bluuebunny/crossref_metadata_2025_split huggingface dataset:
SELECTIVE INHIBITION OF HUMAN MEGAKARYOCYTOPOIESIS IN VITRO BY HIGHLY PURIFIED PLATELET FACTOR 4
Authors: Gewirtz A , Xu W Y , Rucinski B , Niewiarowski S 
Published: August 2018

Platelet (plt) factor 4 (PF4) is an alpha granule protein which can modulate T lymphocyte function. T cells may help regulate megakaryocytopoiesis. Therefore, we hypothesized that T cell-PF4 interactions might play a role in autoregulating marrow megakaryocyte (MEG) production. To test this idea, we studied MEG colony formation in plasma clot cultures containing human serum derived solely from pit poor normal AB plasma, enriched hematopoietic progenitor cells (HPC), autologous T cells, and exogenous PF4. Highly purified PF4 (single band on SDS gel) was prepared from outdated human pits by a combination of heparin-agarose, Sephacryl G-200, and Sephadex G-50 column chromatography. HPC were prepared by depleting normal light density marrow mononuclear cells of adherent monocytes, and T cells. T cells were further fractionated into helper (Leu 3+) and suppressor (Leu 2+) subtypes by solid phase immunoabsorption ("panning"). MEG colonies were enumerated by indirect immunofluorescence with an anti-human platelet glycoprotein antiserum. HPC(5×105/ml) were co-cultured with Leu 3+, or Leu 2+ T cells at target;T cell ratios of 2:1 (n=3; n=4 respectively) and l:l(n=4; n=4 respectively) in the presence of 2.5 μg/ml PF4. Under these growth conditions, MEG colony formation was unchanged (p>0.5) when compared to colonies formed by HPC in the absence of PF4. When the above experiments were repeated (n=2-3/condition) at a higher PF4 concentration [25 μg/ml], MEG colony formation was markedly (>60%) inhibited. To determine if PF4 directly inhibited MEG or erythroid progenitor cell growth (CFU-Meg; CFU-E) in vitro, HPC were cloned in PF4 (25μg/ml) without added T cells. Mean ± SEM of MEG and CFU-E derived colonies formed without vs. with PF4 was as follows:These results suggest that: 1) PF4 may be a non-T cell dependent, lineage specific inhibitor of CFU-MEG, and 2) PF4 may play a role in autoregulating human megakaryocytopoiesis.
A MODEL TO EVALUATE BLOOD CELLS BEHAVIOUR TO CELL ACTIVATION: A DIFFERENCE BETWEEN MEN AND WOMEN
Authors: Hansen J B , Wilsgard L , Olsen J O , Østerud B 
Published: August 2018

This study was carried out to see what kind of response the blood cells have to a weak stimuli of lipopolysacc-harides (LPS), a substance that in small quantities may be a small part of the blood system. The parameters tested under this condition were: thromboxane A2 (TxA2) (produced in platelets), prostacyclin (PGI2) produced in white cells (mainly monocytes) and induced thromboplastin synthesis in monocytes.Heparinized blood from 40 men and 40 women was incubated with 2 ng LPS/ml blood for 2 hours. Blood cells were then either spun down to obtain plasma or mononuclear cells were isolated from the incubated blood followed by the quantitation of thromboplastin. In order to get measureable PGI2 production, liposomes of soya lecithin were added to amplify this production in monocytes (see abstract by Østerud et al. "Monocyte stimulation--". The quantitation of TxB2 in the resultant plasma samples- revealed a highly significant difference in production of TxA2 between men and women in this system. In the group of men a value of 13.0 ± 5.9 ng/ml was found compared to 7.6 ± 5.8 for women (p<0.01). The liposomes had no effect on the TxA2 production. In contrast, the PGI2 production in women was higher than in men. By quantitating 6-keto-PG 1α concent rat i on in the plasma samples it was found that women had 148 ± 53 pg/ml whereas men had 105± 5 pg/rnl (p< 0.001). ft low index of TxA2 is supposed to be beneficial and associated with PGI2 low frequence of coronary heart disease. In the present study this value was estimated to be 51 for women and 124 in men.A weak but not significant higher thromboplastin activity was found in the stimulated monocytes of men as compared to women (91.4 ± 40.8×10−3/106 cells for men and 74.9 45.6×10−3/ 106 cells for women).It is concluded that blood cell activation in women is less harmful than in men and this may reflect the lower rate of CHD in women as compared to men.
BINDING SITE OF VITAMIN K-DEPENDENT PROTEIN S ON C4b-BINDING PROTEIN
Authors: Suzuki K , Nishioka J , Kusumoto H , Deyashiki Y 
Published: August 2018

