bluuebunny/crossref_metadata_2025_split huggingface dataset:
THE EFFECT OF RED BLOOD CELLS ON PLATELET AGGREGATION IN NORMAL SUBJECTS AND MYELOFIBROTIC PATIENTS
Authors: Balduini C L , Bertolino G , Polino G , Gamba G , Sinigaglia F , Ascari E 
Published: August 2018

We investigated the effect of red blood cells (RBC) on "in vitro" platelet aggregation by the use of the "Electronic Whole Blood Aggregometer" (Chrono-Log Corporation). Preliminary experiments, studying platelet aggregation in the same PRP by the simultaneous use of the optical method and the electronic method, demonstrated that the maximum rate of impedance changes corre lated well with both the rate and the extent of platelet aggregation as measured by the optical method. The refore, the maximum rate of impedance increase was cho sen for the measurement of platelet aggregation in the presence of different concentrations of RBC. RBC, both at 40 and 60%, significantly inhibited platelet aggregation stimulated by low and high concentration of ADP and epinephrine. Platelet aggregation stimulated by co llagen was slightly reduced only by the higher RBC con centration. The effect of RBC on platelet aggregation was also investigated in idiopathic myelofibrosis, a pathological condition characterized by both platelet and RBC alterations. While on the basis of PRP studies 5 out of 17 patients had hypo-aggregation and 12 had normal aggregation, whole blood studies evidentiated hypo-aggregation in 3 patients, normal aggregation in 4 and spontaneous platelet aggregation (SPA) in 10. SPA was a consequence of platelet abnormality, since it occurred also when platelets from patients were sti rred with normal RBC.In conclusion, RBC may exert different effects on the aggregation of normal and pathological platelets.
EVALUATION OF THROMBOPOIESIS IN ESSENTIAL THROMBOCYTHAEMIA BEFORE AND AFTER α-INTERFERON TREATMENT
Authors: Gugliotta L , Macchi S , Catani L , Mattioli Belmonte M , Gaggioli L , Tura S 
Published: August 2018

The α-interferon (α-IFN) has been shown efficacious in controlling thrombocytosis in chronic myeloproliferative disorders. In order to better understand the mechanisms by which this effect is produced, the main parameters of thrombopoiesis have been evaluated in 8 patients with Essential Thrombocythaemia (ET)just before and at the end of induction therapy with α-IFN. The patients, 2 males and 6 females, 17-54 years old, at diagnosis or at least 3 months off cytotoxic drugs, received α-IFN (Roferon A-Roche) s.c. at a daily dose of 3 × 106 IU for 6-13 weeks. The baseline platelet count of 993±266×109/1 fell, after treatment, to a value of 377±96×109 /1. The histological analysis of the bone marrow showed that the number of megakaryocytes (MK), initially 5-15 times the normal value (N), decreased to a value of 3-6 × N, while the MK volume resulted always high. The "in vitro" study of megakaryocytopoiesis, by the plasma-clot culture technique, documented a significant decrease of the number of MK colonies (from 73±18 to 29±15; p <0.0001). The half life-span of autologous platelets labelled withulIn-oxine remained unchanged (97±22 and 101±25 hours before and after therapy, respectively), while the platelet function abnormalities (hypo-aggregation, storage pool deficiency, etc) appeared less severe after treatment.It is concluded that in ET the α-IFN therapy is able to normalize the platelet count mainly or exclusively by a decrease of platelet production.The work was supported in part by grant of National Research Council (CNR), special Project Oncology No 85.02200.44.
HETEROGENEITY OF MEGAKARYOCYTE PLOIDY PROFILE IN PATIENTS WITH ITP
Authors: Bussel J , Rabellino E 
Published: August 2018

