The Stress of Lung Aging: Endoplasmic Reticulum and Senescence Tête-à-Tête.

Beyond the structural changes, features including the dysregulation of endoplasmic reticulum (ER) stress response and increased senescence characterize the lung aging. ER stress response and senescence have been reported to be induced by factors like cigarette smoke. Therefore, deciphering the mechanisms underlying ER and senescent pathways interaction has become a challenge. In this review we highlight the known and unknown regarding ER stress response and senescence and their cross talk in aged lung.

Human lung extracellular matrix hydrogels resemble the stiffness and viscoelasticity of native lung tissue.

Chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD) are associated with changes in extracellular matrix (ECM) composition and abundance affecting the mechanical properties of the lung. This study aimed to generate ECM hydrogels from control, severe COPD [Global Initiative for Chronic Obstructive Lung Disease (GOLD) IV], and fibrotic human lung tissue and evaluate whether their stiffness and viscoelastic properties were reflective of native tissue. For hydrogel generation, control, COPD GOLD IV, and fibrotic human lung tissues were decellularized, lyophilized, ground into powder, porcine pepsin solubilized, buffered with PBS, and gelled at 37°C. Rheological properties from tissues and hydrogels were assessed with a low-load compression tester measuring the stiffness and viscoelastic properties in terms of a generalized Maxwell model representing phases of viscoelastic relaxation. The ECM hydrogels had a greater stress relaxation than tissues. ECM hydrogels required three Maxwell elements with slightly faster relaxation times (τ) than that of native tissue, which required four elements. The relative importance (Ri) of the first Maxwell element contributed the most in ECM hydrogels, whereas for tissue the contribution was spread over all four elements. IPF tissue had a longer-lasting fourth element with a higher Ri than the other tissues, and IPF ECM hydrogels did require a fourth Maxwell element, in contrast to all other ECM hydrogels. This study shows that hydrogels composed of native human lung ECM can be generated. Stiffness of ECM hydrogels resembled that of whole tissue, while viscoelasticity differed.

Phenotype and Functional Features of Human Telomerase Reverse Transcriptase Immortalized Human Airway Smooth Muscle Cells from Asthmatic and Non-Asthmatic Donors.

Asthma is an obstructive respiratory disease characterised by chronic inflammation with airway hyperresponsiveness. In asthmatic airways, there is an increase in airway smooth muscle (ASM) cell bulk, which differs from non-asthmatic ASM in characteristics. This study aimed to assess the usefulness of hTERT immortalisation of human ASM cells as a research tool. Specifically we compared proliferative capacity, inflammatory mediator release and extracellular matrix (ECM) production in hTERT immortalised and parent primary ASM cells from asthmatic and non-asthmatic donors. Our studies revealed no significant differences in proliferation, IL-6 and eotaxin-1 production, or CTGF synthesis between donor-matched parent and hTERT immortalised ASM cell lines. However, deposition of ECM proteins fibronectin and fibulin-1 was significantly lower in immortalised ASM cells compared to corresponding primary cells. Notably, previously reported differences in proliferation and inflammatory mediator release between asthmatic and non-asthmatic ASM cells were retained, but excessive ECM protein deposition in asthmatic ASM cells was lost in hTERT ASM cells. This study shows that hTERT immortalised ASM cells mirror primary ASM cells in proliferation and inflammatory profile characteristics. Moreover, we demonstrate both strengths and weaknesses of this immortalised cell model as a representation of primary ASM cells for future asthma pathophysiological research.

Latrophilin receptors: novel bronchodilator targets in asthma.

BACKGROUND: Asthma affects 300 million people worldwide. In asthma, the major cause of morbidity and mortality is acute airway narrowing, due to airway smooth muscle (ASM) hypercontraction, associated with airway remodelling. However, little is known about the transcriptional differences between healthy and asthmatic ASM cells. OBJECTIVES: To investigate the transcriptional differences between asthmatic and healthy airway smooth muscle cells (ASMC) in culture and investigate the identified targets using in vitro and ex vivo techniques. METHODS: Human asthmatic and healthy ASMC grown in culture were run on Affymetrix_Hugene_1.0_ST microarrays. Identified candidates were confirmed by PCR, and immunohistochemistry. Functional analysis was conducted using in vitro ASMC proliferation, attachment and contraction assays and ex vivo contraction of mouse airways. RESULTS: We suggest a novel role for latrophilin (LPHN) receptors, finding increased expression on ASMC from asthmatics, compared with non-asthmatics in vivo and in vitro, suggesting a role in mediating airway function. A single nucleotide polymorphism in LPHN1 was associated with asthma and with increased LPHN1 expression in lung tissue. When activated, LPHNs regulated ASMC adhesion and proliferation in vitro, and promoted contraction of mouse airways and ASMC. CONCLUSIONS: Given the need for novel inhibitors of airway remodelling and bronchodilators in asthma, the LPHN family may represent promising novel targets for future dual therapeutic intervention.