Protein S, a cofactor for activated protein C, reversibly complexes with a regulatory complement component C4b-binding protein (C4bp) in plasma. In plasma of patients with congenital protein S deficiency, most protein S exists as a complex with C4bp, which has no cofactor activity. C4bp (Mw 550,000) is composed of approximately seven subunits with Mw 75,000 which are linked by disulfide bonds near the carboxy1-terminus. We report here about the complex formation between protein S and C4bp particularly on the binding site of protein S on C4bp molecule. Protein S and C4bp were purified from human plasma. Seventeen mouse monoclonal antibodies against C4bp were prepared. Chymotrypsin-digested C4bp was separated on gel filtration into a fragment with Mw 160,000 derived from the carboxyl-terminal core of the intact C4bp and fragments with Mw 48,000 from the amino-terminus. The carboxy1-terminal fragment with Mw 160,000 was found to be composed of approximately seven polypeptides with Mw 25,000, which were linked by disulfide bonds.The experiments using these fragments and the monoclonal antibodies showed that: (1) Protein S bound not only to the intact C4bp, but also to the fragment with Mw 160,000. (2) The fragment with Mw 160,000 inhibited the binding of protein S to C4bp, but the fragment with Mw 48,000 did not. (3) One of the seventeen monoclonal antibodies blocked the inhibition of C4bp on the cofactor activity of protein S. (4) This antibody inhibited C4bp binding to protein S. (5) The antibody bound to the fragment with Mw 160,000. Based on these results, protein S was suggested to lose its cofactor activity for activated protein C by binding to the carboxyl-terminal core of C4bp where seven subunits are linked by disulfide bonds.
RESTRICTION ANALYSIS AND SOUTHERN BLOTTING OF TOTAL HUMAN DNA REVEALS THE EXISTENCE OF MORE THAN ONE GENE HOMOLOGOUS WITH PROTEIN S cDNA
Authors: Ploos van Amstel J K , van der Zanden A L , Reitsma P H , Bertina R M 
Published: August 2018

A deficiency in protein S, the cofactor of activated protein C, is associated with an increased risk for the development of venous thrombosis. It is inherited as an autosomal dominant disorder. To improve the detection of heterozygotes in affected families, we have started to search for restriction fragment length polymorphism (RFLP) in the protein S gene. This study revealed the existence of two genes containing sequences homologous to protein S cDNA.Three non-overlapping fragments of clone pSUL5, which codes for the carboxy-terminal part of protein S and contains the complete 3' untranslated region, were isolated and used as probes in search for RFLP of the protein S gene.Surprisingly the non-overlapping probes shared more than one hybridizing band. The hybridization took place under stringent assay conditions.This observation is contradictory to the intron-exon organization of a gene and suggests the existence of two genes, containing sequences homologous with pSUL5. Both genes could be assigned to chromosome 3 by mapping through somatic cell hybrids. Whether two functional protein S genes are present in the human genome remains to be established.
TWO VON WILLEBRAND FACTOR (vWF) GENE POLYMORPHISMS SEGREGATE WITH VON WILLEBRAND'S DISEASE (vWD) TYPE IIA: ASSIGNMENT OF THE DEFECTIVE GENE LOCUS IN vWD TYPE IIA
Authors: Verweij C L , Quadt R , Briët E , Pannekoek H 
Published: August 2018