Thrombocytopenia in patients with ITP is ascribed to peripheral platelet destruction mediated by antiplatelet antibodies. Patients with a chronic severe form of disease may also have im paired platelet production. Profile of DNA content and distribu tion in marrow megakaryocytes (MK) is a sensitive parameter of megakaryocyte development. In these studies MK derived from clinical aspirates were analyzed by flow cytometry for DMA content and platelet glycoprotein GP IIb/IIIa. Both whole unsenar-ated and MK-enriched marrow were stained using double label staining for GP IIb-IIIa by immunofluorescence (green) and for DNA with propidium iodide (red). 3 different profiles were seen in ITP patients as indicated in the Table:Profile I represented the commonest pattern and was identical to that seen in normals. Profiles II and III demonstrated peak ploidy values of 8C and a bimodal 2C/16C pattern respectively. In order to investigate the relationship of ploidy profile to platelet production, we compared the peak platelet response to IVGG, a treatment which stops platelet destruction, among the 3 groups. The average platelet response to IVGG was 221,000/ul for group I, much greater than both group II (119k) and group III (96k)(I vs II + III, p < 0.05). Heterogeneity of clinical cases of ITP may be caused by differences in ploidy profile of patient marrow megakaryocytes.These profiles may be caused by a heterogeneity of the target antigen of the antiplatelet antibodies in different patients with differing crossreactivity to and effects on megakaryocytes. This heterogeneity appears to be reflected in different responses to therapies suggesting that it may be useful in designing treatment strategies.
FURTHER EVIDENCES OF VON WILLEBRAND FACTOR INVOLVEMENT IN THROMBOTIC THROMBOCYTOPENIC PURPURA (TTP)
Authors: Chediak J , Eldridge J , Sobel D , Maxey B , Baron J , Telfer M C 
Published: August 2018

Laboratory findings of TTP include severe thrombocytopenia and marked anemia due to intravascular hemolysis. The pathogenesis of the thrombocytopenia is poorly understood. Possible mechanisms include the presence of a platelet aggregating factor (JCI 76:1330, 1935), a calcium dependent protease (Blood 63:310a, 1986), abnormal prostaglandin production or metabolism, and an excessive consumption of high molecular weight forms of von Uillebrand factor (VUF). Von Willebrand factor proteins from 12 patients (pts) diagnosed as having TTP were studied. They include 8 females and 4 males; nine were studied during the acute presentation and seven pts also during the remission period. Three pts died during the acute event. None of the pts had a recurrence. Control subjects include both normal individuals and thrombocytopenic pts due to a variety of underlying diseases including marrow aplasia and immune thrombocytopenia, but excluding pts with DIC or suffering infection. Plasmas were tested for VWF antigen and Ristocetin Cofactor (RiCof) activity. The electrophoretic mobility (CIE) of VWF:Ag was also assessed and the ratio VWF:RiCof to VWF:Ag was determined. Statistical analysis including p values will be reported. Results:These results suggest that during the acute event there is an excessive consumption of large VWF multimers and the ratio VWF:RiCof/VWF:Ag could be used to corroborate the diagnosis of TTP and by doing sequential measurements to monitor the response to therapy.
RELAPSING THROMBOTIC THROMBOCYTOPENIA PURPURA (TTP): A CLINICAL STUDY OF 38 PATIENTS
Authors: Eldor A , Rose M 
Published: August 2018

A dramatic change in the prognosis of TTP has taken place since the introduction of plasma therapy in 1977. However, plasma therapy does not always result in a complete cure since TTP manifestation may reappear. We describe 38 patients who were treated for TTP in 15 hospitals in Israel and New York City since 1977. 37 patients received plasma (transfusions and exchanges) and 30 survived. 12 patients (37%) developed relapsing TTP. Infections, pregnancy and surgery frequently preceded the initial episodes and the relapses. To evaluate the clinical course of relapsing TTP, a scoring system was developed based on 4 parameters: neurological findings, renal function impairment, platelet count and hemoglobin value at presentation. Each parameter was marked on a scale of 0 to 2: 0= no abnormality, 1= moderate impairment, 2= severe impairment. The severity of each episode was the sum of the 4 scores. The cases were divided into 3 groups: Group A= patients who died during 1st episode; Group B= patients who had only one episode; Group C= patients who relapsed. The following are the average severity scores:The differences between Groups A and B; A and C; B and C; and B and the relapses are significant (p<0.005)(Chi-square test). The differences between Group C first episode* and subsequent relapses are not significant (paired t-test). Hence, relapsing TTP was manifested by a milder clinical course, however two relapses were fatal, demonstrating that this is not a benign disorder. Therapy consisted of plasma, plasmapheresis, steroids, platelet-inhibitor drugs and 11 patients underwent splenectomy. Addition of vincristin seemd to shorten the duration of the TTP episode. No differences were observed in the treatment of Group A, B and C patients. However, during relapses, patients received less therapy probably due to their milder clinical course. Splenectomy did not prevent relapses, but prolonged the remission duration. Based on our data, we recommend that the severity score be used in determining the intensity of treatment.
INCREASED SENSITIVITY OF A PLATELET-SUBPOPULATION TO CRYOPRESERVATION
Authors: van Marwijk-Kooy M , Dullemond-Westland A C , van Prooijen H C , Riemens M I , Akkerman J W N 
Published: August 2018

