Global longitudinal strain: an early marker for cardiotoxicity in patients treated for breast cancer.

BACKGROUND: Patients treated with anthracyclines and trastuzumab are at increased risk of developing heart failure. Early diagnosis and treatment may prevent irreversible left ventricular (LV) dysfunction. This study investigates whether subclinical deterioration of global longitudinal strain (GLS) is a more reliable early predictor for LV dysfunction than three-dimensional (3D) LV ejection fraction (LVEF). METHODS: Adult patients receiving anthracyclines and trastuzumab for breast cancer who had serial echocardiographic follow-up were included in this retrospective study. The primary endpoint was the necessity to temporarily pause chemo- or immunotherapy due to declining LVEF (decline in 3D LVEF of > 10 percentage points to < 53%). Linear mixed-effects models were used to assess the longitudinal evolution of 3D LVEF and GLS over time. RESULTS: Fifty-one women were included, mean age 54 (50.5-57.6) years, with a total of 216 follow-up echocardiograms (mean follow-up 1.1 ± 0.45 years). GLS and 3D LVEF were significantly correlated (Spearman's rho: -0.36, p < 0.001). A decrease in GLS significantly predicted a lower LVEF on the subsequent echocardiogram [ß -0.6, 95% confidence interval (CI) (-1.0 to -0.2), p < 0.006]. Conversely, prior LVEF did not significantly predict GLS on the subsequent echocardiogram [ß -0.04, 95% CI -0.1 to -0.01, p = 0.12]. Nine patients reached the primary endpoint. On average, patients who reached the primary endpoint had a relative decrease of 15% GLS at day 205 and an absolute 10% decrease of LVEF to LVEF < 53% at day 235. DISCUSSION: GLS is able to identify subclinical LV dysfunction earlier than 3D LVEF measurement in women undergoing treatment for breast cancer with anthracyclines followed by trastuzumab.

Small dense LDL cholesterol in human subjects with different chronic inflammatory diseases.

BACKGROUND AND AIMS: Chronic inflammatory diseases (CID) are associated with a profound increase in cardiovascular (CV) risk resulting in reduced life expectancy. However, LDL-cholesterol is reported to be low in CID patients which is referred to as the "LDL paradoxon". The aim of the present study was to investigate whether LDL-particles in CID exhibit an increased content of the highly atherogenic small-dense LDL subfraction (sdLDL). METHODS AND RESULTS: In this prospective, single center, observational study we enrolled 141 patients with CID (RA n = 59, inflammatory bowel disease (IBD) n = 35, ankylosing spondylitis (SpA) n = 25, Psoriasis n = 22) in 2011 through 2013 to evaluate sdLDL levels before as well as 6 and 26 weeks after initiation of different anti-cytokine therapies (anti-TNFα, anti-IL-6R antibodies). sdLDL levels were compared to 141 healthy individuals in a case control design. Compared to healthy controls, all CID patients displayed a significantly higher sdLDL content within the LDL cholesterol fraction: RA 35.0 ± 9.2% (p < 0.001), SpA 42.5 ± 10.5% (p < 0.001), IBD 37.5 ± 7.1% (p < 0.001), Psoriasis 33.6 ± 4.6% (p < 0.01). Furthermore, the sdLDL/LDL ratio was significantly higher in male compared to female RA subjects (p < 0.05). Neither anti-TNFα nor anti-IL6R medication altered sdLDL levels despite a significant improvement of disease activity. CONCLUSION: In several different chronic inflammatory disease entities, LDL-cholesterol is shifted toward a pro-atherogenic phenotype due to an increased sdLDL content which might in part explain the LDL paradoxon. Since premature CV disease is a major burden of affected patients, specifically targeting lipid metabolism should be considered routinely in clinical patient care. CLINICAL TRIALS: Registration at German Clinical Trial Register (DRKS): DRKS00005285.

Pregnancy in women with hypertrophic cardiomyopathy: data from the European Society of Cardiology initiated Registry of Pregnancy and Cardiac disease (ROPAC).

