels increased dramatically at 1 hr, then returned to baseline at 24 hr after transplantation. Forty-eight hour preserved grafts (0% survival) showed a decreasing expression. To identify possible mediators, the effects of tumor necrosis factor-alpha and interleukin-1beta on GMP-140 expression in primary sinusoidal endothelial cells were analyzed. These cytokines increased both the percentage of stained cells as well as their mean staining fluorescence. The absence of increase in 48-hr grafts suggests that GMP-140 may distinguish viable from nonviable livers.

A new variant of Bernard-Soulier syndrome characterized by dysfunctional glycoprotein (GP) Ib and severely reduced amounts of GPIX and GPV.

We describe a new variant of Bernard-Soulier syndrome characterized by almost normal amounts of GPIb and severely reduced GPIX and GPV. Despite surface expression, GPIbalpha failed to support ristocetin-induced platelet agglutination and to bind two conformation-dependent monoclonal antibodies, suggesting a qualitative defect. Sequence analysis of the gene coding for GPIX revealed a T-to-C substitution at base 1811, leading to a Leu40Pro conversion, whereas no defects were found in the coding region of the GPIbalpha gene. Allele-specific restriction enzyme analysis showed that the propositus and one of his sisters. both with severe bleeding diathesis. were homozygous for the GPIX mutation: the members of the family with mild bleeding diathesis and/or giant platelets in the peripheral blood were heterozygous, whereas the healthy ones were homozygous for the normal allele. Infusion of 1-desamino-8-D-arginine vasopressin normalized bleeding time in the two severely affected patients, although it did not modify ristocetin-induced platelet agglutination or membrane expression of GPIbalpha, GPIX, GPIIb-IIIa and GMP-140. Moreover, in one patient, normalization of bleeding time and rise of von Willebrand factor plasma concentration did not seem to be directly related.

Is complement a target for therapy in renal disease?

Complement deposition in the injured kidney is common, especially in glomerulonephritis. The precise role of the complement system in the mediation of tissue injury in the kidney has been defined in recent years, and this has assumed extra importance with the recent development of specific forms of therapy directed at the complement pathway. As well as the induction of cell lysis, complement has many subtle effects on cell biology, particularly on endothelial cells. Complement components are produced locally in the kidney. Detailed studies of certain rare forms of nephritis have provided evidence that complement activation can directly cause tissue injury. Appreciation of the importance of complement in hyperacute rejection of xenotransplants has given new impetus to the development of complement inhibitors. A narrative review is provided, with a brief overview of the complement pathway and its regulatory mechanisms, mechanisms of complement-induced tissue injury, local complement production, and the renal consequences of complement dysregulation. Currently available forms of therapy aimed at the complement system are reviewed, and possible future therapeutic strategies are suggested. The complement system plays a direct causal role in tissue injury in certain forms of renal disease. Specific forms of therapy are becoming available that can selectively interrupt complement activation or promote its regulation. Much of the drive for the development of these therapies comes from the field of xenotransplantation, but these forms of therapy should also be tested in various primary renal diseases.

MOLECULAR INSIGHTS INTO THE ANTICOAGULANT-INDUCED SPONTANEOUS ACTIVATION OF PLATELETS IN WHOLE BLOOD - VARIOUS ANTICOAGULANTS ARE NOT EQUAL

The spontaneous anticoagulant-dependent platelet activation in vitro may potentially interfere with the determination of haemostatic parameters. The effects of various blood anticoagulants on platelet activation were monitored using flow cytometry. Regardless of a blood anticoagulant used (EDTAK 2, heparin, citrate or PPACK), platelet activation began immediately after blood withdrawal and was most pronounced in the EDTAK 2-anticoagulated blood samples. The progressing expression of GMP-140 antigen was accompanied by the enhanced abundance of the subunit β 3 of the platelet membrane integrin α IIbβ 3 without parallel changes in the fluorescence attributed to the complex form of the integrin α IIbβ 3. The increased expression of GMP-140 was paralleled by the enhanced platelet clumping in the samples anticoagulated with either EDTAK 2 or heparin, and the raised platelet microparticles in blood withdrawn into citrate. The EDTAK 2-induced platelet activation was markedly reduced by methyl 2,5-dihydroxycinnamate, tyrosine kinase inhibitor. The influence of disodium EDTA on platelet membrane dynamics closely mimicked the alterations induced upon the interaction of fibrinogen with platelet GPIIb-IIIa. Thus, the EDTAK 2-induced platelet activation might result from an interference with platelet membrane protein structure and conformation and possibly relate to an “unspecific” trigerring of a signal transduction pathway. Overall, EDTAK 2 and heparin appeared the least suitable anticoagulants, particularly with the regard to the expression of GMP-140 antigen. The failure to recognize the importance of a spontaneous anticoagulant-induced platelet activation may result in misdiagnoses during the monitoring of coagulation parameters.