When cryopreserved human platelets are stimulated with a high dose of collagen (40 μg/ml), about 50% of the cells fail to aggregate, as measured by counting the disappearance of single platelets. This suggests the presence of a subpopulation with increased susceptibility to cryopreservation-induced cell damage. In an attempt to identify this subfraction fresh platelet suspensions were separated into four size-dependent fractions by counterflow centrifugation. Large platelets (MPV 11.1 μm3 ) better preserved their responsiveness than small platelets (4.2 μm3) with intermediate responses for fractions of 5.7 and 8.0 μm3. In contrast, the partial loss of secretion granules (βTG and PF-4) seen during cryopreservation was greater in the large platelets than in the small cells and did not parallel the differences in aggregability.In a second separation technique the unresponsive fraction was separated from the cells that aggregated with collagen. This fraction consisted of intact cells (LDH), and normal secretion granules (βTG-PF-4) but failed to show changes in Pl-cycle and protein phosphorylation, two essential steps in the activation mechanism of platelets. In accordance with this, the total cryopreserved suspension showed a partial reduction in these second messengers with a 35% lower phosphatidic acid formation, 50-55% less protein phosphorylation and a 50-75% reduction (depending on stimulus concentration) of the liberation of intracellular Ca2+ ions, compared with fresh platelets.Hence, cryopreservation disturbs the activation mechanism of a platelet subpopulation consisting of about 50% of the cells, probably as a result of membrane damage, whereas the other half of the population recovers relatively unharmed from the freeze-thawing procedure.Supported by the Praeventiefund (grant 28953), The Hague.
PRODUCTION OF A TISSUE INHIBITOR OF METALLOPROTEINASES BY BOVINE VASCULAR CELLS
Authors: DeClerck Y A , Bock R , Laug W E 
Published: August 2018

Tissue Inhibitor of Metalloproteinases (TIMP) plays an important role in collagen turnover in tissue due to its ability to irreversibly inhibit mammalian collagenases. We have investigated the production of such an inhibitor by various cells of bovine vessels including endothelial cells of arterial, venous and capillary origin and arterial smooth muscle cells. While large amounts of collagenase inhibitor (800 mU/106 cells/24 hr) were produced by vascular smooth muscle cells, smaller amounts were detected in* the medium conditioned by either arterial, capillary or venous endothelial cells (90, 1.7 and 1.1 mU/106 cells/24 hr respectively). An inhibitor with a Mr of 28,500 was purified from serum free medium conditioned by bovine smooth muscle cells using molecular sieve followed by heparin sepharose and carboxy-methylcellulose chromatography. It inhibited several vertebrate collagenases but was inactive against bacterial collagenase. This inhibitor was resistant to treatment with acid and heat but sensitive to trypsin and reduction alkylation. It formed with vertebrate collagenase an enzyme-inhibitor complex resistant to organomercurials or trypsin. This inhibitor, therefore, is similar to a collagenase inhibitor produced by human fibroblasts and a tissue inhibitor of metalloproteinases extracted from human amniotic fluid and rabbit bone.The production of TIMP by bovine vascular smooth muscle cells markedly increased during cell proliferation. In addition, when endothelial cells were grown on a preformed layer of smooth muscle cells, the production of TIMP was more than additive suggesting an enhancing effect of endothelial cells on vascular smooth muscle cells.These data suggest that the large amount of TIMP produced by vascular muscle cells may be responsible for the accumulation of collagen characteristically observed in conjunction with smooth muscle cells hyperplasia in atherosclerotic plaques.
NUCLEOTIDE SEQUENCE OF THE STAPHYLOCOAGULASE GENE FROM STAPHYLOCOCCUS AUREUS STRAIN BB
Authors: Kaida S , Miyata T , Kawabata S , Morita T , Yoshizawa Y , Igarashi H , Iwanaga S 
Published: August 2018