AIMS: We report the maternal and foetal outcomes at birth and after 6 months in a cohort of pregnant women with hypertrophic cardiomyopathy (HCM). Although most women with HCM tolerate pregnancy well, there is an increased risk of obstetric and cardiovascular complications. METHODS AND RESULTS: All pregnant women with HCM entered into the prospective worldwide Registry of Pregnancy and Cardiac disease (ROPAC) were included in this analysis. The primary endpoint was a major adverse cardiovascular event (MACE), which included death, heart failure (HF), thrombo-embolic event, and arrhythmia. Baseline and outcome data were analysed and compared for patients with MACE vs. without MACE and for patients with obstructive HCM vs. non-obstructive HCM. Sixty pregnant women (mean age 30.4 ± 6.0 years) with HCM (41.7% obstructive) were included. No maternal mortality occurred in this cohort. In 14 (23%) patients at least one MACE occurred: 9 (15.0%) HF and 7 (12%) an arrhythmia (6 ventricular and 1 atrial fibrillation). MACE occurred most commonly during the 3rd trimester and postpartum period. In total, 3 (5.0%) women experienced foetal loss. Women with MACE had a higher rate of emergency Caesarean delivery for cardiac reasons (21.4% vs. 0%, P = 0.01). No significant differences in pregnancy outcome were found between women with obstructive and non-obstructive HCM. NYHA functional class of ≥II and signs of HF before pregnancy, were associated with MACE. CONCLUSION: Although most women with HCM tolerated pregnancy well, cardiovascular complications were not uncommon and predicted by pre-pregnancy status facilitating pre-pregnancy counselling and targeted antenatal care.

Insights into the genetic architecture of morphological traits in two passerine bird species.

Knowledge about the underlying genetic architecture of phenotypic traits is needed to understand and predict evolutionary dynamics. The number of causal loci, magnitude of the effects and location in the genome are, however, still largely unknown. Here, we use genome-wide single-nucleotide polymorphism (SNP) data from two large-scale data sets on house sparrows and collared flycatchers to examine the genetic architecture of different morphological traits (tarsus length, wing length, body mass, bill depth, bill length, total and visible badge size and white wing patches). Genomic heritabilities were estimated using relatedness calculated from SNPs. The proportion of variance captured by the SNPs (SNP-based heritability) was lower in house sparrows compared with collared flycatchers, as expected given marker density (6348 SNPs in house sparrows versus 38 689 SNPs in collared flycatchers). Indeed, after downsampling to similar SNP density and sample size, this estimate was no longer markedly different between species. Chromosome-partitioning analyses demonstrated that the proportion of variance explained by each chromosome was significantly positively related to the chromosome size for some traits and, generally, that larger chromosomes tended to explain proportionally more variation than smaller chromosomes. Finally, we found two genome-wide significant associations with very small-effect sizes. One SNP on chromosome 20 was associated with bill length in house sparrows and explained 1.2% of phenotypic variation (VP), and one SNP on chromosome 4 was associated with tarsus length in collared flycatchers (3% of VP). Although we cannot exclude the possibility of undetected large-effect loci, our results indicate a polygenic basis for morphological traits.

Differences and similarities between mothers and fathers of premature children: a qualitative study of parents' coping experiences in a neonatal intensive care unit.

BACKGROUND: The aim of this study was to explore and describe the coping experiences of parents to children admitted to a neonatal unit. METHODS: A qualitative research approach was chosen, using in-depth interviews with eight fathers and eight mothers. RESULTS: The main findings were that parents with previous complicated births had more difficulties in coping compared to those parents with no experience with complications. Coping seemed easier where parents' opinions were heard regarding their baby's care and when both parents were present in the neonatal intensive care unit (NICU). The main similarities between mothers and fathers were the reluctance to speak their opinions on childcare, and both experienced a sense of alienation and problems in bonding with the baby. They also needed a limitation on the number of visitors in the NICU. Differences between mothers and fathers were that fathers tried hard to be the strong partner in the relationship, and were more concerned with the mother if she was seriously ill postpartum, while mothers were more concerned for their baby. Mothers' postpartum period was felt as more stressful if the father was not present, but mothers were also better at welcoming support from the health personnel. CONCLUSION: This study highlights the parent's coping experiences in NICUs. Coping seemed easier where parents' opinions were heard. Nurses in the NICU should take the former experiences of the parents into consideration when nursing in the NICU and planning for discharge.