Dual effects of diclofenac on human platelet adhesion in vitro.

The effect of two non-steroidal anti-inflammatory drugs on the adhesion function of human platelets was evaluated. Platelets isolated from healthy human subjects were treated for 10 min with the indicated drugs and then incubated in fibrinogen-coated microwell plates in the absence or in the presence of ADP (10 microM) and thrombin (0.05 U/ml). After 1 h of incubation, adherent platelets were measured using an enzymatic assay. ADP- and thrombin-stimulated adhesion was significantly inhibited by high doses ( > 500 microM) of diclofenac, while doses ranging from 50 to 300 microM stimulated adhesion in the absence of agonists (resting platelets). A similar stimulatory effect on platelet adhesion was observed also with 200-500 microM flurbiprofen. Moreover, immunocytofluorimetry demonstrated that diclofenac dose-dependently (100-500 microM) induced the expression of GMP-140 and increased the expression of GPIIb/IIIa on the membrane of unstimulated platelets. High doses ( > 500 microM) of this drug inhibited thrombin-stimulated expression of GPIIb/IIIa and GMP-140.

Plasmin accelerates platelet-dependent prothrombinase formation without activating the platelets.

Patients with acute myocardial infarction who undergo thrombolytic therapy may shortly thereafter present evidence for increased platelet activation and thrombin activity, and recurrent thrombosis. This study investigated whether plasmin activates platelets and prothrombin in recalcified platelet-rich plasma (RPRP) to cause (at least in part) these side-effects of thrombolytic therapy. Plasmin (0.1 and 1.0 CU/ml) addition to RPRP with microM r-tick anticoagulant peptide (the latter a factor Xa inhibitor which abrogates prothrombin activation by prothrombinase at the concentration used) resulted in no change in the concentration of prothrombin fragment 1 + 2, or in the expression of GMP-140, the resting and activated GP IIb-IIIa conformers, and GPIb on platelets. Thus, plasmin neither activates platelets nor prothrombin in RPRP. However, plasmin accelerated platelet activation and secretion, and prothrombin fragment 1 + 2 production in RPRP. When combined with 1 microM r-tick anticoagulant peptide and 1 or 10 mM alpha-thrombin to RPRP, plasmin also increased the number of GMP-140 molecules expressed/platelet without enhancing alpha-thrombin binding to the platelets. Additionally, plasmin accelerated prothrombin activation when it was added to washed platelets resuspended in factor V depleted plasma simultaneously with 10 mM CaCl2, 10 nM alpha-thrombin for 10 s (to activate platelets and platelet factor V), followed by 4 microM hirudin and 1 nM factor Xa. Thus, plasmin potentiates the platelet release reaction in response to alpha-thrombin (probably by increasing the availability of factor V on the platelets) to enhance prothrombin activation in RPRP. These actions of plasmin may contribute to the increased platelet activation and thrombotic side-effects that can occur after thrombolytic therapy.

Cell surface expression of lysosome‐associated membrane protein‐2 (lamp2) and CD63 as markers of in vivo platelet activation in malignancy

Platelets may become activated in vivo in a number of prethrombotic conditions including cancer. In the present study, cell surface expression of lysosome-associated membrane protein-2 (lamp2) and CD63 (lamp3) were examined by flow cytometry in 15 healthy volunteers and 5 patients with metastatic cancer to determine their utility as markers of in vivo platelet activation. Unstimulated platelets from controls had low levels of lamp2 expression, in contrast to cancer patients, who had significantly elevated levels (3.79 +/- 1.48% vs 33.9 +/- 5.6%). Upon stimulation with collagen, a greater than two-fold increase in the number of platelets expressing detectable levels of lamp2 was seen only among controls and not in cancer patients. Stimulation with collagen also resulted in a nearly two-fold increase in the proportion of platelets expressing CD63 in the control group, and a less than 1.5-fold increase in CD63 was seen in the patient group. The results suggest that cell surface lamp2 and CD63, like cell surface expression of GMP-140, may be good indicators of in vivo platelet activation and may be potentially useful in identifying patients with prethrombotic disorders.

