bluuebunny/crossref_metadata_2025_split huggingface dataset:
PSEUDO-THROMBOCYTOPENIA ASSOCIATED WITH AN ANTICOAGULANT INDEPENDENT IgM PLATELET AGGLUTININ IN A PATIENT WITH PROLONGED BLEEDING TIME
Authors: Horellou M H , Capelle C , Lecompte T , Kaplan C , Lecrubier C , James J M , Le Menn R , Muller J Y , Samama M 
Published: August 2018

An original observation of platelet agglutination was found in a 63 y.o. man during 5 years following, without spontaneous bleeding. Low platelet counts (10 × 109/liter) and large agglutinates were observed on blood specimens collected into EDTA (1 to 15 mg/ml whole blood WB), citrate (1/10 volume citrate 0.11 M), sodium heparin (100 units/ml WB), citrate plus acetyl salicylic acid (5 to 100 mg/ml WB), citrate plus prostacyclin (Upjohn 10−5 to 10−6 M)? and buffering anticoagulant (0.8 M citrate, pH 4.65). Low platelet count and large agglutinates were also observed in capillary blood obtained by finger puncture kept at 22° or 37°C. All techniques revealed significant clumping making assessment of overall platelet number impossible. Bleeding time Ivy horizontal incision is longer than 20 minutes.Patient's serum induced normal platelets agglutination up to a 1/512 dilution at 22°and 37°C temperature. Lack of agglutination after treatment of the serum by IgM and indirect immunofluorescence tests identified the IgM nature of the agglutinating factor. This antibody reacted with Glanzmann thrombasthenia platelets ruling out the IIb/IIIa complex as the target of this agglutinin.There was no evidence of platelet activation following agglutination of autologous or alldgenic platelets : electronic microscopy of native blood platelets clumps did not show any sign of activation, and plasma level of B-thromboglobulin was normal in this patient. Normal platelets agglutination by patient's serum was not accompanied with ATP secretion (luciferin-luciferase).This IgM agglutinin was associated with elevation of IgM (700 mg/dl) without clinical or biological signs of auto-immune disease.No similar case with irreversible platelet agglutination in both capillary or venous blood has been reported before. The significance of the observation remains obscure.
CARRIER DETECTION AND PRENATAL DIAGNOSIS IN HAEMOPHILIA A BY GENE ANALYSIS
Authors: Moodie P , Peake I R , Liddell M B , Bloom A L 
Published: August 2018

Restriction fragment length polymorphism (RFLP) analysis has been used to perform family studies, including prenatal diagnosis, in 21 haemophilia A kindred.Two intragenomic RFLPs were studied in conjunction with one linked RFLP. The intragenomic BgII RFLP,situation 3' to exon 26 was detected with cDNA probe C (Genetics Institute) giving bands of 20kb (17% of X chromosomes) and 5kb (83%), and the intragenomic Bell RFLP, situated 3' to exon 18, was detected with the genomic DNA probe pi 14.12 from Genentech. The frequency of this RFLP in the local population was 23% (1.1 kb allele) and 77% (0.88kb allele). The linked probe DXS15 (DX13) was used to detect a Bglll RFLPwith alleles of 5.8kb (45%) and 2.8kkb (55%). A recombination rate of approximately 5% has been estimated between the factor VIII and DXS15 lociCarrier studies were performed in 15 kindreds. 25 obligate carriers were identified and of these, 20 were potentially informative (heterozygous and phase known) for at least 1 RFLP (9 for Bgll, 9 for Bell and 7 for BgIII). 34 possible carriers were studied, of which 13 were diagnosed as normal (6 by BgII, 6 by Bell and 5 by BgIII). 17 were diagnosed as carriers (2 by Bgll, 12 by Bell and 10 by Bglll) and diagnosis was not possible in a further 4 cases. Of these diagnosed as carriers 3 were non-informative for all RFLPs, and 14informative for at least one RFLP (3 by Bgll, 8 by Bell and 8 with BgIII).Prenatal diagnosis was attempted by analysis of DNA extracted by chorionic villussampling in 6 cases of male fetuses at risk of havinghaemophilia A. 1 fetus was diagnosed as being affected (Bell) and was electively terminated. Three otherfetuses were diagnosed as normal by the BgIII/DXS15 RFLP, but the two intragenomic RFLPs were non-informative. Because of the possibility of a crossover allthree patients opted for mid-trimester fetoscopy andmeasurement of fetal factor VIII at Kings College Hospital, London (Dr Reuben Mibashan), where the diagnoses were confirmed. In the 4th case a normal fetus was diagnosed by the Bgll RFLP analysis, but a spontaneous abortion at 12 weeks prevented confirmation of this result. In the final case of twin male fetuses, none of the RFLPs was informative and both were diagnosed as normal by fetal blood sampling at fetoscopy.
RECURRENT MUTATIONS AND AN UNUSUAL DELETION IN HEMOPHILIA A
Authors: Youssoufiän H , Patel A , Phillips D , Kazazian H H , Antonarakis S E 
Published: August 2018