The wingless-related integration site-5a/secreted frizzled-related protein-5 system is dysregulated in human sepsis.

Sepsis and type 2 diabetes exhibit insulin resistance as a common phenotype. In type 2 diabetes we and others have recently provided evidence that alterations of the proinflammatory wingless-related integration site (wnt)-5a/anti-inflammatory secreted frizzled-related protein (sFRP)-5 system are involved in the pathogenesis of insulin resistance. The aim of the present study was to investigate whether this novel cytokine system is dysregulated in human sepsis, which may indicate a potential mechanism linking inflammation to metabolism. In this single-centre prospective observational study, critically ill adult septic patients were examined and proinflammatory wnt5a and wnt5a inhibitor sFRP5 were measured in serum samples by enzyme-linked immunosorbent assay (ELISA) at admission to the intensive care unit (ICU) and 5 days later. Sixty sepsis patients were included, and 30 healthy individuals served as controls. Wnt5a levels were found to be increased significantly in septic patients compared to healthy controls (2·21 ± 0·33 versus 0·32 ± 0·03 ng/ml, P < 0·0001). In contrast, sFRP5 was not altered significantly in septic patients (19·72 ± 3·06 versus 17·48 ± 6·38 ng/ml, P = 0·07). On admission to the ICU, wnt5a levels exhibited a significant positive correlation with the leucocyte count (rs = 0·3797, P = 0·004). Interestingly, in patients recovering from sepsis, wnt5a levels declined significantly within 5 days (2·17 ± 0·38-1·03 ± 0·28 ng/ml, P < 0·01). In contrast, if sepsis was worsening, wnt5a levels increased in the same time-period by trend (2·34 ± 0·59-3·25 ± 1·02 ng/ml, P > 0·05). sFRP5 levels did not change significantly throughout the study period. The wnt5a/sFRP5 system is altered in human sepsis and might therefore be of interest for future studies on molecular pathophysiology of this common human disease.

Peripartum cardiomyopathy: Euro Observational Research Program.

Peripartum cardiomyopathy is a rare but potentially life-threatening form of heart failure affecting women late in pregnancy or in the first months after delivery. Peripartum cardiomyopathy is difficult to diagnose and its onset and progression are variable between individuals. The pathophysiology remains poorly understood, hence treatment options are limited and possibly harmful to the foetus. Furthermore, geographical incidence varies greatly and little is known about the incidence in Western countries. To gain further understanding of the pathophysiology and incidence of peripartum cardiomyopathy, the European Society of Cardiology initiated a study group to implement a registry. This review provides an overview of current insights into peripartum cardiomyopathy, highlights the need for such a registry and provides information about this Euro Observational Research Program.

Immunochemical evidence for protein abnormalities in platelets from patients with Glanzmann's thrombasthenia and Bernard-Soulier syndrome.

Crossed immunoelectrophoresis of Triton X-100 solubilized proteins from normal and abnormal platelets was performed with rabbit antibodies raised against normal platelets. In Bernard-Soulier platelets protein 13 was not detected, and neither the amphiphilic (probably GP Ib) nor the hydrophilic (glycocalicin) glycocalicin-related proteins were seen when monospecific antiglycocalicin antiserum was used. The most prominent precipitate, 16, and platelet fibrinogen, 24 were not detected in platelets of two patients with type I thrombasthenia, whereas in one patient with type II thrombasthenia fibrinogen was clearly detected, but the amount of protein 16 remained severely reduced. Protein 16 was heavily labeled after lactoperoxidase-catalyzed (125)I iodination of normal platelets, and was precipitated by IgG-L, an alloantibody from a polytransfused thrombasthenic patient. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or protein 16 cut out from immunoplates showed two (125)I-labeled glycoprotein bands, which migrate as GP IIb and GP IIIa. SDS-PAGE of (125)I-labeled type I thrombasthenic platelets showed no periodic acid-Schiff bands or peaks of radioactivity in the GP IIb and GP IIIa regions, whereas in the GP I region both the periodic acid-Schiff band intensity and the radiolabeling were within the normal range. Autoradiography after crossed immunoelectrophoresis of iodinated thrombasthenic platelets showed that the bulk of radioactivity was bound to protein 17. This glycoprotein, which was also present in normal and Bernard-Soulier platelets, migrates in the GP I region on SDS-PAGE. Thus, the bulk of radioactivity observed in the GP I region after SDS-PAGE is associated with protein 17 and not with glycocalicin.