A comprehensive hematologic study in calves with total artificial hearts.

Thromboembolism and infection remain potential threats for long-term circulatory assist and replacement devices. The alteration of the hemostatic system and of blood cell functions caused by device implantation may predispose the recipient to these complications. Many sensitive coagulation assays and the technology of flow cytometry would be powerful tools for this investigation. The availability of such immunologic technologies for animal species other than humans has yet to be established. In a series of in vitro tests we found that the following assays, among others, are usable in calves: TAT, TxB2, platelet surface glycoprotein IIbIIIa, and membrane aminophospholipid. F1.2, D-dimer, beta TG, PF-4, and platelet surface expression of GMP-140 and receptors for fibronectin, thrombospondin, and vWF were not measurable. A sustained mild decrease in hematocrit levels in six calves with the Cleveland Clinic-Nimbus total artificial heart for 11-120 days was attributed to an increase in circulating blood volume, but not to red blood cell damage. Whole blood platelet aggregation was suppressed only for the first 3 post operative days, with decreased GPIIbIIIa expression. Polymorphonuclear phagocytosis, chemotaxis, and superoxide anion production were not altered. Device infection and thromboembolism occurred in one of 13 cases overall.

Morphologic and ultrastructural evidence of interleukin-6 induced platelet activation.

The in vitro effect of IL-6 on platelet activation was investigated. When human platelets were incubated with high (1,000 ng/ml) or low (1 ng/ml) dose IL-6, expression of GMP-140 was enhanced by 42% (N = 6; P < 0.009) and 46% (N = 6; P < 0.061) in 1 hr low and high dose IL-6-platelet incubations, respectively, as assessed by flow cytometry. In platelet specimens incubated with high dose IL-6 for 3 hr, a 70% (N = 6; P < 0.009) increase in GMP-140 expression over control was observed. Parallel high dose IL-6 incubations subjected to scanning electron microscopic studies revealed a 3.4-fold increase (N = 6; P < 0.001) in spheroid morphologic platelet forms in 1 hr incubations in comparison to control platelet preparations, whereas in 3 hr IL-6-platelet incubations, a 96% increase in dendritic platelet forms was observed (N = 6; P < 0.001). Significant increases in platelet ATP levels were observed in both 1 min and 1 hr high dose and low dose IL-6 platelet incubations. In 3 hr high dose-IL-6 platelet incubations, a significant 18% (N = 8; P < 0.001) decrease in platelet ATP was parallelled by a significant 40% increase (N = 8; P < 0.014) in plasma ATP in the same specimens. This increased plasma ATP was highly correlated with a reduction in platelet ATP when analyzed by bivariate regression analysis. Lastly, transmission electron microscopic analysis demonstrated a significant reduction in dense granule number and ratio of dense granule surface area/cell surface area in 3 hr high dose IL-6 incubations. These findings suggests that IL-6 activates platelets in vitro.

Original Article: Evaluation of Platelet Function by Flow Cytometric Measurement of Ligand Binding

Rapid and relevant evaluation of platelet function is often clinically important. By means of fluorescent labelled chicken antibodies (which do not bind to Fc-receptors) against fibrinogen and von Willebrand factor and flow cytometry, we have determined the time course of ligand association to platelets after stimulation with adenosine 5'-diphosphate and ristocetin respectively. The expression of guanosine 5'-phosphate (GMP)-140 was also measured. We have applied this technique to evaluate platelet function during platelet storage and cardiopulmonary bypass. There was a significant reduction of the binding of fibrinogen and von Willebrand factor and significantly increased expression of GMP-140 after 9 days of storage. Changes in metabolic variables such as lactate accumulation, glucose consumption and decrease in pH confirm that the functional impairment is due to a large extent to a deteriorated platelet metabolism. No significant differences were found between samples taken before and during cardiopulmonary bypass, but there was a tendency towards increased ligand binding as well as increased expression of GMP-140 at the end of cardiopulmonary bypass. The flow cytometric technique that is described may be useful for evaluation of platelet function and platelet activation in vivo.