Patients with autosomal, dominant von Willebrand's disease (vWD) type IIA display a decreasedristocetin cofactor activity and lack the large and intermediate size von Willebrand factor (vWF) multimers. As yet the cause for this abnormal vWF protein is not known. In this study we determined whether vWD type IIA is due to a mutation in the vWF gene or by a defect in another gene involved, in for example, vWF processing.Restriction fragment-length polymorphisms (RFLP's), using the enzymes BgIII and XbaI in conjunction with human vWF cDNA probes, have been described. Restriction endonuclease analysis of genomic vWF DNA revealed that these genetic marker are located within the vWF gene. The vWF gene was determined to comprise about 160 kb and harbors at least 20 exons. The RFLP's were applied to study the segregation of alleles associated withvWD type IIA in a comprehensive, affected family. It is demonstrated that both RFLP's are completely linked with the vWD type-IIA trait. From this finding, we conclude that the defect, causing the vWD type IIA, is most likely due to a mutation in the vWF gene and not to a mutation in another gene involved in, for example, post-translational processing of the vWF protein.
BUTYRATE SPECIFICALLY STIMULATES TISSUE-TYPE PLASMINOGEN ACTIVATOR SYNTHESIS IN CULTURED HUMAN ENDOTHELIAL CELLS
Authors: Kooistra T , van den Berg J A , Tüns H A M , Platenburg G , Rijken D , van den Berg E A 
Published: August 2018

In a search for compounds that can enhance tissue-type plasminogen activator (t-PA) synthesis in cultured human endothelial cells we found that dibutyryl cyclic AMP (at a concentration of 2 mM) led to a several-fold increase in t-PA production by endothelial cells over a 24 h incubation period. Further researchshowed that this stimulating effectcould be explained by the slow liberation of butyrate, as the effect could be reproduced by addition of free butyrate to the medium, but notby addition of 8-bromo cyclic AMP. With butyrate, an accelerated accumulation of t-PA antigen in the conditioned medium (CM) was observed after a lag period of about 6 h. Increasing amounts of butyrate caused anincreasingly stimulatory effect, reaching a plateau with 5 mM butyrate. The relative increase in t-PAproduction in thempresence of 5 mM butyrate varied among different endothelial cell cultures from 6 to 25-fold in 24 h CM. The butyrate-induced increase in t-PA production wasaccompanied by increased t-PA mRNAlevels.Analysis of radiolabelled CM andcell extracts by SDS-PAGE and autoradiography indicated that the potent action of butyrate is restricted to a limited group of proteins. We found that theaccumulation of PA-inhibitor activity in CM from butyrate-treated cells increased only moderately.In our study of the relationshipbetween structure and stimulatory activity we found that butyrate wasby far the most effective inducer oft-PA synthesis. Shortening or lengthening the carbon chain by one carbon atom decreased the stimulatory effect after 24 h of incubation by 50-70%. Further changes in the length of the carbon chain almost completely suppressed the stimulatory activity. Similarly, alterations of the aliphatic chain by introduction of functional groups, a double bond or a branched structure rendered butyrate ineffective in increasing t-PA synthesis. Thus, a straight-chain C4 monocarboxylate structure with a methyl group at one end and a carboxyl moiety at the other seems to be required for the rather specific induction of t-PA synthesis in cultured human endothelialcells.
PERSISTENT DECREASED FIBRINOLYTIC ACTIVITY IN CYCLOSPORIN A (CyA) TREATED RENAL ALLOGRAFT RECIPIENTS
Authors: Cohen H , Mackie I J , Patel R , Neild G , Machin S J 
Published: August 2018