Beyond TGFß--novel ways to target airway and parenchymal fibrosis.

Within the lungs, fibrosis can affect both the parenchyma and the airways. Fibrosis is a hallmark pathological change in the parenchyma in patients with idiopathic pulmonary fibrosis (IPF), whilst in asthma or chronic obstructive pulmonary disease (COPD) fibrosis is a component of the remodelling of the airways. In the past decade, significant advances have been made in understanding the disease behaviour and pathogenesis of parenchymal and airway fibrosis and as a result a variety of novel therapeutic targets for slowing or preventing progression of these fibrotic changes have been identified. This review highlights a number of these targets and discusses the potential for treating parenchymal or airway fibrosis through these mediators/pathways in the future.

Doxycycline inhibits matrix metalloproteinase-2 secretion from TSC2-null mouse embryonic fibroblasts and lymphangioleiomyomatosis cells.

BACKGROUND AND PURPOSE: Lymphangioleiomyomatosis (LAM) is characterized by the abnormal growth of smooth muscle-like cells (LAM cells) and cystic destruction of the lung parenchyma. LAM cell-derived matrix metalloproteinases (MMPs) are thought to play a prominent role in the tissue destruction. The aim of this study was to determine whether doxycycline, a known MMP inhibitor, can inhibit LAM cell proliferation or mitochondrial function and/or modulate MMPs and their tissue inhibitors (TIMPs). EXPERIMENTAL APPROACH: Wild-type and tuberous sclerosis complex-2 (TSC2)-null mouse embryonic fibroblasts (MEFs) were cultured in DMEM containing 10% fetal bovine serum (FBS). Human LAM cells were derived from the lungs of LAM patients and airway smooth muscle cells from control subjects. Cells were stimulated with FBS with or without doxycycline for up to 9 days. Proliferation was assessed by manual cell counts and MTT assay, MMP production by zymography and ELISA, and TIMP production using elisa. KEY RESULTS: Doxycycline did not change FBS-induced proliferation in MEFs or human cells. However, doxycycline did reduce metabolic activity of both wild-type and TSC2-null MEFs and LAM cells, but had no effect on control cells. Furthermore, doxycycline reduced MMP-2 from MEFs and decreased active-MMP-2 from LAM cells but had no effect on TIMP-1 and TIMP-2 from human LAM cells. CONCLUSIONS AND IMPLICATIONS: Doxycycline decreased MMP levels and cell metabolic activity, which raises the possibility of therapeutic efficacy in LAM.

CD40 and OX40 ligand are differentially regulated on asthmatic airway smooth muscle.

BACKGROUND: CD40 and OX40 Ligand (OX40L) are cell-surface molecules expressed on airway smooth muscle (ASM) that can enhance inflammatory cell activation and survival. The aim of this study was to examine the effect of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) on ASM CD40 and OX40L expression. METHODS: CD40 and OX40L expression on human ASM cells from asthmatic and nonasthmatic donors following stimulation with TNF-alpha and/or IFN-gamma was measured using cell-surface enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Involvement of signalling pathway was investigated with pharmacological inhibitors. Soluble TNF receptor levels were quantified by ELISA. RESULTS: Interferon-gamma and TNF-alpha synergistically increased CD40 expression to a greater extent on asthmatic than on nonasthmatic ASM. In contrast, IFN-gamma reduced TNF-alpha-induced OX40L expression to a similar extent in both cell types. TNF-alpha and IFN-gamma induced CD40 via nuclear factor-kappaB (NF-kappaB) and signal transducer and activator of transcription-3 in both cell types and modulated OX40L via NF-kappaB and c-Jun N terminal kinase in nonasthmatic cells. Similar effects on the induction of OX40L in asthmatic cells were seen with NF-kappaB, but these were not statistically significant. The reduced OX40L expression with TNF-alpha and IFN-gamma involved extracellular regulated kinase 1/2 activation. CONCLUSION: Asthmatic ASM may modulate airway inflammation locally by increasing CD40 and OX40L expression in response to cytokines. IFN-gamma may regulate ASM pro-inflammatory actions by differentially modulating ASM CD40 and OX40L expression.

