bluuebunny/crossref_metadata_2025 huggingface dataset:
Impact of clumping on core-collapse supernova radiation
Authors: Dessart L. , Hillier D. J. , Wilk K. D.
Published: August 2018

There is both observational and theoretical evidence that the ejecta of core-collapse supernovae (SNe) are structured. Rather than being smooth and homogeneous, the material is made of over-dense and under-dense regions of distinct composition. Here, we have explored the effect of clumping on the SN radiation during the photospheric phase using 1D non-local thermodynamic equilibrium radiative transfer and an ejecta model arising from a blue-supergiant explosion (yielding a Type II-peculiar SN). Neglecting chemical segregation, we adopted a velocity-dependent volume-filling factor approach that assumes that the clumps are small but does not change the column density along any sightline. We find that clumping boosts the recombination rate in the photospheric layers, leading to a faster recession of the photosphere, an increase in bolometric luminosity, and a reddening of the SN colors through enhanced blanketing. The SN bolometric light curve peaks earlier and transitions faster to the nebular phase. On the rise to maximum, the strongest luminosity contrast between our clumped and smooth models is obtained at the epoch when the photosphere has receded to ejecta layers where the clumping factor is only 0.5 – this clumping factor may be larger in nature. Clumping is seen to have a similar influence in a Type II-Plateau SN model. As we neglected both porosity and chemical segregation, our models underestimate the true impact of clumping. These results warrant further study of the influence of clumping on the observables of other SN types during the photospheric phase.
Möglichkeiten zur Optimierung der Implantatstabilität bei osteoporotischen Frakturen der Wirbelsäule
Authors: von der Höh Nicolas , Spiegl Ulrich-J. , Jarvers Jan-Sven , Völker Anna , Josten Christoph , Heyde Christoph-Eckhard
Published: August 2018

Versorgungspflichtige osteoporotische Wirbelkörperfrakturen stellen heutzutage mehr denn je eine Herausforderung an Behandler und Gesellschaft dar. In dieser Übersichtsarbeit werden Lösungsansätze und Möglichkeiten zur Optimierung der Implantatstabilität bei osteoporotischen Frakturen aufgezeigt und mit einigen Beispielen illustriert.
Advances in Understanding the Immune Imbalance between T-Lymphocyte Subsets and NK Cells in Recurrent Spontaneous Abortion
Authors: Zhao Xiaoxuan , Jiang Yuepeng , Wang Lin , Li Zhihao , Li Qiang , Feng Xiaoling
Published: August 2018

Recurrent spontaneous abortion is a global problem, and unexplained recurrent abortion triggered by immunological factors is an important focus of current research. Helper T lymphocytes (Th cells) and regulatory T lymphocytes (Treg cells) are central in human immune regulation and play a complex role in pregnancy. Natural killer cells (NK cells) exist in the endometrium and cooperate with T lymphocytes to create immune tolerance at the maternal-fetal interface, which is essential for successful pregnancy. This review has analyzed studies on Th17 cell, Treg cell and NK cell dysfunction and cellular imbalances which may contribute to unexplained recurrent spontaneous abortion to suggest a possible direction for future immunotherapies.
Endometriosis and Beta-hCG > 775 IU/l Increase the Risk of Non-tube-preserving Surgery for Tubal Pregnancy
Authors: Nicolaus Kristin , Jimenez-Cruz Jorge , Bräuer Dominik , Lehmann Thomas , Mothes Anke , Runnebaum Ingo
Published: August 2018

Introduction Tubal pregnancy is the most clinically relevant form of ectopic pregnancy. Surgery consisting of laparoscopic salpingotomy is the therapeutic gold standard. This study looked at risk factors for non-tube-preserving surgery. The aim was to determine a cut-off value for beta-hCG levels, which could be used to predict the extent of tubal surgery.

Materials and Method 97 patients with tubal pregnancy who underwent primary salpingotomy in the Department of Gynecology and Obstetrics of Jena University Hospital between 2010 and 2016 were retrospectively analyzed. A prior medical history of risk factors such as adnexitis, ectopic pregnancy, tubal surgery, treatment for infertility and intrauterine pessary was included in the analysis. The study population was divided into two subgroups: (1) a group which underwent laparoscopic linear salpingotomy, and (2) a group which had laparoscopic partial tubal resection or salpingectomy. Risk factors for salpingectomy were determined using binary logistic regression analysis. Statistical analysis was done using SPSS, version 24.0, to identify risk factors for non-tube-preserving surgery.

Results 68 patients (70.1%) underwent laparoscopic salpingotomy and 29 patients (29.9%) had laparoscopic salpingectomy. The two groups differed with regard to age (p = 0.01) but not with regard to the parameters ‘gestational age’, ‘viability and rupture status of the ectopic pregnancy’ or ‘symptoms at presentation’. Patients who were known to have endometriosis prior to surgery or who were diagnosed with endometriosis intraoperatively were more likely to undergo salpingectomy (OR: 3.28; 95% CI: 0.9 – 10.8; p = 0.05). Calculated mean beta-hCG levels were higher in the salpingectomy group compared to the group who had tube-preserving salpingotomy (3277.8 IU/l vs. 9338.3 IU/l, p = 0.01). A cut-off beta-hCG value of 775 IU/l prior to surgery was predictive for salpingectomy with a true positive rate of 86.2% and increased the probability that salpingectomy would be necessary (OR: 5.23; 95% CI: 0.229 – 0.471; p = 0.005).