Galactosyl transferase activity in human transitional cell carcinoma lines and in benign and neoplastic human bladder epithelium.

Cultured cells of human transitional cell carcinoma line MGH-U1, in suspension, were assayed for galactosyl transferase by measurement of the transfer of [3H]galactose from uridine diphosphate:[3H]galactose to desialylated ovine submaxillary mucin. The assay was optimized with respect to time and to protein, uridine disphosphate:galactose, desialyated ovine submaxillary mucin, and Triton X-100 concentrations. This assay was then applied to fresh specimens of benign, inflamed, and neoplastic bladder epithelium from 33 patients who under went cold-cup biopsies at cytoscopy. Transitional cell carcinoma specimens gave values in the range of 24.7 to 184.8 cpm [3H]galactose transferred per microgram protein per hr [72.0 +/- 44.7 (S.D.); n = 25]; normal and inflamed specimens ranged from 0.8 to 46.1 cpm/microgram protein per hr [8.3 +/- 8.4 (S.D.); n = 35]. By using a known method of cell rupture, cell ghosts, representing cell-surface membranes, were isolated both from the cultured cell line and from two biopsy specimens of transitional cell carcinoma. Although a complete enzymatic and electron microscopic analysis was not undertaken, the coincidence of an enzyme marker with the cell ghost fraction containing the elevated galactosyl transferase made it appear probable that this enzyme is located in the cell surface.

Effects of thrombin on washed, human platelets: changes in the subcellular fractions.

Pressure homogenization and subcellular fractionation has been performed on washed, human platelets and platelets treated with thrombin to undergo the so-called release reaction. Electron microscopy revealed that the particulate zones obtained from the control sample corresponded to membrane vesicles (B), small storage granules (D) as well as mitochondria and larger storage granules (E). Only a few storage granules could be observed in the particulate zones isolated from thrombin-treated platelets. Visual comparison of the sucrose gradient patterns revealed that one granule fraction (D) had disappeared from the thrombin-treated sample. Sodium dodecysulfate-polyacrylamide gel electrophoresis showed a major protein band (mol. wt 145 500 plus or minus 1000) in the extracellular phase (supernatant after removal of the platelets) of the thrombin-treated sample and in the granule fractions (D and E) of the control (mol. wt 147 000 plus or minus 1000). Incubation of whole, washed platelets with thrombin for 5 min at 37 degrees C followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis of the isolated membrane fraction revealed no reproducible differences in the protein band pattern compared to membranes isolated from control platelets. However, after treatment with thrombin for 30 min, a protein band (mol. wt 183 000 plus or minus 3500) had disappeared. The distribution of protein and beta-N-acetylglucosaminidase activity among the subcellular fractions were measured. Both were mainly recovered in the soluble fraction (greater than 77%). The granule fractions, D and E of the control contained 3.0% plus or minus 0.8% and 6.4% plus or minus 1.3% of the total amount of beta-N-acetylglucosaminidase in the gradient. Fraction E of the thrombin-treated cells contained 3.3% plus or minus 1.0% of total while fraction D was lacking.

Studies on subcellular fractions of human platelets by the lactoperoxidase-iodination technique.