Treating asthma means treating airway smooth muscle cells.

Asthma is characterised by airway hyperresponsiveness, airway inflammation and airway remodelling. Airway smooth muscle cells are known to be the main effector cells of airway narrowing. In the present paper, studies will be discussed that have led to a novel view of the role of airway smooth muscle in the pathogenesis of asthma in which airway hyperresponsiveness, remodelling and inflammation are, at least in part, attributable to airway smooth muscle. Furthermore, how this new view may lead to a change in the phenotyping and treatment of patients with asthma will be discussed.

Airway epithelium-derived transforming growth factor-beta is a regulator of fibroblast proliferation in both fibrotic and normal subjects.

BACKGROUND: In the healthy lung, airway epithelial cells (AEC) regulate fibroblast proliferation through release of soluble factors, such as prostaglandins and proteins. Fibroproliferative diseases and airway remodelling may result from an inadequate generation of suppressive factors by AEC or the inability of fibroblasts to respond to them appropriately. OBJECTIVE: The aim of this study was to study the effect of primary human AEC on the proliferation of fibroblasts obtained from healthy and fibrotic lungs in an interactive cell culture model. RESULTS: Conditioned medium (CM) from 14 out of 16 AEC lines significantly inhibited proliferation of normal human lung fibroblasts by 51.2+/-6.0%. The proliferation of fibroblasts derived from patients with lung fibrosis was equally inhibited by CM of AEC. The inhibitory effect of AEC-CM was completely reversed when fibroblasts were pre-incubated with 2.5 microm indomethacin. Furthermore, primary human AEC, but not fibroblasts, secrete TGF-beta, and the inhibitory effect of the AEC-CM was blocked by neutralizing anti-TGF-beta antibodies. CONCLUSION: These results demonstrate that AEC actively inhibit the proliferation of both normal and fibrotic fibroblasts via TGF-beta, which induces the prostaglandin E(2) synthesis in fibroblasts. The data indicate that proliferative lung diseases may be treated using the epithelial cell as the target of medication.

Rhinovirus exposure impairs immune responses to bacterial products in human alveolar macrophages.

BACKGROUND: Rhinovirus infection is responsible for considerable morbidity and mortality as the major cause of exacerbations of asthma, and is also known to induce exacerbations of cystic fibrosis and chronic obstructive pulmonary disease. Exacerbations of these diseases are also frequently associated with bacterial and atypical bacterial infection. Alveolar macrophages are the major immune cells in the airways and are important in defence against bacterial infections. METHODS: The authors investigated whether rhinovirus modifies cytokine release, the pattern recognition receptor expression and phagocytosis by human alveolar macrophages in response to bacterial products. RESULTS: Viable rhinovirus was detected in macrophages up to 3 days after exposure and viral RNA expression persisted for 10 days. Infectious but not UV inactivated rhinovirus increased tumour necrosis factor alpha (TNFalpha) and interleukin (IL)8 release by macrophages. In contrast, infectious rhinovirus impaired lipopolysaccharide and lipoteichoic acid induced TNFalpha and IL8 secretion by macrophages. Rhinovirus induced impairment of macrophage antibacterial immune responses did not involve IL10, prostaglandin E(2) or downregulation of Toll-like receptor 2. Furthermore, the macrophage phagocytic response to labelled bacterial particles, but not to latex beads, was impaired. CONCLUSION: The authors have identified impairment of cytokine responses to bacterial lipopolysaccharide and lipoteichoic acid by alveolar macrophages in response to infectious rhinovirus. Virus induced impairment of antibacterial host defence has important implications in the pathogenesis of exacerbations of respiratory diseases.