We have identified 15 mutations of the factor VIII (F8) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects byrestriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22, and exon 24; a single CG to TG transition was identified in exon 23; and a CG to CA transition was identified in exon 24. In addition, a Taq I site alteration in intron 4 was identified in a patient with mild hemophilia, which arose dg. S23&in a grandpaternal germ cell. Cloning and sequencing of this region suggests the generation of a newsplice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. Six different deletions were also characterized. In one family, the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.Of the 15 mutations, 6 occurred de novo within 2 generations: 4 in males and 2 in females. In these djg.novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man.
MODULATION OF ANTITHROMBIN III ACTIVITY AND ANTITHROMBIN III-THROMBIN COMPLEXES BINDING TO CULTURED CELLS BY MONOCLONAL ANTIBODIES AGAINST ANTITHROMBIN III
Authors: Knoller S , Savion N 
Published: August 2018

Two monoclonal antibodies (mAb's) against antithrombin III (ATIII) were characterized with respect to their ability to interfere with ATIII activity. AT III activity was measured by its ability to inhibit the amidolitic activity of thrombin on the substrate BCP-100. Incubation of 150 ng of ATIII with 28pg mAb A36R2 prior to addition of 50 ng thrombin totally abolishes the inhibitory effect of ATIII on thrombin. Incubation of 200ng of ATIII with 10 μg of mAb B26R4 prior to addition of 75 ng thrombin raises the inhibitory effects of ATIII from 37% to 100%. We examined the effect of these mAb's on binding of antithrombin III-thrombin (ATIII-Th) complexes to bovine corneal endothelial cells. 120 pg/ml mAb's are reacted with 2 μg/ml ATIII-Th complexes prior to their addition to the cells. mAb A36R2 completely blocks ATIII-Th complexes binding. In contrast, mAb B26R4 enhances binding up to 250% of the control binding.We conclude that mAb A36R2 prevents binding of thrombin to ATIII by recognizing an epitope on ATIII close to thrombin binding site or that its binding to ATIII induces a conformational change in the thrombin binding site thus it no longer recognizes thrombin. mAb B26R4 has a heparin-like effect on ATIII: Its binding to ATIII induces conformational changes which improve thrombin binding to ATIII. There is a correlation between inhibition and enhancement of thrombin binding to ATIII and of ATIII-Th complexes binding to cells by the two mAb's. These mAb's may provide a new tool to control the activity of ATIII and to identify the cellular binding site on the ATIII-Th complex.This research was supported by a grant from the National Council for Research and Development, Israel and G.S.F. München, Germany.
MECHANISM OF PLASMINOGEN ACTIVATION AT LOW TEMPERATURE IN PLASMA SAMPLES CONTAINING THERAPEUTIC CONCENTRATIONS OF t-PA
Authors: Rijken D C , Seifried E , Barrett-Bergshoeff M M , Kluft C 
Published: August 2018

It is known that plasminogen activation in blood samples taken during thrombolytic therapy with tissue-type plasminogen activator (t-PA) may continue during plasma handling, leading to artificially low fibrinogen (Fbg) and α2-antiplasmin (AP) values. Addition of D-Phe-Pro-Arg-CH2Cl or quenching antibodies against t-PA prevents this phenomenon, but these additions do not allow measurement of t-PA activity. The question of this study is, why the in vitro effects occur, even during freezing of the samples. Normal plasma was supplemented with various amounts of two-chain melanoma or recombinant t-PA and stored at -20°C, with and without a prior snap-freeze procedure. AP consumption (chromo-genic substrate assay) and Fbg degradation (Clauss method), measured in thawed samples, were most pronounced in the non snap-frozen samples. As it took a relatively long time before these samples were really frozen, the time course of the effects was studied at different temperatures. Plasma samples containing 1000 IU t-PA per ml were incubated at 37, 25,10, 0 and -8°C between 0 and 120 min. AP reduction was most rapid at 37°C (50% after 13 min), was less at 25°C (50% after 30 min), but did not further decrease at lower temperatures. The AP reduction at temperatures between 25 and -8°C corresponded to the effect of 40% t-PA activity at 37°C. The Fbg values gave a similar picture: the most rapid reduction occurred at 37°C, a slower reduction at 25°C, but no further reduction (even a small increase) was found from 25 to -8°C. The experiments were repeated in a purified system, consisting of t-PA, plasminogen, Fbg and AP. In contrast to the plasma system, AP reduction gradually decreased from 37 to 0°C. The apparent t-PA activity at 0°C was 6% of the activity at 37 °C.It is concluded that the in vitro effects in plasma samples containing t-PA can be, at least partially, explained by an abnormally strong plasminogen activation around 0°C. A normal temperature dependency in the purified system strongly suggests that unknown plasma factors enhance plasminogen activation at low temperatures.
Thrombin stimulation of human platelets:Phosphoinositides as the only source of the diacylglycerol moiety in phosphatidic acid
Authors: Tysnes O-B , Verhoeven AJ M , Holmsen H 
Published: August 2018