Lactoperoxidase-catalyzed 125I iodination and sodium dodecyl sulphate-polyacrylamide gel electrophoresis have been performed on whole, washed platelets as well as on isolated platelet membranes and granules. Electrophoresis of the whole platelets demonstrated two major radioactive peaks, corresponding to glycopolypeptides of estimated molecular weights of 120 000 and 100 000. A small, but consistent amount of radioactivity was also associated with a 147 000 dalton glycopolypeptide. The membranes showed the same pattern of radioactivity as the whole platelets, whereas only negligible amounts of labeled material was found in the soluble and granule fractions. Practically all the polypeptides were labeled in membranes iodinated after their isolation. A glycopolypeptide of 147 000 molecular weight was observed also in the soluble and the granule fractions, but no radioactivity was associated with these substances. In unreduced form, the granule glycopolypeptide penetrated only slightly into the polyacrylamide gel. Thrombin induced the relase of this granule-located substance from whole platelets, as observed by gel electrophoresis of the supernatant after release reaction (secretion). The granule glycoproteins were only partly exposed on the granule membrane since about 50% of the acid-hydrolyzable sialic acid could be liberated by neuraminidase treatment of isolated granules. In whole, iodinated granules the bulk of the radioactivity was associated with a polypeptide of estimated molecular weight 46 000 (possibly actin). This polypeptide was not seen in the supernatant after removal of the thrombin-degranulated platelets by centrifugation, which indicates that the granule membrane is retained with the platelets during the secretion process.

Characterization of the proteins of isolated human platelet alpha-granules. Evidence for a separate alpha-granule-pool of the glycoproteins IIb and IIIa.

The protein composition of a well-defined alpha-granule preparation isolated from human platelets has been studied. Crossed immunoelectrophoresis against polyspecific platelet antibodies revealed more than 20 immunoprecipitates. The glycoprotein IIb-IIIa complex represented a major antigen in the Triton X-100-solubilized alpha-granule preparation and cross-reacted with the corresponding platelet membrane antigen. Furthermore, after lactoperoxidase-catalyzed 125I-iodination of whole platelets it was not labelled, in contrast to its membrane-located counterpart. This indicates an intracellular location of glycoproteins IIb and IIIa, probably as constituents of the alpha-granules. Fibrinogen, platelet factor 4, albumin, factor VIII-related antigen and the main granule glycoprotein (thrombinsensitive protein, thrombospondin) were identified in the alpha-granule preparation by the crossed immunoelectrophoresis technique. Crossed affinity immunoelectrophoresis using lectins revealed the presence of at least seven glycoproteins, and six sialoglycoproteins were identified by their altered electrophoretic mobility after neuraminidase treatment. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of reduced samples of the alpha-granules revealed at least 15 Coomassie Brilliant Blue-staining polypeptide bands, one of which comigrated with myosin heavy chain. No prominent band was observed in the actin region. Five glycopolypeptide bands were observed after periodic acid-Schiff staining. The dominant three represented the main granule glycoprotein, glycoprotein IIb and glycoprotein IIIa, respectively. More glycoproteins seem to be present in the alpha-granules than was previously recognized.

Platelet glycocalicin. Its membrane association and solubilization in aqueous media.

Glycocalicin has been extracted from human platelets by 3 M KCl and purified using affinity chromatography on columns of Sepharose-coupled wheat germ agglutinin as the most efficient step. Rabbit antiserum to the purified protein agglutinated human platelets and inhibited the agglutination induced by bovine Factor VIII-related protein. Crossed immunoelectrophoresis of Triton X-100 extracts of platelets in Triton X-100-containing agarose revealed the presence of two glycocalicin-related components of different electrophoretic mobilities giving a continuous double-peak immunoprecipitate with this antiserum. The fast-moving component, which represented the minor peak of the immunoprecipitate, corresponded to purified soluble glycocalicin. Crossed hydrophobic interaction immunoelectrophoresis did not demonstrate binding of the purified glycocalicin or the fast-moving component to phenyl-Sepharose CL-4B as hydrophobic matrix. The slow-moving component, which represented the major peak of the immunoprecipitate, showed a strong binding to the hydrophobic matrix. Immunoelectrophoretic quantitation of glycocalicin present in the aqueous media demonstrated that the presence of EDTA, N-ethylmaleimide and iodoacetamide during lysis of platelets significantly reduced the solubilization of glycocalicin. At the same concentrations these inhibitors strongly inhibited the calcium-activated protease of platelet sonicates. Sialic acid determination after acid hydrolysis of aliquots from the soluble fractions showed that their content of sialic acid was considerably higher when lysis was performed in the absence, rather than in the presence, of EDTA and that glycocalicin contributes significantly to the total platelet sialic acid.