CyA administration isssociated with histological evidenceof vascular injury. Since vascularendothelial cell components are known to modify fibrinolytic activity, we have examined fibrinolytic changes in renal allograft recipients onCyA and, in addition, markers of vascular endothelial cell damage: plasma vWFRiCof activity and AT-III activity.Parameters of fibrinolytic activity have been assessed serially in 21 renal allograft recipients for 1 year post-transplantation by study of the following: euglobulin clot lysis time (ELT), plasminogen activator activity (PAA) by fibrin plate assay, tissue plasminogen activator (t-PA) by ELISA and fast-acting t-PA inhibitor (t-PAI) by chromogenic assay.From 1 month onwards, fibrinolytic activity (ELTs and fibrin plates) is significantly decreased, p<0.002 and p<0.002 respectively, compared to normal controls, and this decrease persists at oneyear post-transplantation. Mean ELTs (mins) and ranges at 1 and 6 months and 1 year are 363 (120-1740), 598 (135-1610) and 538(80-870) respectively. Mean PAA (% of pooled normal plasma) and ranges at these times are 104 (70-154), 79 (48-118) and 83 (65-108) respectively.In contrast, serial t-PA levels (%of pooled normal plasma) are significantly increased (p<0.02), 428 (86-3686), 297 (81-1130) and 198 ( 44-637 ) at 1 and 6 months and 1 year respectively.These results suggest that the decreased fibrinolytic activity is due to increased levels of t-PA inhibitor, too high to be surpassed by the amount of t-PA released. vWFRiCof activity and AT III activity aresimilarly increased.In conclusion, CyA-treated renal allograft recipients show evidence of chronic vascular endothelial damage and a prothromboticstate.
IN VITRO NEUTROPHIL ACTIVATION BY PLASMA OF STORED PLATELET CONCENTRATES
Authors: Muylle L , Van Brussel C , van Bockstaele D R , Peetermans M E 
Published: August 2018

A previous study showed an effect of storage time of platelet concentrates (PLC) on the frequency and severity of reactions to platelet transfusion.As most of the leukocytes were removed from the PLC prior to transfusion, it was suggested that at least part of the reactions were caused by the transfusion of cell products, accumulated during conservation of the PLC.This idea is supported by the finding of high histamine levels in PLC after 5 days storage.In order to test for the presence of substances causing neutrophil activation, samples from 7 PLC were taken at various storage times and incubated for 30 minutes at 37°C with neutrophilic granulocytes obtained from healthy donors. Binding of a monoclonal antibody specific for a neutrophil-associated membrane glycoprotein (gp 170) was analyzed by indirect immunofluorescence. (CLB 13.9,kindly given by P. Tetteroo).It is known that the expression of gp 170 is enhanced by stimulation of the neutrophil.Fluorescence intensity was quantified by flow cytometry (Ortho cytofluorograf 50-H).Results were expressed as an activation ratio (AR) (ratio of the median fluorescence intensity over that of negative control neutrophils).At day 0 no activation was recorded (AR = 1±0.09,n = 7). In contrast, the supernatant plasma of PLC stored for 7 days caused an elevated AR in 4/7 PLC (AR = 3.20±1.20, n = 4).For comparison the AR of the positive control was 3.66±0.63 (n = 5).In 2 PLC activation was already observed after 1 day storage.The increased expression of gp 170 was not correlated with the histamine level, the lactate dehydrogenase level, the leucocyte or platelet count in the PLC.These preliminary results indicate the presence of factors causing in vitro activation of neutrophils in the supernatant plasma of some stored PLC.Further investigations are needed to confirm these results, to identify the responsible factor(s) and to study the clinical relevance of this finding.
INDIUM-111 PLATELET SURVIVAL STUDIES ON PLATELET CONCENTRATES (PCVSTORED FOR UP TO 14 DAYS
Authors: Holme S , Heaton W A , Hartman P 
Published: August 2018