Recent reports on various cell types including platelets have concluded in the heterogenous production of phosphatidic acid (PA) during the stimulus-response coupling. Human platelets were pulse-labelled simultaneously with [32P] P. and [32H]glycerol. Extracts were analyzed for masses and radioactivities of ATP and phosphoinositides. When the cells were stimulated with low concentrations of thrombin, the production of [32P] PA was evident without measurable production of [3H] PA. At higher doses of the agonist,[3H] PA was formed, but distinctly later than [32P] PA.This suggested a heterogenous production of PA upon thrombin stimulation of platelets. The specific H-radioactivity of PA in unstimulated cells was about 50% of that of the phosphoinositides. Upon l80 sec of stimulation with 0.5 U/ml of thrombin, the specific [3H] PA radioactivity increased to the level of the phosphoinositides which remained constant during platelet activation. Since other phospholipids incorporate [3H] glycerol much slower than the phosphoinositides, these latter remain the only possible source of the diacylglycerol moiety of PA. The specific 32P-radioactivity of PA in unstimulated cells was only 4% of that of α-ATP and similar to the specific 32P-radioactivity of phosphatidylinositol in unstimulated platelets. After Jyijin of stimulation with 0.5 U/ml of thrombin, specific [32P] PA was similar to that of γ-ATP. The descrepancy in [32P] Pi. and [3H] glycerol incorporation into PA upon thrombin stimulation of platelets is therefore mainly due to a thrombin-induced shift inspecific 32P-radioactivity in PA.
WHOLE BLOOD AGGREGOMETER IN THE ASSESSMENT OF PLATELET HYPER-AGGREGABILITY
Authors: Abbate R , Boddi M , Favilla S , Costanzo G , Paniccia R , Paniccia G F 
Published: August 2018

The aim of this study has been to investigate the reliability of platelet aggregation in whole blood in some clinical conditions associated to thromboembolic complications.18 healthy subjects, 15 patients affected by ischemic heart disease (IHD) and 15 patients affected by insulin independent diabetes, free of vascular complications, were studied. Collagen induced (2.5 mg/L f.c.) platelet aggregation was evaluated both in whole blood (WB) by using impedance whole blood aggregometer (Chrono-Log) and in platelet rich plasma (PRP) by Born aggregometer. Aggregation was significantly higher in whole blood than in PRP in all the groups investigated (p < 0.01). No significant difference was found in PRP aggregation among the three groups, whereas WB aggregation was significantly higher in the two patient groups (IHD 79.5 + 14.2%, Diabetes 81.3 + 17.6%) than in controls (64.8 ± 14.1%) (p < 0.01 for both comparisons). No relationship was found between WB aggregation and Hct or platelet number in any of the groups studied. A slight relationship was found between megathrombocyte count and WE aggregation values (r=0.31, p < 0.05).Collagen platelet aggregation in WB seems to be provided with higher sensibility than PRP aggregation in detecting hyper-aggregability, probably because it does not imply the selection of platelet populations with loss of larger platelets and of other blood cells.
SITES IN TISSUE-TYPE PLASMINOGEN ACTIVATOR INVOLVED IN THE INTERACTION WITH FIBRIN, PLASMINOGEN AND LOW MOLECULAR WEIGHT LIGANDS
Authors: Verheijen J M , Caspers M P M , de Munk G A W , Enger-Valk B E , Chang G T G , Pouwels P H 
Published: August 2018