Conclusion Endometriosis and a beta-hCG value of more than 775 IU/l significantly increased the risk for non-tube-preserving surgery in women with tubal pregnancy.
MEASUREMENT OF CYTOPLASMIC [MG++] IN PLATELETS BY NMR AND NULL-POINT TITRATION WITH ATOMIC ABSORPTION SPECTROSCOPY
Authors: Smith M , Ware J A , Fossel E T , Salzman E W
Published: August 2018

Magnesium is a cofactor for phosphatases, kinases, and synthetases in all cells, and thus the cytoplasmic [Mg++] influences most intracellular processes. The concentration of cytoplasmic free Mg++ varies considerably among different cell types and has been measured in platelets, as most techniques for [Mg++] measurement are suitable only for cells large enough to microinject. Incorrect estimates of this value could markedly affect many Intracellular investigations, including calibration of platelet [Ca++] with aequorin and other Ca++ -sensitive probes. We measured cytoplasmic free [Mg++] by two methods in washed, gel-filtered human platelets suspended in modified Tyrode's buffer: NMR spectroscopy of intracellular inorganic phosphates in intact platelets, and a modified null-point titration method in platelets selectively permeabilized with digitonin, using atomic absorption spectroscopy. The phosphorus-31 NMR spectra were obtained at 145.7-5Mhz and the cytoplasmic [Mg++] was calculated from the chemical shift values of ATP resonances; platelet free Mg++ was found to vary between 0.05 and 0.15mM, depending on the Kd used for Mg-ATP in the calculation. The null-point titration method gave a range of 0.1 to 0.35 mM for platelet cytoplasmic Mg++. Although these values are considerably lower than those reported in lymphocytes (ImM) they are consistent with those reported in erythrocytes (0.1-0.3mM) and hepatocytes (0.35mM) by similar methods. As an example of the importance of these determinations, the new [Mg++] values were used to construct a [Ca++] calibration curve for aequorin that proved to indicate lower resting and stimulated [Ca++] values than those obtained from previous curves based on assumed values for [Mg++] of 1.25 mM (by analogy with lymphocytes). This finding largely resolves the discrepancy between resting [Ca++] as determined by aequorin or by fura-2 and indo-1.
CALPAIN-MEDIATED CYTOSKELETAL DEGRADATION CORRELATES WITH STIMULATION OF PLATELET PROCOAGULANT ACTIVITY
Authors: verhallen P F J , Bevers E M , Comfurius P , Linkskens W M A , Zwaal R F A
Published: August 2018

We have shown earlier that the negatively charged phospholipid phosphatidylserine (PS), which becomes translocated from the inner surface to the outer surface of the plasma membrane upon platelet activation, is responsible for platelet procoagulant activity. Studies with erythrocytes have suggested a role for cytoskeletal proteins in the regulation of transmembrane asymmetry of PS. The possibility that platelet cytoskeletal proteins are involved in the loss of transmembrane asymmetry of PS, was explored by correlative investigations of both platelet prooagulant activity and activity of calpain, an endogenous Ca 2+ -dependent thiol-protease, known to hydrolyze major cytoskeletal proteins (e.g.: filamin, talin, myosin). Platelet procoagulant activity was assayed by determination of the prothrombinase activity under conditions at which the catalytic PS-surface was rate-limiting. Calpain-activity was monitored by the appearance of known degradation products of major cytoskeletal proteins. The following results were obtained: (1) The ability of thrombin, collagen, collagen & thrombin, or the Ca -ionophore A23187 to stimulate platelet procoagulant activity closely correlated with their ability to stimulate platelet calpain-activity (2). Generation of platelet procoagulant activity upon platelet stimulation by collagen & thrombin or by A23187 exhibited a time course identical to the development of calpain-activity. In addition, the local anesthetics dibucaine and tetracaine, shown to gradually stimulate calpain activity, were able to generate platelet procoagulant activity with a similar time course. (3) Using a Ca2+ buffering system and A23187 to equilibrate intracellular- and extracellular free Ca2+ , it was found that the Ca2+ -response relationship of both platelet calpain- and pro-coagulant-activity was identical. From these findings we conclude that the degradation of cytoskeletal proteins destroys their putative interactions with PS, enabling this lipid to participate in transbilayer movement, leading to the formation of a procoagulant outer surface of the platelet.
ROLE OF THE HIGH-AFFINITY PENTASACCHARIDE IN HEPARIN ACCELERATION OF ANTITHROMBIN III INHIBITION OF THROMBIN AND FACTOR Xa
Authors: Olson Steven T , Bjork Ingemar , Craig Paul A , Shore Joseph D , Choay Jean
Published: August 2018

The high-affinity heparin pentasaccharide (H5) and an 8000 Mr high-affinity heparin (H26) have been compared with respect to their interaction with antithrombin III (AT) and their accelerating effect on AT inhibition of thrombin (T) and factor Xa by rapid kinetic and equilibrium binding studies at pH 7.4, 25°C. Kds of .068 μM at I 0.15 and 0.57 μM at I 0.3 were determined for tne AT-H5 interaction, which were 5 and 2.5-fold weaker, respectively, than affinities determined for H26. Comparison of the kinetics of binding of H5 and H26 to AT at I 0.15 under pseudofirst order conditions ([H]o>> [AT]o) demonstrated a saturable dependence of the observed rate constant for both reaction with indistinguishable limiting rate constants of 700 +/-120 s-1 and 520 +/-90 s-1 , but somewhat different Kds for the initial binding interaction of 20 and 29 μM for H5 and H26, respectively. These results indicate that H5 induces the same conformational change in AT as the larger heparin, but that the rate of reversal of this conformational change is greater for H5 which is the basis for its weaker AT affinity. Bimolecular rate constants for neutralization of factor Xa and thrombin by AT-H5 and AT-H26 complexes were determined by p-aminobenzamidine displacement under pseudo-first order conditions([AT-H] >> [T]o or [Xa]o). I-in-dependent values of .62 μM-1 s-1 were obtained for Xa inhibition by AT-H5 at I 0.15 and 0.3, compared to I-dependent values of 1.4 and 0.91 μM-1 s-1 for AT-H26. For thrombin inhibition by AT-H5, and I-independent enhancement of 1.6-fold in the bimolecular rate constant from .0098 to .016 μM-1 s-1 was observed, in sharp contrast to the marked I-independent enhancement by AT-H26 of the bimolecular rate constant ranging from 4000 to 200-fold at I 0.15 and 0.3, respectively. These results are consistent with a primary ionic strength-independent contribution of the AT conformational change to heparin enhancement of factor Xa but not thrombin neutralization by AT, with an ionic strength-dependent component for both reactions, compatible with a differential role for a protease-heparin interaction. Supported by grant HL-30237
VON WILLEBRAND FACTOR RELEASED FROM WEIBEL-PALADE BODIES BINDS MORE AVIDLY TO EXTRACELLULAR MATRIX THAN THAT SECRETED CONSTITUTIVELY
Authors: Sporn L A , Marder V J , Wagner D D
Published: August 2018