The effect of extended storage on platelet in vitro and in vivo viability was investigated to determine the reliability and efficacy of Indium-111-oxine radiolabeling of long-term stored platelets. The fitness of various mathematical models for measurement of platelet survival and recovery was also evaluated.33 CPDA-1 PC were prepared and stored for various periods of time (see below) according to standard blood bank methods. The volume of the PC was adjusted to 65-75 mL to prevent pH fall with extended storage. After storage, samples were taken for in vitro assays and for radiolabeling witn Indium-111-oxine using standardized methods. None of the PC had pH<6.3. Afxer reinfusion of the labeled platelets, samples for measurement of radioactivity were taken at three hours and daily for seven days.The uptake {%) of Indium-111 and loss of platelets during the labeling showed no correlation with storage time; the means of the 33 studies were 71±8% and 35±6%, respectively. There was no significant correlation between the parameters and recovery or survival which suggests reliable labeling of nonviable platelets.
Excellent fit of the survival data to the multiple hit gamma model was found forpall studies as determined by the coefficient of determination, r 2. The survival curves of platelets stored for one and five days were quite linear as shown by r2 ; with longer storage periods they became more exponential as shown by an increasingly better fit (r2 ) to the log model.Using the gamma model, both in vivo recovery and survival showed a progressive loss with increasing storage duration. This was less evident with the linear model. The loss of in vivo viability paralleled closely a decrease in the in vitro , parameters of platelet quality (discoid shape, hypotonic shock response, ATP content, and oxygen consumption) during storage.In conclusion, this study indicates that extended storage not only yields platelets with reduced recovery, but also platelets with shortened survival.
COAGUIATION, FIBRINLYSIS AND KALLIKREIN ACTIVATION IN SEVERE INFECTION AND SEPSIS : RELATION TO OUTCOME
Authors: Blamback M , Hesselvik F , Brodin B , Maller R , Gaffney R 
Published: August 2018

Fatal multiple organ failure following severe infection may be related to early activation of protease cascade systems. The study aimed to relate changes in the below mentioned components to shock and outcome. Of 53 patients with severe infection, 30 did not develop shock (group I); 12 survived septic shock (groupII); and 11 died from organ failure after septic shock (groupIII). No patient had overt DIC. During the first 3 days after admission, blood was sampled daily for assay of: platelet count, fibrinogen, prothrombin complex, F XII, F VIIIiC, vWF:Ag, F VII, F V, anti thrombin, protein C, plasminogen, antiplasmin, plasminogen activator inhibitor (PAi), X-oligcmers, D-dimers, prekalli-krein, functional kallikrein inhibition (fKl), and fibronectin, by chramogenic substrate and inmunochemical techniques. The Proenzyme functioned, index (PFI) ves calculated combining the results of anti thrombin, plasminogen, antiplasmin, prekallikrein and fKL (Aasen, Acta Chir Scand 1985; 522: 211).Low (p<.001) initial values for F XII, prothrombin complex, F VII, antithrcmbin, protein C, prekallikrein, and fibronectin were seen in all groups. The shock groups (I-III) had in addition significant decreases in platelet count, antiplasmin, and plasminogen. Fibrinogen, F VIII :C, vWF:Ag, X-oligcmers, and D-dimers were significantly higher than normal in all groups. Shock patients had higher X-oligcmers and D-dimers, but lower fibrinogen than non-shock patients. PAi was within the normal range in survivors (I-II), but was elevated ten-fold and increased progressively over 3 days in the non-survivors. vWF:Ag showed a similar progressive increase in non^survivors; these two variables ware the best early indicators of non-survival. PFI was significantly lower in shock patients (II-III), but did not discern between survivors and non-survivors during days 1-3. The results indicate a marked activation of coagulation in patients with severe infection, with more fibrin formation and fibrinolysis in the shock groups. High vWF:Ag and PAi in non-survivors may indicate nmore endothelial damage, and potentially harmful fibrinolysis inhibition.
COMPARISON OF TO BIOLOGICAL EFFECT, PHARMACOKINETICS AND REPRODUCIBILITY OF INTRANASAL AND INTRAVENOUS ADMINISTRATION OF DDAVP
Authors: Lethagen S , Harris A S , Nilsson I M 
Published: August 2018