Tissue-type plasminogen activator (t-PA) activates the proenzyme plasminogen to the active protease plasmin which degrades fibrin. The unique properties of t-PA, fibrin binding and stimulation of activity by fibrin make it an interesting molecule for specific thrombolysis. t-PA is thought to consist of five structural regions designated finger (F), growth factor (G), kringle 1 (Kl), kringle 2 (K2) and protease (P). Previous studies have shown that the interaction of t-PA with fibrin is mediated by the F and K2 regions.Mutated t-PA cDNA molecules were expressed in Chinese hamster ovary cells and t-PA analog proteins were purified from serum free culture media using affinity chromatography with immobilized monoclonal antibodies. Besides FGK1K2P (native t-PA) the following analogs were used GK1K2P, klK2P, K2P, P, FP and FGKlk2P (kl and k2 have partial deletions of the kringle). All the molecules comprising K2P could be stimulated in plasminogen activation activity by fibrinogen fragments comparable to normal t-PA. The activities of FP and FGKlk2P were only slightly influenced by these fragments. It was shown that the fibrin binding site in K2 was plasminogen dependent whereas that in F was not. K2 was found to contain a binding site for lysine, 6-amino-hexanoic acid but also 6-amino-hexane and thus to differ from the high affinity lysine binding sites in plasminogen.Chemical modification of lysine and arginine residues in t-PA with citraconic anhydride and cyclohexanedione respectively, revealed no involvement of these residues in interaction with lysine or analogs nor in stimulation of activity by fibrinogen fragments. Arginine modification led to inhibition of plasminogen activation activity, both in the presence and absence of fibrinogen fragments, but the amidolytic activity as measured with a tripeptide paranitroanilide was not changed. The involvement of one or more arginine residues in interaction of t-PA with plasminogen seems likely.
CHANGES IN PROTEOGLYCAN AND SULFATED PROTEIN SYNTHESIS DURING MEGAKARYOCYTE MATURATION IN VIVO
Authors: Schick B P , Walsh C J , Jenkins-West T 
Published: August 2018

We investigated changes in sulfated proteoglycan (PG) and sulfated protein synthesis during megakaryocyte (MK) maturation in vivo by characterizing the (35S)-labeled molecules in MKs and platelets (PLTs) obtained daily from 3 hr to 5 days after injection of guinea pigs with (35S)sulfate. Radioactivity in macromolecules was maximal in MKs 3 hr and in PLTs 3 days after the injection. The cells were solubilized in 8M urea/50mM Tris/0.2% Triton X-100/0.1M NaCl, and PGs and sulfoproteins were separated by DEAE-Sephacel chromatography. PGs (65% of cell 35s) were eluted as two fractions, one (PG-1, 87%) with 4M Gdn HC1 and another (PG-2, 13%) with 4M Gdn HCl/2% TX-100. The Kav of PLT PG-1 on Sepharose CL-6B shifted gradually from 0.18 to 0.10 from 1-5 days after (35S) injection, and the smaller and larger PG-1 species were resolved on SDS-PAGE by fluorography. The size of PG-1 molecules was a function of glycosaminoglycan (GAG) chain length. The appearance of the different size PG-1 molecules in PLTs was accounted for by their disappearance from MKs over the same time period. Thus the size of the PG-1 synthesized by MKs decreased with MK maturation. The (35S)-PG-2 appeared in PLTs only 2-3 days after (35S) injection, had Kav 0.07 on CL-6B, but had GAGs of the same average size as those of PG-1. The hydrophobic character of PG-2 suggests that it might be the membrane PG. PG-1 and PG-2 were separated by SDS-PAGE and identified by fluorography. The core proteins of PG-1 and PG-2 were obtained by chondroitinase digestion and identified by SDS-PAGE and fluorography. The GAGs of PG-1 and PG-2 were almost entirely chondroitin-6-sulfate. The average size of PG-1 was 200,000 and its GAGs about 45,000.The sulfated proteins (20-25% of total cell 35S) eluted in the wash-through of the DEAE-Sephacel column and with 0.23M NaCl. Their isoelectric points were 4.0-6.5. They eluted as a small peak near the V0 and a major broad peak from Kav 0.3-0.6 on CL-6B columns, and could be identified as at least 8 distinct bands on SDS-PAGE by fluorography. Digestion with NaOH/NaBH4, Pronase or papain released small (35S)-labeled fragments, and the (35S) appeared to be associated with oligosaccharides. The sulfoproteins appeared in PLTs primarily 2-4 days after (35S) injection, and different proteins were labeled at different time points.
FACTOR XIII (FXIIIa) OF BLOOD COAGULATION NORMALIZES COLLAGEN SYNTHESIS OF SCLERODERMA FIBROBLASTS
Authors: Paye M , Krieg T , Lapière Ch M 
Published: August 2018