Large multimers of von Willebrand factor (vWf) are released from the Weibel-Palade bodies (WPB) of cultured endothelial cells following treatment with a secretagogue, whereas predominantly dimeric forms are secreted constitutively. These two pools of vWf were used to compare binding of the various multimeric forms of vWf to the extracellular matrix (ECM), the in vitro model of the basement membrane. The released multimers and an equal number of subunits of constitutively secreted vWf were placed, for 72 hours, on cultures of human foreskin fibroblasts (HFF) grown on glass coverslips, then fixed and stained by fluorescence using anti-vWf antiserum. Constitutively secreted vWf produced only a trace of matrix decoration, whereas the released large multimers bound more extensively. In order to determine if increased binding of released vWf was due to the presence of another component in the releasate, releasate from which vWf was adsorbed was combined with constitutively secreted vWf, and this mixture was overlaid onto HFF. The presence of the adsorbed releasate did not promote binding of constitutively secreted vWf. Therefore, it appears that the enhanced binding observed was due to the large multimeric size of vWf stored in the WPB. To further substantiate this, iodinated plasma vWf which was presumably constitutively secreted from endothelial cells was overlaid on.to HFF for 72 hours, labeled vWf was removed, and cells were washed extensively and lysed. Samples of iodinated plasma vWf (starting material) and cell lysates were e1ectrophoresed, non-reduced on an agarose gel. Densitometric scans of starting material and of bound vWf revealed that the large multimeric forms bound preferentially. It appears that multivalency is likely an important property in vWf interaction with the ECM, just as has been shown for vWf interaction with platelets. The pool of vWf contained within the WPBs, therefore, is not only especially suited for platelet interaction, but also for interaction with the ECM.
MAPPING OF FUNCTIONAL DOMAINS OF HUMAN HIGH MOLECULAR WEIGHT (HMW) AND LOW MOLECULAR WEIGHT (LMW) KININOGENS BY USING MURINE MONOCLONAL ANTIBODIES (MAbs)
Authors: Ishiguro H , Higashiyama S , Namikawa C , Ohkubo I , Sasaki M
Published: August 2018

It has been widely known that HMW and LMW kininogens are the large potential sources of kinin in human blood, and that HMW kininogen also functions as a cofactor in the contact activation of blood coagulation. Recently, it has been demonstrated that the heavy chains of kininogens strongly inhibited a number of cysteine proteinases such as calpains, cathepsins, papain and ficin. We made an attempt at mapping of functional domains on the molecules of both kininogens by using MAbs.

Thirty four MAbs raised against human HMW and LMW kininogens were screened by ELISA. By using HMW kininogen, kinin-free HMW kininogen, kinin and fragment 1.2 (fr 1.2)-free HMW kininogen, LMW kininogen, kinin-free LMW kininogen, heavy chain of LMW kininogen and light chains of both kininogens, the MAbs were characterized and.classified into four groups; [1] 20 MAbs reacted with heavy chain, a common region of HMW and LMW kininogens. These MAbs possessed the specificity for domain 1 (2 MAbs), domain 2 (2 MAbs), domain 3 (7 MAbs), and both domains 2 and 3 (7 MAbs) of the heavy chain; [2] 7 MAbs recognized the fr 1.2, a unique histidine-rich region; [3] 5 MAbs reacted with the light chain of HMW kininogen; [4] 2 MAbs recognized the light chain of LMW kininogen.

Two MAbs, designated HKG H7 and H12, effectively inhibited the cysteine proteinase inhibitor activity of HMW and LMW kininogens and the others did not affect it. Further, the MAbs, which recognized the fr 1.2 or light chain of HMW kininogen, suppressed the clotting activity. Especially, 2 MAbs, named HKG L2 and L5, effectively suppressed the clotting activity of HMW kininogen. The former, which neutralized about 70% of the clotting activity, reacted specifically with fr 1.2 region of HMW kininogen, and the latter, which neutralized more than 90% of it, recognized the light chain of HMW kininogen. In the results of competition ELISA, fr 1.2 specific MAbs could be classified into 5 kinds of MAbs for recognition sites, and the light chain (HMW kininogen)-specific MAbs also could be classified into 3 kinds of MAbs. Further, 2 light chain (LMW kininogen)-specific MAbs were thought to recognize an identical antigenic site.
HUMAN VASCULAR SMOOTH MUSCLE CELLS (HVSMC) PRODUCE PLASMINOGEN ACTIVATOR INHIBITOR 1 (PAI-1) AND PROTEASE-NEXIN (PN)
Authors: Laug Walter E
Published: August 2018

The arteriosclerotic lesion is characterized by myointimal proliferation. The pathological growth of SMC may alter the biological function of the overlaying endothelial cells.