In vitro studies of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is associated with abnormal airway smooth muscle that leads to the characteristic pathology of lung nodule formation and destruction of lung tissue. The current authors have previously identified abnormal behaviour of airway smooth muscle cells from patients with asthma. In this study, cells and tissue sections derived from patients with LAM (n=7), asthma (n=8), and nonasthmatic controls (n=9) were compared. The presence of the antigen human melanosome (HM)B-45 was investigated, along with the proliferation and release of extracellular matrix proteins, release of endogenous prostaglandin E2 (PGE2), vascular endothelial growth factor and connective tissue growth factor, and the expression of integrins. Positive HMB-45 staining was found in all LAM patients and no controls. Proliferation of LAM cells was not different from control cells nor was its inhibition by beta-agonists, corticosteroids, rapamycin or PGE2. However, endogenous PGE2 levels were markedly decreased in LAM cells, and this was associated with decreased expression of the inducible form of cyclooxygenase (COX-2). The increased levels of connective tissue growth factor seen in asthma cells were not observed in LAM. Elastin mRNA in response to transforming growth factor-beta stimulation was markedly lower in LAM cells than either asthma or control cells. In conclusion, lymphangioleiomyomatosis cells exhibit abnormal properties in vitro that may contribute to pathophysiology and symptomatology in patients with lymphangioleiomyomatosis.

Molecular mechanisms of drug-induced thrombocytopenia.

A wide range of drugs can induce thrombocytopenia. Molecular mechanisms for the formation of specific epitopes for all the drug-dependent antibodies appear to be very similar. A restricted set of glycoproteins on the platelet surface interacts with the drugs to form neoepitopes, to which the drug-dependent antibodies bind. Molecular mapping of antigenic sites may help characterize genetic polymorphisms that predispose to the formation of the antibody binding sites. Identification of antibody binding sites will enhance our understanding of the pathogenesis of immune drug-induced thrombocytopenia.

Airway smooth muscle cell proliferation is increased in asthma.

UNLABELLED: Increased airway smooth muscle (ASM) within the bronchial wall of asthmatic patients has been well documented and is likely to be the result of increased muscle proliferation. We have for the first time been able to culture ASM cells from asthmatic patients and to compare their proliferation rate with that of nonasthmatic patients. Asthmatic ASM cell cultures (n = 12) were established from explanted lungs and endobronchial biopsies. Nonasthmatic ASM cells (n = 10) were obtained from explanted tissue from patients with no airway disease, emphysema, carcinoma, and fibrosing alveolitis. Cell counts, tritiated thymidine incorporation, and cell cycle analysis were conducted over 7 d. Asthmatic ASM cell numbers at Days 3, 5, and 7 were significantly higher than corresponding values for nonasthmatic cells (p < 0.05). Tritiated thymidine incorporation was increased 3.2-fold in asthmatic cells compared with nonasthmatic cells within the first 24 h (p = 0.026). Flow cytometric analysis of DNA content on Days 1 and 2 revealed that a significantly greater percentage of asthmatic ASM cells were in the G2 + M phase (p < 0.05). This study shows for the first time that proliferation of ASM cells is increased in patients with asthma and provides evidence for an intrinsic abnormality in the ASM cell in this disease. KEYWORDS: asthma; human airway smooth muscle; cell culture; cell proliferation; hyperplasia

Gene expression studies using microarrays.

1. The rapid progression of the collaborative sequencing programmes that are unravelling the complete genome sequences of many organisms are opening pathways for new approaches to gene analysis. As the sequence data become available, the bottleneck in biological research will shift to understanding the role of each of these genes. Techniques that enable the analysis of large sets of genes in one experiment will elucidate the interactions of genes in diverse biological systems. 2. Microarrays, which consist of large numbers of cDNA or oligonucleotides spotted onto a glass microscope slide, are one such technology. RNA isolated from two populations of cells, one control and one altered by experimental treatment or disease, is labelled with two different fluorochromes before being hybridized to the microarray. After a standard hybridization reaction, a scanner records the intensity of the two fluorochromes. The data can be analysed using special software that enables clustering of genes that have similar expression patterns. 3. Such powerful analysis techniques will provide information about genes whose functions are currently unknown and enhance our understanding of how genes interact to provide molecular control. This increase in knowledge about gene function will allow new targeted approaches for the development of drugs and/or gene therapies.

Mapping quantitative trait loci for serum insulin-like growth factor-1 levels in mice.