Progressive systemic scleroderma (PSS) fibroblasts display, in some cases, an excessive collagen production which leads to fibrosis of the skin and internal organs. Administration of FXIIIa has been reported to be beneficial to some of the PSS patients. The effect of FXIIIa on collagen synthesis by PSS fibroblasts was studied in vitro in two different culture conditions: in a confluent monolayer on plastic and in a three-dimensional collagen lattice.Proteins and collagen synthesis was measurfed by metabolic labeling for 24 h with 3H-proline in absence or presence of FXIIIa (1 U/ml) in dermal fibroblasts from an active lesion (PSS forearm), from an uninvolved area of the skin of the same patient (control abdomen) and from the skin of a normal subject.Proteins synthesis was similar for the three strains under both culture conditions while collagen synthesis was strongly increased in PSS forearm fibroblasts as compared to the uninvolved and to the normal skin fibroblasts. The addition of FXIIIa repressed collagen synthesis of PSS forearm synthesis to the level observed in the abdominal skin fibroblasts of the patient and the normal cells in absence of FXIIIa. When cultured in a collagen lattice collagen synthesis was repressed in a similar proportion in all fibroblasts. Addition of FXIIIa further reduced collagen biosynthesis in the active PSS fibroblasts. The addition of FXIIIa in the lattice largely increased the degradation of newly synthesized collagen in all the strains of fibroblasts.The action of FXIIIa on collagen biosynthesis was also tested in monolayer in 3 other strains of PSS fibroblasts and 3 controls. In all fibroblasts, collagen biosynthesis was reduced by 75 % after addition of FXIIIa.Our results suggest that the beneficial effect of FXIIIa administration in PSS patient might be related to a reduction o1 excessive collagen production and perhaps to an increased degradation of newly synthesized molecules.
FUNCTIONAL CHARACTERIZATION OF HUMAN β-THROMBIN
Authors: Bezeaud A , Guillin M C 
Published: August 2018

Autolysis or tryptic hydrolysis converts β-thrombin (α-T) t β-thrombin ( β -T), and subsequently β-T to y-thrombin (γ-T). Human β-T differs from native α-T by the loss of a unique ll-re-sidues peptide arising from the B chain. Unlike its bovine counterpart, human β-Tisa transient intermediate and its enzymatic properties had not yet been investigated using purified materiaL After 3 min incubation of human β-T with trypsin-sepharose, the resulting β-T was separated from α- and γ-T by chromatography on Biorex 70 with a gradient from 10 mM to 500 mM phosphate at pH 8. No major differences were found between human α- and β-T regarding the kinetic parameters (Km, kcat, kcat/Km) on S 2238, nor the rate of inactivation by TLCK. In contrast, inhibition of β-T by DFP was slower (k = 426 ±[; 10.8 M-1 min−1) compared to α-T (764.5 ± 19.5 M−11min−1and the inhibition constant for benzami-dine was higher with β-T (Ki = 11.2 ± x00B1;.2 10−4 M) compared to α-T (Ki = 2.86± 0,06 10−4 M). The drastic reduction in the clotting activity of β-T (25 u mg−1 versus 3000 u mg−1 for α-T) was further explored by measuring the affinity of β-T for fibrinogen and fibrin. Human fibrinogen was used as a competitor in the inactivation of thrombin by DFP : 10 μM fibrinogen prevented the inhibition of α-T by DFP but failed to modify the inactivation rate of α-T. Binding of thrombin to fibrin was studied using fibrin monomers covalently linked to sepharose 4B, equilibrated in 50mM Tris, pH 7.5, 50 mM NaCl : β -T did not bind to the resin, whereas α-T was retained and eluted upon application of a NaCl gradient.In conclusion, the loss of the peptide extending from lie (63) to Arg (73) in the thrombin B chain is responsiblefor multiple defects in thrombin enzymatic activity. Although, the three active site residues Ser (205), His (43), Asp (99) remain in an active configuration, subtle changes are induced in the microenvironment of the catalytic Ser (205), and in particular, in the primary binding pocket. In addition, the results presented in this study indicate thatthe loss of clotting activity is mainly the result of a decreased affinity for fibrinogen and fibrin, suggesting that the structural changesaffect both the fibrinopeptide groove and the anionic binding site involved in fibrinogen/fibrin recognition.