Thus we have found that HVSMC produce large amounts of plasminogen activator inhibitor (PAI) activities, which neutralize the PA activities elaborated by endothelial cells. Most ( 95%) of the PA activities detectable by reverse fibrin autography bind to heparin affigel at low salt concentration (0.05 m NaCl). Two peaks with PAI activities are eluted with a linear NaCl gradient from 0.05 m to 1.0 m. The first peak eluted at NaCl concentrations of 0.05 m to 0.3 m while the second peak was found in the range of 0.5 m to 0.7 m NaCl. Both PAI had an approximate molecular weight of 45,000 to 50,000 as estimated by SDS PAGE followed by either reverse fibrin autography or silver stain. The pooled fractions of the first peak did not bind 125I-thrombin while the protein of the second peak formed SDS resistant complexes with it like PN.

The pooled fractions of the first PAI peak were further purified by SDS gel electrophoresis followed by electro-blotting onto glass fiber paper. Partial amino acid microsequencing demonstrated homology with the amino acid sequence of PAI-1. In addition, positive reaction with specific antiserum to PAI-1 was demonstrated in Western blots. Oligonucleotide probes were synthesized from the amino acid sequences obtained and PAI-1 gene probes were isolated from a human placenta cDNA library. Subsequently PAI-1 gene expression in HVSMC was demonstrated with Northern blots.

These studies show that PAI-1 binds to heparin affigel at low salt concentrations which facilitates considerably its purification. In addition, the production of PAI-1 and PN by HVSMC may be of importance for the pathogenesis of thrombotic complications in arteriosclerotic vessels.
ISOLATION OF cDNA FOR TYPE-2 PLASMINOGEN ACTIVATOR INHIBITOR
Authors: Ny T , Hansson L , Åstedt B
Published: August 2018

The placental type plasminogen activator inhibitor (PAI-2) has been purified from extracts of human placenta and from a histiocytic lymphoma cell line. It is mainly an uPA inhibitor but it also inhibits the two-chain form of tPA.

In order to determine the factors regulating PAI-2 gene expression and thereby clarify the physiological role of PAI-2 we have undertaken the molecular cloning of PAI-2 cDNA. A λgt11 expression library prepared from placental mRNA, was screened, immunologically using a monoclonal antibody probe developed against PAI-2 purified from human placenta. When 1.7×105 recombinant phages were screened six positive clones were obtained. Hybridization experiments and comparison of restriction enzyme cleavege pattern revealed that the DNA inserts of the six clones were, related. To identify the clones as coding for PAI-2, a lysogen made from one of them was induced, and the proteins were separated by SDS-PAGE. In immuno-blotting wxperiments the recombinant fusion protein and purified PAI-2 were recognized by the monoclonal antibody and a monospecific polyclonal antibody against PAI-2, revealing an immunological similarity. The nucleotide sequence of the largest cDNA was determined. It was found to code for a protein with extensive sequence homology with members of the serine protease inhibitor family (serpins) Alignment of the active center region with other serpins indicates that PAI-2 is an arg-serpin, as expected for an inhibitor of plasminogen activators.
EVIDENCE FOR A PLASMINOGEN ACTIVATOR INHIBITOR BINDING PROTEIN IN PLASMA
Authors: Wiman B , Carlsson T , Chmielewska J
Published: August 2018

For several years it has been known that plasminogen activator inhibitor in plasma behaves as a high molecular weight compound on gelfiltration, in spite of that the molecular weight is only 50,000 in the presence of sodium dodecylsul-phate. The reason for this has so far been unknown. On gelfiltration of plasma, to which purified latent PAI from HT 1080 cells was added, the PAI antigen gel-filtered as a 50,000 Mr protein. However, if the latent form of PAI was reactivated by guanidinium chloride prior to the gel-filtra-tion experiment, an apparent molecular weight of about 250.000 for PAI antigen and activity was observed. If more than 10,000 U of PAI activity was added/mL of normal human plasma, excess PAI occurred at 50,000 Mr on gel-filtration. Human normalplasma was subjected to gel-filtration on sepha-cryl S-300 or Sepharose 6B and the fractions were checked for capacity to transform low Mr functional PAI to high Mr functional PAI. This capacity was only found in the 150 - 200,000 Mr region of the chromatogram. These data suggest that human plasma contains a protein that binds active forms of PAI. The complex of this protein and PAI could be dissociated by gelfiltration in the presence of 3 mol/L guanidinium chloride or 0.1 % (w/v) sodium dodecylsulphate. The physiological or pathophysiological role of the PAI-binding protein is not known. Work with purification of the protein is in progress. Considerable purification have so far been obtained by precipitation with polyethylenglycol 6000 (0-6%), gel-filtration on Sephacry1 S-300, followed by affinity chromatography on heparin-Sepharose.
INTRACELLULAR REGULATION OF TRANSGLUTAMINASE IN INTACT AND H202 INJURED CLONED BOVINE ENDOTHELIAL CELLS
Authors: Korner G , Bjornsson Thorir D
Published: August 2018