Serum insulin-like growth factor-1 (IGF-1) and femoral bone mineral density (BMD) differ between two inbred strains of mice, C3H/HeJ (C3H) and C57BL/6J (B6), by approximately 30% and 50%, respectively. Similarly, skeletal IGF-1 content, bone formation, mineral apposition, and marrow stromal cell numbers are higher in C3H than in B6 mice. Because IGF-1 and several bone parameters cosegregate, we hypothesize that the serum IGF-1 phenotype has a strong heritable component and that genetic determinants for serum IGF-1 are involved in the regulation of bone mass. We intercrossed (B6 x C3H)F1 hybrids and analyzed 682 F2 female offspring at 4 months of age for serum IGF-1 by radioimmunoassay and femoral BMD by peripheral quantitative computerized tomography (pQCT). Genomic DNA was assayed by polymerase chain reaction (PCR) to determine alleles for 114 Mit markers inherited in F2 mice at average distances of 14 centimorgans (cM) along each chromosome (Chr). Serum IGF-1 levels in the F2 progeny were relatively normal in distribution, but showed a greater range than either progenitor, indicating that serum IGF-1 level is a polygenic trait with an estimated heritability of 52%. Serum IGF-1 correlated with femoral length (r = 0.266, p < 0.0001) and femoral BMD (r = 0.267, p < 0.0001). Whole genome scans for main effects associated with serum IGF-1 levels revealed three significant QTLs (in order of significance) on mouse Chrs 6, 15, and 10. The QTL on Chr 6 showed a significant reduction in IGF-1 associated with increasing C3H allele number, whereas the Chr 15 and Chr 10 loci showed additive effects with increasing C3H allele number. A genome-wide search for interacting marker pairs identified a significant interaction between the Chr 6 QTL and a locus on Chr 11. This interactive effect suggested that when the Chr 11 locus was homozygous for C3H, there was no effect of the Chr 6 locus on serum IGF-1; however, the combination of C3H alleles on Chr 6 with B6 alleles on Chr 11 was associated with reduced serum IGF-1 concentrations. To test this in vivo, we tested congenic mice carrying the Chr 6 QTL region from C3H on a B6 background (B6.C3H-6). Both serum IGF-1 and femoral BMD were significantly lower in female congenic than progenitor B6 mice. In summary, we identified three major QTLs on mouse Chrs 6, 10, and 15, and noted a major locus-locus interaction between Chrs 6 and 11. We named these QTLs IGF-1 serum levels (Igf1sl1 to Igf1sl4). Functional isolation of the Igf1sl1 QTL on Chr 6 for IGF-1 in B6.C3H-6 congenic mice demonstrated effects on both the IGF-1 and BMD phenotypes. The genetic determinants of these Igf1sl QTLs will provide much insight into the regulation of IGF-1 and the subsequent acquisition of peak bone mass.

New developments in the analysis of gene expression.

An understanding of the relationship between gene expression, protein expression and the influences of genetic responses upon gene function is vital before we can understand the complexity of genomes. Traditional methods for the study of gene expression are limited to studying small groups of genes at a time and a source of pure starting material has been difficult to obtain. Recent technological advances have enabled large numbers of genes, from specific cell populations, to be studied in a single experiment. Laser capture microdissection (LCM) and microarray technology are providing the next revolution in the study of gene expression. LCM-based molecular analysis of histopathological lesions can be applied to any disease process that is accessible through tissue sampling. Examples include: (i) mapping the field of genetic changes associated with oxidative stress; (ii) analysis of gene expression patterns in atherosclerotic tissues, sites of inflammation and Alzheimer's disease plaques; (iii) infectious micro-organism diagnosis; and (iv) typing of cells within disease foci. Microarray hybridisation glass chips spotted with sets of genes can then be used to obtain a molecular fingerprint of gene expression in the microdissected cells. The variation of expressed genes or alterations in the cellular DNA that correlate with a particular disease state can be compared within or between individual samples. The identification of gene expression patterns may provide vital information for the understanding of the disease process and may contribute to diagnostic decisions and therapies tailored to the individual patient. Molecules found to be associated with defined pathological lesions may provide clues about new therapeutic targets in the future.

Physical proximity and functional association of glycoprotein 1balpha and protein-disulfide isomerase on the platelet plasma membrane.

Platelet function is influenced by the platelet thiol-disulfide balance. Platelet activation resulted in 440% increase in surface protein thiol groups. Two proteins that presented free thiol(s) on the activated platelet surface were protein-disulfide isomerase (PDI) and glycoprotein 1balpha (GP1balpha). PDI contains two active site dithiols/disulfides. The active sites of 26% of the PDI on resting platelets was in the dithiol form, compared with 81% in the dithiol form on activated platelets. Similarly, GP1balpha presented one or more free thiols on the activated platelet surface but not on resting platelets. Anti-PDI antibodies increased the dissociation constant for binding of vWF to platelets by approximately 50% and PDI and GP1balpha were sufficiently close on the platelet surface to allow fluorescence resonance energy transfer between chromophores attached to PDI and GP1balpha. Incubation of resting platelets with anti-PDI antibodies followed by activation with thrombin enhanced labeling and binding of monoclonal antibodies to the N-terminal region of GP1balpha on the activated platelet surface. These observations indicated that platelet activation triggered reduction of the active site disulfides of PDI and a conformational change in GP1balpha that resulted in exposure of a free thiol(s).

Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies.

The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1) it immunoprecipitated drug-dependently the former but not the latter glycoprotein complex and (2) the MAIPA assay showed strong rifampicin-dependent antibody binding when anti-GPIb-IX monoclonal antibodies (mAbs) (AK2 and FMC25) but not anti-GPIIb-IIIa mAbs (AP2, SZ21, and SZ22) were used to capture the antigen. The antibody binding site was further localized to the GPIX subunit of the GPIb-IX complex because flow cytometric analysis revealed drug-dependent antibody binding to L cells transfected with human GPIbbeta and GPIX complementary DNA (L betaIX cells) but not with human GPIbalpha and GPIbbeta complementary DNA (L alphabeta cells). Finally, in the MAIPA assay, the rifampicin-dependent antibody almost completely cross-blocked the binding of the anti-GPIX mAb (SZ1) to platelets. Similar cross-blocking of SZ1binding to platelets by the quinine-dependent antibodies was also observed. This finding not only confirms that the epitope of the rifampicin-dependent antibody is on GPIX but it is also identical to or located in close proximity to that of the quinine-dependent antibody and SZ1. Further characterization of the epitopes of these antibodies may have important implications for a general understanding of the mechanism of drug-induced thrombocytopenia. (Blood. 2000;95:1988-1992)

Quinine-dependent antibodies bind a restricted set of epitopes on the glycoprotein Ib-IX complex: characterization of the epitopes.

Severe immune thrombocytopenia is an idiosyncratic complication of quinine therapy. Although in most cases the responsible antibody is directed against platelet membrane glycoprotein (GP) Ib-IX, specificity for GPIIb-IIIa or both epitopes has also been reported. The objective of this study was to characterize the binding site of GPIb-IX-specific quinine-dependent antibodies. Antibody binding to Chinese hamster ovary cells or mouse L cells stably transfected with various combinations of the three genes (Ibalpha, Ibbeta, or IX) that encode this complex was detected using flow cytometry, monoclonal antibody-specific immobilization of platelet antigens assay, and differential adsorption studies. IgG in sera from 15 patients with quinine-induced thrombocytopenia binding to the cells, in the presence of quinine, showed three distinct patterns. Group 1 sera contained at least two antibody populations, one which binds to GPIbalpha and another which recognizes GPIX. Group 2 sera contained an antibody which binds drug dependently to GPIX, and Group 3 sera contained an antibody which recognizes a quinine-dependent epitope on GPIbalpha. Thus, the quinine-dependent antibodies fall into two distinct populations that bind to GPIbalpha and GPIX independently. Using proteases which cleave GPIbalpha at specific sites, we have shown that the GPIbalpha-specific antibody binds to an 11-amino acid (283 to 293) region. Peptide inhibition studies provide confirmatory evidence that this region contains the epitope for the GPIbalpha-specific quinine-dependent antibody.

The platelet proaggregating and potentiating effects of unfractionated heparin, low molecular weight heparin and heparinoid in intensive care patients and healthy controls.

Heparin binds to platelets and can cause platelet proaggregating and potentiating effects, possibly causing thrombocytopenia, particularly in patients in intensive care with hyperaggregable platelets. In this study we compared the platelet proaggregating and potentiating effects of unfractionated heparin (UH), 2 low molecular weight (LMW) heparins, enoxaparin and dalteparin, and a heparinoid, danaparoid sodium (orgaran), to platelets of an ICU patient population and a normal control group. In both populations UH caused platelet aggregation in a dose-dependent manner. This occurred in the therapeutic range of the drug, with as little as 0.5 U/ml UH. The LMW heparins caused less and the heparinoid least platelet aggregation. Generally, the aggregation observed in ICU patients was greater than in the normal population. The potentiating effects of the 4 drugs in association with physiological agonists was examined. Similar patterns of potentiation were observed in both populations, with UH causing significant enhancement of platelet aggregation, the LMW heparins intermediate and heparinoid least enhancement. There was substantial variability in the individuals' platelets' reactions to the drugs, in particular to UH. Our findings suggest that UH has the greatest effect, the low molecular weight heparins an intermediate effect and the heparinoid the least propensity to cause platelet activation.