Demonstration of 125I-labelled thrombin binding platelet proteins by use of crossed immunoelectrophoresis and autoradiography.

A possible receptor for thrombin on the platelet membrane has been identified. Whole platelets were treated with 125I-labelled thrombin followed by washing of the platelets, solubilization in Triton X-100, crossed immunoelectrophoresis and autoradiography. A heavily labelled antigen which migrated slightly more slowly than albumin was observed. No corresponding arc was seen on the same immunoplate when stained with Coomassie brilliant blue, indicating that the antigen possessed weak antigenic properties and/or was present in very small amounts. When 125I-labelled thrombin that had been inactivated by phenylmethylsulphonyl fluoride was used, no such labelled arc was seen. The radiolabelled immunoprecipitate does not represent any of the antigens identified hitherto in the immunoelectrophoretic patterns obtained with platelets or platelet material. The electrophoretic mobility of the antigen was influenced neither by neuraminidase treatment of the platelets prior to the 125I-labelled thrombin exposure nor by inclusion of concanavalin A, wheat-germ lectin or lentil lectin in the gel during the first-dimension electrophoresis. This suggests that the antigen does not represent a glycoprotein. Upon subcellular fractionation the radioactively labelled arc was observed in the cytosol fraction following crossed immunoelectrophoresis and autoradiography. Analysis of the secreted proteins after induction of the release reaction with 125I-labelled thrombin revealed labelling of immunoprecipitates representing thrombospondin, albumin and the 'line' form of platelet factor 4. This confirms that stable complexes of 125I-labelled thrombin and platelet proteins can exist in the presence of Triton X-100 and during electrophoresis.

Evidence for release of soluble, but not of membrane-integrated, proteins from human platelet alpha-granules.

Proteins released from stimulated platelets were compared to those of a well-defined preparation of alpha-granules and the soluble cytoplasm by crossed immunoelectrophoresis. Nearly all releasable proteins were detected in the alpha-granule, whereas the true proteins of the soluble cytoplasm were not released. The released glycoproteins interacted with lectins similarly to their alpha-granula-located counterparts. The alpha-granules were divided into soluble contents and membranes by ultrasonication followed by ultracentrifugation. The proteins of the soluble content corresponded to those released from the stimulated platelets. This observation was also supported by SDS-polyacrylamide gel electrophoresis. The results indicate that the bulk of the proteins released from stimulated platelets originate from the soluble content of the alpha-granules. Two major alpha-granule antigens as well as the myosin heavy chain were not released and recovered in the alpha-granule membrane. These results support the hypothetical exocytosis mechanism for the release of alpha-granule proteins from platelets.

Dissociation of the glycoprotein IIb-IIIa complex in isolated human platelet membranes. Dependence of pH divalent cations.