Vascular endothelial cells are normally in a quiescent, nonproliferating state. After intimal injury, they are likely to undergo proliferation. We found that cloned bovine aortic endothelial cells (EC) in culture contain high activity of tissue-type transglutaminase (TG). The enzyme was Ca++ dependent, the Km and vmax for putrescine were 0.203 and 18.5 nmol/min/mg protein. Primary amines were capable of inhibiting its activity but not methylated dansylcadaverine. EC and TG molecular weight estimated by gel filtration or by SDS-PAGE, was 88±5,000 Kd. Immunologically it was cross-reactive with purified rat liver TG. TG activity and antigen were found to increase significantly, when the cultures reached confluence and even more when cultures arrested at G0/G1 state by serum depletion. The half-life of EC-TG in G0/G1 arrested cultures was 128 minutes, determined in the presence cycloheximide; no decrease was observed in confluent cultures, indicating active accumulation of TG at the non-pro-liferative state. Most of cellular TG activity was found in the 15,000 g soluble fraction, with the crude membrane fraction containing 4-22% of the total TG activity, depending on the state of cell proliferation. However, immunoblots revealed that the membrane fraction contained similar amounts of TG antigen as did the soluble fraction. When the membranes were treated with detergents (0.1% SDS, 0.5 M NaCl, 75 mM KSCN, 75 mM dithio-threitol), the activity increased significantly, resulting in as much as 4- to 7-fold increases above control. Similar treatment of the soluble fraction did not increase TG activity. This data strongly suggests that the cell membranes contain a latent or cryptic form of TG that can undergo activation. Indeed, when cultures were injured by exposing them to H2O2 (10-10 mM) , a rapid 75-200% increase in TG activity was observed. One hour later, the activity was at control level. If protein synthesis inhibitors were present, no decrease was noted up to 8 hours. It is proposed that significant amounts of TG is stored in an inactive form in endothelial cell membranes, and that under different conditions, e.g., cell injury, activation and translocation of the enzyme can occur, out of and back into the cells membrane.
ACTIVATED HUMAN PLATELETS AND NEUTROPHILS COOPERATE FOR THE FORMATION OF PAF-ACETHER (PLATELET-ACTIVATING FACTOR)
Authors: COEFFIER e , Delautier D , Le Couedic J-P , Chinqnard M , Benveniste J
Published: August 2018

Interactions between platelets and neutrophils have been reported for the production of several mediators of inflammation such as hydrogen peroxides or leukotrienes. Another potential mediator of thrombosis and inflammation is paf-acether which is synthesized by activated platelets and neutrophils.Since platelets form and release large amounts of the paf-acether precursor lyso paf-acether, platelets and neutrophils cooperation for paf-acether biosynthesis was investigated. Purified human neutrophils (4 × 106 /ml) stimulated by opsonized zymosan (ZC, 1 mg/ml) formed 4.5 ± 2.5 ng/ml paf-acether. Human washed platelets (3 × 108 /ml) stimulated with thrombin (l IU/ml) formed 0.60 ± 0.43 ng/ml paf-acether. Platelets and neutrophils, incubated together and both stimulated by their specific agonist, formed more than twice as much paf-acether as platelets and neutrophils separately (10.90 ± 4.25 ng/ml, n = 6, p < 0.001). The formation of lyso paf-acether and the release of lysozyme and LDH were unchanged under the cooperation conditions. The formation of paf-acether almost doubled(10.24 ± 8.79 ng/ml paf-acether versus 5.30 ± 5.23, p < 0.05, n = 4) when ZC-stimulated neutrophils were incubated with supernatants from thrombin-stimulated platelets as well as with synthetic lyso paf-acether. Extracted and purified lyso paf-acether from thrombin-stimulated platelets led to increase of biosynthesis of paf-acether by neutrophils (13.86 ± 3.92 ng/ml paf-acether versus 5.76 ± 0.66, p < 0.05, n = 3). These results indicate that cooperation between platelets and neutrophils is likely to occur via acetylation by neutrophils of excess lyso paf-acether originating from platelets. Thus neutrophils can increase their biosynthesis of paf-acether by acetylating lyso paf-acether either synthetic or released from other cells.
HEREDITARY HYPOFIBRINOGENEMIA WITH HEPATIC STORAGE OF FIBRINOGEN
Authors: Calles F , de Vos R , del Bono R , Tortora O , Albertini A , Henschen A , Desmet V J
Published: August 2018

An Italian family suffering from a novel hereditary disease, i.e. hypofihrinogenemia connected with hepatic storage of fibrinogen, has been identified. A similar disorder has previously been described in two German families. The discovery originated from the finding of a massive hepatic storage of fibrinogen in a liver biopsy specimen from a 65 years old female who during a laparotomy unexpectedly turned out to display cirrhosis. Identification of the stored material as fibrinogen was carried out by specific immunostaining applied on both light and electron-microscopic level. The stored fibrinogen appeared in the form of eosinophilic, pale, irregularly shaped inclusions within the cytoplasm of the hepatocytes. E.M. showed that the inclusions correspond to dilated cisternae of the RER filled by densely packed, curved, tubular structures arranged in a fingerprint-like fashion. Similar features of fibrinogen storage were not detected in any of 500 consecutive liver biopsy specimens examined for this purpose by appropriate techniques. Plasma fibrinogen (thrombin clottable fibrinogen) was found to be 35 mg/dl (n.v. 100-300 mg/dl), and remained persistently very low (20-1-0 mg/dl) over a follow-up period of two years. The immunoreactive fibrinogen was found to be two-three times as high as the clottable. The patient showed no clinical defect of hemostasis or impaired wound healing. Eight out of 19 family members also had persistently low plasma fibrinogen levels (U5-80 mg/dl) over the same follow-up period. Hypofibrinogenemia with hepatic storage of fibrinogen seems to represent a further endoplasmic reticulum storage disease in addition to a-1-antitrypsin deficiency, the plasma deficiency being due to defective secretion of the protein from the hepatocytes. A genetically determined molecular abnormality may be responsible for the defective intracellular transport and subsequent storage at the primary site of protein synthesis, the RER. Hepatic storage of fibrinogen is likely to predispose to development of liver cirrhosis, but neither to defective hemostasis nor impaired wound healing.
THE DYNAMIC STRUCTURE OF FIBRINOGEN PROBED BY SURFACE LABELLING AND CHEMICAL CROSS-LINKING
Authors: Apap-Bologna Angela , Webster Ailsa , Raitt Fiona , Kemp Graham
Published: August 2018

There is much controversy regarding the conformation of fibrinogen. Several models have been proposed ranging from an almost linear trinodular arrangement to a globular conformation. Consequently, it has been suggested that fibrinogen has a flexible structure where the actual conformation is influenced by its environment - one major factor being calcium concentration. Although the importance of tightly bound calcium ions (Kd ∼ luM) to fibrinogen structure is well established, the role of the larger number of low affinity sites (Kd∼lmM) is still a matter of debate.