DDAVP has previously mostly been given by the parenteral route but the development of intranasal delivery has made it possible for the patients to treat themselves at home without delay. The effect of intranasal administration of DDAVP as drops using a rhinyle catheter or a pipette has been unpredictable. The development of a spray has improved the intranasal administration of DDAVP with well controlled doses and better absorption.In this study we conpared intranasal administration of 300 pg DDAVP by spray, with intravenous administration of 0.2, 0.3 and 0.4 pg DDAVP/kg in 10 healthy volunteers. We measured the effect on VIII:C, VIII:Ag and vW:Ag and also followed plasma levels of DDAVP before and 10' , 30' , 45' , 60' , 90' , 2 h, 4 h, 6 h and 8 h after administration.We also studied the reproducibility of the spray effect. 10 healthy volunteers were tested before and 1 h after the administration of 300 pg DDAVP intranasally by spray on 5 different occasions with an interval of at least 1 week between the tests.The highest response was obtained after 0.3 μg/kg i.v. The response to 0.4 μg/kg i.v. was less than 0.3 μg/kg indicating that maximum stimulation was reached with 0.3 μg/kg. The effect of spray approximates the 0.2 μg/kg response.The reproducibility of the effect of the spray dose on VIII:C was 25% (coefficient of variation) and 33% for the intra-individual and inter-individual variation respectively. This corrpares favourably with the inter-individual variation after intravenous administration of 0.2 pg/kg (30%), 0.3 μg/kg (22%) and 0.4 μg/kg (35%). It was of interest to note that the intra- and inter-individual variation of pre-treatment basal levels of VIII:C was 14% and 17% respectively.Intranasal DDAVP (300 μg) is as effective as 0.2 μg/kg intravenously and provides an accurate, reproducible and convenient alternative to parenteral administration.
IMMUNOASSAY FOR FACTOR VIII-HEAVY CHAIN. AN INDICATOR FOR IMMUNE COMPLEXES DURING HIGH DOSE FVIII INHIBITOR TREATMENT
Authors: Nordfang O , Ezban M , Knudsen J B 
Published: August 2018

Specificity studies have shown that most hemophilia A inhibitor antibodies are directed towards the light chain of coagulation factor VIII (FVIII). Thus, conventional immunoassays for FVIII-antigen (FVIII:Ag) presumably have reactivity for FVIII-Light Chain (FVIII-LC) . Our sandwich FVIII :Ag assay has been shewn to be specific for only FVIII-LC. We have now developed a specific immunoassay for FVIII-Heavy Chain (FVIII-HC) . This has made it possible to investigate the FVIII-HC content in hemophilia A plasma, and to study the expression of FVIII-HC in culture medium frcm transfected cell lines.By adding purified FVIII-LC and FVIII-HC in coagulation inhibition assay, plasma frcm one of seven hemophilia A inhibitor patients was found to be reactive with both FVIII-LC and FVIII-HC. IgG frcm this plasma was used for a FVIII-HC specific inhibition radioimmunoassay. The polyspecific antibodies were coated to microplates with removable wells. The coated wells were incubated with test sample and with purified 125I-FVIII-HC. When normal human plasma pool contains 1 U/ml of FVIII-HC, the sensitivity of the assay was 0.004 U/ml.For normal plasma and plasma frcm non inhibitor hemophilia A patients, FVIII-HC measurements correlated with FVIII:C and FVIII-LC measurements. However, after FVIII injection hemophilia A inhibitor patients in high dose FVIII treatment showed a much higher FVIII-HC content (1-5 U/ml) than FVIII-LC and FVIII:C (< 0.05 U/ml). These patients have previously been shown to have antibodies towards FVIII-LC. Therefore the antigen measurements indicate that inhibitor patients in high dose FVIII treatment have FVIII/anti-FVIII-LC immune complexes. These circulating immune complexes may be the mediator of an antibody dependent immune tolerance, during the high dose FVIII treatment.