The role of metal corrosion in inflammatory processes: induction of adhesion molecules by heavy metal ions

Prosthetic devices undergo corrosion processes after implantation including the release of certain amounts of metal ions into the adjacent tissues. On reaching the bloodstream, a systemic influence of those ions may be envisaged. Cell adhesion molecules (CAMs) are recognized as an essential component of the mechanisms of endothelial damage. To study the influence of selected heavy metals on human umbilical vein endothelial cells (HUVEC) EIA methods were used to evaluate cellular expression of E-selectin, ICAM-1, VCAM-1 and GMP-140 under the influence of high (cytotoxic) very low (non-cytotoxic) concentrations of Zn, Ni, Co and Cr. The de novo synthesis of CAMs was studied with the help of mRNA analysis. Intermediate voltage immuno electron-microscopical imaging was performed to detect the localization on the cell surface of the adhesion molecules E-selectin and ICAM-1 under the influence of cytokines, which represent important factors in inflammatory processes. Very low concentrations of metal ions, which gave no significant influence on cell morphology, elicited a significant expression of CAMs on endothelial cells in vitro. Thus, for example, zinc, nickel and cobalt ions in concentrations of 1×10-9 M increased the expression of endothelial E-selectin, compared to the control after a 5 h incubation. Similar findings were established for zinc, nickel and cobalt ions also with regard to ICAM-1, VCAM-1 and GMP-140. Northern blot analysis gave an increased ELAM-1 and ICAM-1 mRNA expression after incubation with high concentrations of zinc and nickel ions. The results should draw attention to possible effects of very low concentrations, which are released during processes of metal corrosion on prosthetic devices.

Exposure of Anionic Phospholipids upon Platelet Activation Permits Binding of β2 Glycoprotein I and Through it that ofIgG Antiphospholipid Antibodies. Studies in Platelets from Patients with Antiphospholipid Syndrome and Normal Subjects

Patients with antiphospholipid syndrome, whether primary or secondary to systemic lupus erythematosus, may have thrombocytopenia. Their antibodies to anionic phospholipids might bind to phospholipids on the platelet wall but anionic phospholipids are asymmetrically located in the inner leaflet. In addition, antibodies to anionic phospholipids may require beta 2 glycoprotein I (beta 2GPI) as a cofactor in order to bind to phospholipids. In turn, beta 2GPI has high affinity for anionic phospholipids. Loss of this asymmetry occurs upon platelet activation and could thus permit such antibody-beta 2GPI-platelet interaction. We studied this by flow cytometry using purified beta 2GPI-FITC labelled and similarly labelled affinity-purified polyclonal antibodies to cardiolipin or phosphatidylserine (aPL) obtained from sera of patients with primary antiphospholipid syndrome. Five percent of resting platelets were bound by aPL in the presence of beta 2GPI. Such binding increased when we activated platelets with various agonists, reaching 31% with the concurrent use of thrombin and the calcium ionophore A23187. Platelet activation resulted in the expression of GMP140 but this did not correlate with aPL binding. This probably reflects that the expression of GMP140, which depends on their secretion of alpha granules, has different agonist responses and occurs at different times than do microvesicle formation and expression of prothrombinase activity which coincide with the loss of phospholipid asymmetry on the platelet wall. When we studied the binding of purified beta 2GPI we also found that it binds preferentially to activated platelets and that it seems to be a prerequisite for the binding of aPL onto them. Our findings indicate that aPL from patients with antiphospholipid syndrome may bind to activated platelets through beta 2GPI.

Platelet-activating factor-induced endothelial cell expression of adhesion molecules and modulation of surface glycocalyx, evaluated by electron spectroscopy chemical analysis.

PAF synthesized by or added to the endothelial cell monolayer has as first target the endothelium itself, inducing the expression of GMP-140 on the cell surface and qualitative alterations in the composition of glycocalyx. As seen by ESCA, sulfated chemical groups were significantly reduced after PAF stimulation. This reduction depended on selective loss of sulfated proteoglycans that were released into the supernatant. The alteration of glycocalyx may favor functional exposure of GMP-140 and of PAF on the surface of endothelial cells and may reduce charge repulsive forces between cells. PAF associated with endothelial cell surface has as second target PMNs and stimulates in cooperation with GMP-140, the functional activation of PMN CD11-CD18 adhesion molecules.