We have utilised the techniques of radio-active photoaffinity surface labelling and chemical cross-linking to probe the molecule's conformation under different conditions. Studies were carried out in an attempt to provide information on: (1) The relative locations of the major domains within the fibrinogen molecule (2) The regions of the chains which are exposed on the surface (3) The dependence of the conformation on the solvent composition with particular reference to the effect of calcium concentration. Our results indicate that the central, or E domain of the molecule is partially buried and that the conformation of fibrinogen is certainly influenced by changes in solvent composition. Increasing calcium concentration in the millimolar range results in an increase in the proportion of intermolecular cross-linking, mainly through the [A]α chains. There have been several reports that the C-terminal regions of the [A]α chains are in close association, forming a fourth domain within the molecule. Our results suggest that calcium ions promote the dissociation of this domain.
DIRECTED MUTAGENESIS OF HUMAN FIBRINOGEN: Aα CHAIN SUBSTITUTIONS THAT ALTER THROMBIN CLEAVAGE AND ANTIBODY RECOGNITION
Authors: Lord S T
Published: August 2018

The initial event in fibrin clot formation is the thrombin catalized cleavage of the Aa chain of fibrinogen between Argl6 and Glyl7, releasing fibrinopeptide A. Previous data indicate that most of the information required for thrombin recognition and cleavage of the Aa chain lies within the amino terminal 51 residue CNBr fragment. In order to use protein engineering techniques to study the interaction of thrombin with the Aa chain, we have constructed a plasmid expression vector which encodes a tripartite protein consisting of amino acids 1-50 of the Aa chain of human fibrinogen followed by 60 amino acids of chicken collagen, and the beta-galactosidase protein from Escherichia coli. The codons for an initiator methionine and amino acids 1-50 were assembled from 7 oligonucleotides. Protein blot analysis of bacterial lysates of cells induced to synthesize this tribrid protein show a single band (MW = 125,000) crossreactive with a monoclonal antibody, Y-18, which recognizes the Aa chain of fibrinogen but not the products of thrombin cleavage. When these lysates are incubated with thrombin, fibrinopeptide A is released as demonstrated both by protein blot analysis and radioimmunoassay. By including one heterogeneous oligonucleotide in the assembly process, we have constructed plasmids which encode specific amino acid substitutions within residues 1-23. One of these substitutions, Glyl4 to val, significantly alters both cleavage by thrombin and recognition by Y-18. Substitution of ilu for Arg23 alters neither thrombin cleavage nor monoclonal recognition while substitution of leu for Argl6 alters thrombin cleavage, but not recognition by Y-18.
DEMONSTRATION OF Cl-INHIBITOR COMPLEXES IN PLASMA BY HIGHLY SENSITIVE RADIOIMMUNOASSAYS USING A MONOCLONAL ANTIBODY AGAINST A NEODETERMINANT ON COMPLEXED Cl-INHIBITOR
Authors: Nuijens J H , Huijbregets C C M , Thijs L G , Hack C E
Published: August 2018

Levels of factor XIIa- and kallikrein-Cl inhibitor (Cl-Inh) complexes in plasma reflect activation of the contact system in vivo. Here, we report the development of radioimmunoassays (RIAs) for these complexes using a monoclonal antibody (mAb K0K12) that reacts with a neodeterminant exposed on Cl-Inh after interaction with proteases. mAb K0K12 was obtained by a fusion experiment with spleen cells of a mouse hyperimmunized with Cl-Inh complexes.

Experiments with purified Cl-Inh incubated with either Cls or elastase revealed that the determinant for mAb KOK12 is exposed on complexed as well as proteolytically inactivated (modified) Cl-Inh.

Radioimmunoassays (RIAs) for the detection of factor Xlla-Cl-Inh and kallikrein-Cl-Inh complexes were performed as follows: mAb K0K12 was coupled to Sepharose and incubated with the sample to be tested. Binding of Cl-Inh complexes was detected by a subsequent incubation with 125I-antibodies against factor XII or (pre)kallikrein.

With these RIAs, activation of 0.1% of factor XII or prekal-likrein in plasma is easily detected.

Optimal conditions for blood sampling and processing were established, i.e. conditions that prevented any in vitro activation of factor XII and prekallikrein. Levels of factor XIIa-Cl-Inh and kallikrein-Cl-Inh complexes in plasma samples from normal donors were less than 0.1 U/ml (100 U/ml is the maximal amount of Cl-Inh complexes generated in pooled plasma by DXS). Considerably higher, and fluctuating levels were observed in patients with diseases such as septicaemia. These highly sensitive RIAs will facilitate studies concerning the role of the contact system in human pathophysiology.
INTACT MICROTUBULES ARE NECESSARY FOR COMPLETE PROCESSING, STORAGE AND REGULATED SECRETION OF VON WILLEBRAND FACTOR BY ENDOTHELIAL CELLS
Authors: Sinha S , Wagner D D
Published: August 2018

The importance of intact microtubules in the processing, storage and regulated secretion of von Willebrand factor (vWf) from Weibel-Palade bodies in endothelial cells was investigated. Human umbilical vein endothelial cells treated for one hour with colchicine (10-6M) or nocodozole (10-6M) lost their organized microtubular network. Stimulation of these cells with secretagogues (A23187, thrombin) produced only 30% release of vWf in comparison to control cells containing intact microtubules. The nocodazole treatment was reversible. One hour incubation in the absence of the drug was sufficient for microtubules to reform and to restore the full capacity of the cells to release vWf.