The platelet membrane glycoproteins IIb and IIIa normally exist as a complex which forms a predominant immunoprecipitate after crossed immunoelectrophoresis of Triton-X-100-solubilized platelets. Dissociation of the complex occurs by solubilization in the presence of EDTA or EGTA at pH 8.7 and is readily verified by crossed immunoelectrophoresis. Incubations of isolated membranes with EDTA or EGTA at various pH levels were performed. Removal of the chelators and solubilization showed no dissociation of the glycoprotein IIb-IIIa complex in membranes incubated at pH below 8.0. At pH above 8.0 a dissociation which increased with increasing pH was seen. Under these conditions, dissociation appears to take place already in the intact membranes. The tendency of the glycoprotein IIb-IIIa complex to become dissociated with EDTA or EGTA at increasing pH seems to be due to increased chelating capacity of the chelators concomitant with a decreased chelating capacity of glycoprotein IIb and IIIa. The divalent cations Ca2+ and Mg2+, but not Cu2+, Zn2+, Mn2+ or Sr2+, in molar concentrations below that of EGTA were able to prevent the dissociation of the glycoprotein IIb-IIIa complex by the chelator at pH 9.0, indicating that Ca2+ as well as Mg2+ can be used to keep the complex together. In some experiments it was possible to reverse the dissociation in the membranes after removal of EDTA. At pH 7.5 reassociation occurred within 15 min whether divalent cations were added or not. At pH 9.0. reassociation occurred within 2 h provided Ca2+ was present. The tendency of glycoprotein IIb and IIIa to form a complex thus appeared to be most pronounced over the physiological pH range and to be a rapid process in platelet membranes under such conditions.

A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

Demonstration of a new glycoprotein Ib-related component in platelet extracts prepared in the presence of leupeptin.

The water-soluble protein glycocalicin is generated during platelet lysis by a proteolytic attack on the integral membrane glycoprotein GP Ib. However, only small amounts of glycocalicin are formed when platelets are solubilized by 1% Triton X-100. Crossed immunoelectrophoresis of such extracts using an antiserum to glycocalicin, shows a continuous immunoprecipitate consisting of two peaks, one representing glycocalicin and the other GP Ib. When leupeptin was present during solubilization, subsequent immunoelectrophoresis revealed yet another GP Ib-related component represented by a third, slow-migrating peak of the immunoprecipitate. During incubation of platelets with dibucaine followed by solubilization in the presence of leupeptin, a gradual transformation of this new form of GP Ib into the previously defined one took place prior to the formation of glycocalicin. An increase followed by a decrease in the agglutination response of the platelets to bovine von Willebrand factor occurred concomitant with these transformations. SDS-polyacrylamide gel electrophoresis of Triton X-100 extracts of platelets did not reveal any difference in the size of GP Ib whether or not leupeptin had been present during the solubilization.

Electroimmunochemical characterization of endothelial cell proteins: antigenic relationship with platelet and erythrocyte membrane proteins.

Human endothelial cells isolated from umbilical cords and cultured in primary cultures were solubilized in Triton X-100 and examined by crossed immunoelectrophoresis using rabbit antiserum against endothelial cells. Endogeneous labelling of the endothelial cell proteins with 35S-methionine or 14C-mannose followed by crossed immunoelectrophoresis and autoradiography revealed about 30 or 8 immunoprecipitates, respectively. Antigenic relationship between endothelial cell proteins and proteins in human platelets or erythrocyte membranes was demonstrated by use of the corresponding antisera and by antigen addition experiments. One of the endothelial cell proteins cross-reacted with antiserum against erythrocyte membranes and showed a partial antigenic identity reaction with the band 3 protein complex of erythrocyte membranes. The same protein showed antigenic relationship also with a platelet protein. In addition, endothelial cells contain at least 7 proteins antigenically related to platelet proteins, of which at least 5 were labelled with 14C-mannose and thus were glycoproteins. Three of these glycoproteins were antigenically related to proteins from isolated platelet membranes and three were related to the release products obtained after thrombin treatment of platelets. The present study demonstrated numerous platelet and endothelial cell proteins that were antigenically related, more than previously anticipated.

Further studies on the interaction between thrombin and GP Ib using crossed immunoelectrophoresis. Effect of thrombin inhibitors.

The platelet surface protein GP Ib (glycocalicin-related protein) has been shown to be retarded by thrombin-Sepharose 4B in a crossed immunoelectrophoresis system. The interaction between GP Ib and thrombin was abolished when thrombin was blocked either at the active serine site with tosyl-lysine-chloromethyl-ketone (TLCK) or phenylmethylsulfonylfluoride (PMSF) or at the fibrinogen binding site (macromolecular binding site) with N-bromosuccinimide (NBS) or heparin, indicating that both sites have to be freely accessible for the retention of the glycocalicin-related protein by thrombin.