Effects of Magnesium on Platelet Aggregation and Adhesion

Magnesium deficiency and its association with platelet hyperreactivity has been well recognised in a variety of diseases including myocardial infarction, preeclampsia, and diabetes. In order to investigate potential effects of intravenous Mg2+ supplementation, platelet function was studied by measurements of in vitro bleeding time (BT) and of fibrinogen (Fg)-mediated aggregation of washed platelets. In addition, the effect of Mg2+ on platelet adhesion onto immobilised Fg, on Fg binding to activated platelets, and on surface expression of GMP-140 or GP53 was evaluated. Mg2+ (4 mM) prolonged in vitro BT by 30% and inhibited Fg-mediated aggregation significantly, independent of the agonist used to initiate platelet aggregation (ADP, collagen, epinephrine, thrombin, phorbol ester). Adhesion of resting platelets to immobilised Fg was reduced by 50% in the presence of 2 mM Mg2+. Moreover, Mg2+ reduced Fg binding to ADP- or collagen-stimulated platelets as well as surface expression of GMP-140 with an IC50 of approximately 3 mM. Intravenous administration of Mg2+ to healthy volunteers inhibited both ADP-induced platelet aggregation (p < 0.05) by 40% and binding of Fg or surface expression of GMP-140 by 30% (p < 0.05). Thus, pharmacological concentrations of Mg2+ effectively inhibit platelet function in vitro and ex vivo.

Effect of prestorage leukocyte reduction on proteins of platelets obtained by apheresis.

The effect of prestorage leukocyte reduction was evaluated on platelet concentrates (PCs) obtained by apheresis using the 'surge' technique. Two hours after collection, the PCs were divided into 2 equal units. One unit was filtered through a Sepacell PL-10a, producing a filtered PC (FPC). The second unit constituted a non-filtered PC (NFPC). FPCs and NFPCs were stored at room temperature in 1,400-ml CLX bags on a horizontal agitator up to 7 days. We analyzed platelet samples obtained during storage from NFPCs and FPCs at days 1, 3 and 7. The expression of membrane glycoproteins (GP)Ib and GPIIb/IIIa (assessed by flow cytometry), platelet response to thrombin and ristocetin (aggregometry) and global platelet protein pattern (studied by high-resolution two-dimensional gel electrophoresis) remained stable over the 7 days of storage in NFPCs as well as in FPCs. However, in both preparations, the expression of GMP-140 (flow cytometry) progressively increased during storage. Our in vitro study indicates that early leukocyte reduction by filtration of apheresis PCs does not induce modifications in platelet GPs and protein patterns.

Lectin binding to chronic inflammatory gingival tissue: possible adhesion mechanisms based on lectin-carbohydrate interactions.

In this study, we analyzed the expression of different leukocyte surface antigens, of the adhesion molecules ELAM-1 and GMP-140 and binding of various lectins and neoglycoproteins in inflamed gingival tissue. Cell suspensions from collagenase-digested gingiva were analyzed by flow cytometry in a FACScan. The expression of ELAM-1, GMP-140, carbohydrate structures and lectins in gingival specimens was also studied by immunohistochemistry. Gingival tissue of patients with active periodontal disease contained between 5% and 50% CD45+ mononuclear cells, consisting mainly of CD19+ cells (B lymphocytes). CD62, resembling GMP-140, and ELAM-1 were strongly expressed on endothelial cells of these patients. Control subjects usually contained almost no CD45+ cells in their gingiva and no CD62+ or ELAM-1-positive endothelial cells could be found in 5 of 6 control persons. Analysis of the glycosylation pattern revealed staining of infiltrating cells by peanut agglutinin (PNA; specificity for galactose), whereas soy bean agglutinin (SBA; specificity for N-acetyl-galactosamine) bound to epithelial cells. An endogenous lactosyl-specific lectin could be detected on endothelial cells by binding of lactosyl-BSA. Ulex europeus I agglutinin (UEA-1, specific for fucose) showed selective staining of endothelial and epithelial cells. Expression of a fucose-binding lectin, demonstrated by binding of fucosylated BSA, could be found on infiltrating cells. The adhesion molecules ELAM-1 and GMP-140 seem to be involved in cell adhesion during chronic inflammation of the gingiva. Interaction of other carbohydrate residues with endogenous lectins might resemble additional adhesion mechanisms in inflamed gingiva.

Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies

During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the alpha-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15,000 to 20,000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11,000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages.

Expression of GMP-140 on the surface of vascular endothelial cells is the initial and essential response to ischemia-reoxygenation

Expression of GMP-140 on the surface of vascular endothelial cells is the initial and essential response to ischemia-reoxygenation