Long-term incubation (24 hours) of endothelial cells with microtubule destabilizing agents had a profound effect on vWf distribution. In control cells vWf was localized to organelles in the perinuclear region (i.e. endoplasmic reticulum and Golgi apparatus) and to Weibel-Palade bodies. In drug-treated cells vWf staining was dispersed throughout the cytoplasm and Weibel-Palade bodies were absent. The vWf synthesized in the absence of microtubules contained significantly less large multimers than that produced by control cells. This was not due to possible side effects of the drugs on the cells because the presence of lumicolchicine, an inactive analogue of colchicine, had no effect on vWf processing. Since Weibel-Palade bodies specifically contain the large multimers (Sporn et al, Cell 46:185-190, 1986), we hypothesize that the structural defect in vWf secreted by cells in the absence of microtubules is due to the lack of Weibel-Palade bodies in these cultures.

In summary, the intact microtubular cytoskeleton in the endothelial cells in culture, appeared to be crucial for normal release of Weibel-Palade bodies after stimulation with secretagogues, for reformation of new Weibel-Palade bodies and for the efficient intracellular multimerization of vWf dimeric molecules.
THE ROLE OF GPIb IN PLATELET ACTIVATION
Authors: Wyler B , Clemetson K J
Published: August 2018

Platelet membrane glycoprotein lb (GPIb) is known to contain a receptor for von Willebrand factor (vWf) and for thrombin. The role of GPIb in platelet activation was investigated by comparing the effect of removal of the binding sites by specific proteolysis with that of blocking them using specific antibodies. Specific removal of the 45 kDa outer part of GPIb by treatment of platelets with human leukocyte elastase caused loss of platelet agglutination response to von Willebrand factor (vWf) but also a weaker activation by thrombin similar to that found with Bernard-Soulier syndrome platelets which lack GPIb. Removal of the major part of the external region of the GPIba chain, including the 45 kDa domain, by calcium activated protease or by chymotrypsin, produced similar effects. The 45 kDa region of GPIba was purified by tryptic cleavage of isolated glycocalicin followed by gel filtration. Antibodies to this fragment were produced in rabbits and showed high specificity and affinity for the 45 kDa polypeptide. Fab fragments of IgG prepared from this anti serum affect platelet response to vWf and thrombin in a dose-dependent way. In both protease-treated and antibody-treated platelets the reduced response to thrombin, but not to vWf could be overcome by increasing the dose of thrombin. In contrast to removal of the 45 kDa domain, antibody treatment affected not only the platelet response to vWf and thrombin but also to collagen, ADP, PAF and arachidonic acid. Only the response to the calcium ionophore A23,187 was unaltered.

Though not the essential receptor GPIb is clearly involved in platelet response to thrombin. A possible mechanism could involve a conformational change in the cytoplasmic/inter-membranous part of GPIb induced by binding of thrombin to the 45 kDa domain. Binding of antibodies might affect platelet activation by other agonists via a general down regulation effect. These results support the two receptor models for thrombin activation of platelets as proposed for other cell -types. In platelets the first receptor appears to be GPIb but the second receptor has not yet been identified.
CHARACTERIZATION OF THE VON WILLEBRAND FACTOR-BINDING DOMAIN OF PLATELET MEMBRANE GLYCOPROTEIN Ib
Authors: Handa M , Titani K , Takio K , Ruggeri Z M
Published: August 2018

We have previously obtained immunochemical evidence that the von Willebrand factor (vWF)-binding domain of the platelet membrane glycoprotein (GP) Ib is located near the amino terminus of the a subunit (Journal of Biological Chemistry 261: 12579-12585, 1986). We have now determined the complete amino acid sequence of the 45 kDa tryptic fragment of glycocalicin that contains this domain. Purified glycocalicin was subjected to limited digestion with trypsin and the proteolytic fragments were separated by size-exclusion high-pressure liquid chromatography. Two fragments of 45 kDa and 84 kDa, respectively, were obtained under nonreducing conditions. After reduction and S-carboxymethylation, the 84 kDa fragment was unchanged, while the 45 kDa fragment yielded two new fragments, one of 35 kDa and the other of 7 kDa. This finding proves the existence of a trypsin cleavage site within a disulfide loop. Two primary sets of overlapping fragments were obtained by cleavage of the carboxymethylated protein at methionyl and lysyl bonds following treatment with cyanogen bromide and Achromobacter protease I, respectively. Additional fragments were obtained by treatment of glycocalicin with Staphylococcus aureus V8 protease and Serratia marcescens protease. Analysis of all these fragments provided data that allowed determination of the sequence of the amino terminal 299 residues of the GP Ib a-chain. This includes the 45 kDa tryptic fragment containing the vWF-binding domain. This 299-residue sequence, corresponding approximately to two thirds of the α-chain polypeptide, is largely hydrophobic and contains only two N-linked and one O-linked carbohydrate chains. A hydrophilic region exists between residues 215-299, with a cluster of ten negatively charged residues at 269-287. This area is likely to attract positively charged molecules. The hydrophilic, highly glycosylated (at Ser/Thr residues) region corresponding to the previously described "macroglycopeptide" begins at residue 292. The determined sequence of glycocalicin contains a region with seven repeats, indicative of gene duplication, and is highly homologous to human leucine-rich α2-glycoprotein.
EFFECT OF CACL2 ON ANTI-Xa ACTIVITIES OF HEPARIN AND LMW HEPARINS
Authors: Barrowcliffe T W , Le Shirley Yvonne
Published: August 2018

Assays of anti-Xa activity of heparin are generally carried out using plasma or AT III, in the absence of Ca++ ions. We studied the effect of physiological (3 mM) Ca++ on the relative anti-Xa activities of heparin and LMW heparin, using human AT III, bovine Xa and S-2222.

Potencies of a LMW heparin standard vs. the International Standard (IS) for unfractionated heparin (UEH) were decreased by 45% in the presence of CaCl2. This was shown to be due to a differential effect of CaCl2 on the two substances, with a 93% potentiation in activity of the UEH standard, and only 7% increase for the LMW standard. Addition of CaCl2 to 8 other LMtf heparins gave increases in anti-Xa activity ranging from 0 to 60%. Studies on heparin fractions ranging from 4,000 to 30,000 M.Wt., prepared by gel filtration, showed that a minimum M.Wt. of > 6,000 is required for CaCl2 potentiation of anti-Xa activity.

In kinetic studies, the influence of UEH on second order rate constants was increased by 55% in the presence of CaCl2. However, CaCl2 had no effect on the rate enhancement by three high-affinity oligosaccharides, of 5, 10 and 20 saccharide units.

These studies show that the effect of CaCl2 on the anti-Xa activity of heparin is highly dependent on M.Wt. Anti-Xa activities of LMW heparins determined in the absence of CaCl2 could be misleading.
INFLUENCE OF THE SUBENDOTHELLAL BASEMENT MEMBRANE (BM) COMPONENTS ON FLBRLN ASSEMBLY: EVLDENCE FOR A FLBRLNOGEN BLNDLNG SLTE ON TYPE IV COLLAGEN
Authors: Jones M R , Gabriel D A
Published: August 2018

Effective repair of vascular injury requires assembly of a secure fibrin patch at the injury site. A secure fibrin patch must be anchored to the injury site, else it becomes an embolus. Using enzyme linked immunoassay (ELISA), we have evaluated the binding of fibrinogen (F) to single as well as combinations of the subendothelial BM components including laminin (L), type IV collagen (C), and dermatan sulfate (D). Tests were performed using plates coated over night at either 4°C or 37°C for one hour with individual BM or combinations or BM components. Results indicate that F strongly binds to both D and type IV collagen. Binding of F to L was approximately 50% compared to C or D. Combinations of C, L and D were studied with mole ratios of L and D normalized to C. Thus, when the mole ratio of L was increased from 0.1 to 10 relative to C, the amount of F bound to the plate coated with C and L decreased by 30%. When both D and L were added to C and the binding of F to the coated plate evaluated, increasing the mole ratio of D from 1 to 10 relative to C and L (C:D:L=1:1:0-10), increased the binding of F two fold. If the mole ratio of L was increased by a factor of 10 compare to C, and D varied form 0 to a 10 mole excess compared to C, (C:L:D=1:10:0-10) the increase in F binding was only increased by 1/3. Therefore, L decreases the binding of F by C, while D strongly increases the binding of F. These data suggest that an important interaction between the endothelial basement membrane and fibrin exists and the mole ratio between CLD is critical in determining the extent of the interaction. The interaction between F and C,L,D may underlie the mechanism by which a fibrin clot is anchored and stabilized to the injury site.
ASPIRIN ACETYLATES FIBRINOGEN AND ENHANCES FIBRINOLYSIS IN VIVO. FIBRINOLYTIC EFFECT IS INDEPENDENT OF CHANGES IN PLASMINOGEN ACTIVATOR LEVELS
Authors: Thorir D , Bjornsson M D , Berger Henry
Published: August 2018

In addition to its antiplatelet effect, aspirin has been reported to have fibrinolytic and hypoprothrombinémie effects. The objective of this study was to investigate possible mechanisms underlying the enhanced fibrinolysis observed after aspirin. Five healthy subjects received 650 mg of aspirin ql2hr for five days. Blood samples were collected before aspirin (control) and immediately before (0 hr) and two hours after (2 hr) the last dose for determinations of clot lysis time, time course of thrombin-induced fibrin aggregation, tissue plasminogen activator (tPA), intrinsic pathway fibrinolytic activity (IPFA), plasminogen, fibrinogen, aspirin and salicylic acid, and the coagulation tests activated partial thromboplastin time, thrombin time and prothrombin time. Clot lysis time was shorter after aspirin, control: 9.1±12.4 min (mean±s.d.), 0 hr: 4.6±4.0 min, 2 hr: 5.7±6.2 min (p:0.04), and the fibrin aggregation curves showed increased turbidity (expressed as AUC over 10 min), control: 72.7±17.8 mnumin, 0 hr: 94.6±1.6 mnumin, 2 hr: 112.8±45.1 mnumin (p:0.02). Control values of tPA (0.11±0.04 IU/ml), IPFA (2.20±0.59 IU/ml), plasminogen (10.9±1.0 mg/dl), fibrinogen (288±37 mg/dl), and the coagulation tests were not differnet from those after aspirin. Plasma aspirin concentrations were below detection limits at 0 hr and averaged 1.63±0.97μg/ml at 2 hr. In vitro studies using fibrinogen-free plasma and added acetylated fibrinogen sfiowed an inverse relationship between the extent of acétylation and clot lysis time. Studies using 14C-acetyl-labeled aspirin and fibrinogen showed that fibrinogen is acetylated to form ε-N-acetyl-lysine on both D and E domains of the molecule (50.6±2.0 and 49.4±2.0%, respectively) and on α β and γchains of the molecule (34.4±1.6, 30.7±3.4 and 34.9±4.7%, respectively), with preferential acétylation on the E domain. On the average, 2.88±1.49 ε-N-acetyl-lysyl residues were formed on each fibrinogen molecule. These results suggest that N-acetylation of lysyl residues of fibrinogen is responsible for the increased susceptibility of fibrin clots to lysis after aspirin.