Staphylocoagulase (SC) is a secretary protein produced by several strains of Staphylococcus aureus (S. aureus). This protein forms a molecular complex ("staphylothrombin") with human prothrombin in a molar ratio of 1:1. The complex displays the ability to clot fibrinogen and to hydrolyze the synthetic tripeptide substrates for α-thrombin. The formation of staphylothrombin does not require proteolytic cleavage of the prothrombin molecule, and this mechanism differs markedly from the activation process by either blood-clotting factor Xa or snake venom procoagulant.In the present studies, a pAT153 library containing partial Mbo I-digested DNA prepared from aureus strain BB has been screened with a fibrin gel formation method. The identity of these clones with SC was confirmed by DNA sequence analysis and by comparison of the derived amino acid sequence with that determined for the purified SC protein. One of the positive colonies was isolated and 3.1 Kb of the insert DNA was determined by the dideoxy chain termination method. The results indicated that the insert DNA consists of 148 bp 5' flanking region, protein coding region of 715 amino acids and 746 bp 3' flanking region, and that SC from strain BB is synthesized as a precursor with a signal peptide of 26 amino acids. Thus, the mature form was composed of 689 amino acids with a molecular weight of 77,337- The NH2-terminal sequence (324 amino acids) of SC isolated from S. aureus strain 213 (S. Kawabata et al. (1986): J. Biol. Chem. 261, 527-531) was compared with that of SC derived from strain BB. The sequence homology between them was found to show 57 %. It was also found that SC derived from strain BB was composed of 8 tandem repeats (27 amino acid residues in length) in the COOH-terminal region, although their functions are not known.
EXPRESSION IN ONTOGENESIS OF HUMAN BLOOD COAGULATION FACTORS
Authors: Hassan H J , Leonardi A , Chelucci C , Guerriero R , Mannucci P M , Peschle C 
Published: August 2018

We have analyzed the expression of several blood coagulation factors (IX, VIII, X, fibrinogen chains) and inhibitors (antithrombin III, protein C) in human embryonic and fetal livers, obtained from legal abortions at 6-11 week post-conception. The age was established by morphologic staging and particularly crown-rump lenght measurement.Total cellular RNA was isolated from partially purified hepatocytes or total liver homogenate using the guanidine isothiocyanate method. Poly(A)+ RNA was selected by oligodT cellulose chromatography. The size and the number of the embryonic and fetal transcripts are equivalent to those observed in adult liver, as evaluated by Northern blot analysis of total or poly(A)+ RNA hybridized to human cDNA probes.The level of coagulation factor transcripts in embryonic and fetal liver was evaluated by dot hybridization of total RNA (0.5-10 ug), as compared to RNA extracted from normal adult liver biopsies. The expression of blood coagulation factors in embryos is generally reduced for all factors, but at a different degree. In 5-11 wk liver, the level of factor IX is 5-10% of that observed in adults, while fibrinogen, protein C, antithrombin III RNA level rises from 25 to 50% and factor X is expressed at a level comparable to that observed in adult liver.We conclude that during these stages of development blood coagulation factors are expressed according to three different time, curves, possibly due to the effect of different types of regulatory mechanisms.
THE ACTIVITY OF A SINGLE CHAIN rt-PA MUTANT IN A PRIMATES AND RABBITS
Authors: Hotchkiss A , Knitter G , DeGuzman L , Rsfino C 
Published: August 2018

Recombinant tissue-type plasminogen activator(rt-PA) contains a plasmin sensitive peptide bond domain between amino acids (AAs) 275 and 276. Since the single chain form of rt-PA is rapidly clipped to the two chain form during thrombolysis, one way to determine the relative activities of single chain and two chain rt-PA is to produce a mutant which no longer has this clip site; therefore, a single AA substitution was made at position 275, replacing Glu for Arg (Tate, et al., Biochemistry 1987, in Press). The thrombolytic activity of this mutant (Glu275) was compared to the activity of the wild type rt-PA (Arg275) in a new primate thrombolysis model.An arterio-venous shunt model (Hotchkiss et al, this meeting) was used. Various doses of the rt-PAs were infused over a 90 min period with 10% of the total dose administered as an initial bolus. The rate and extent of thrombolysis were quantitated by the measurement of radioactivity remaining in the thrombus at 15, 30, 60 and 90 min, using an external probe. Data were collected from 5 cyncmolgus monkeys (male 5 to 6 kg). The dose response curves were used to determine the ED40 (dose for 40% clot lysis). The ED40 for Arg275 and Glu275 wire 870 and 680 (ugAg)r respectively. The plasna concentrations of rt-PA were measured at plateau for various doses and the regression of the plasma concentrations (Y,ng/ml) versus the dose rates (X, ug/kg/min) was calculated. The regression equations were; Y=28+51X and Y=39+113X for Arg275 and Glu275, respectively. This indicates that the clearance rate of Glu275 is 2.2 fold slower than Arg275.These experiments were also performed in rabbits. The ED40 for Arg275 and Glu275 were 740 and 660 (ug/kg) respectively. The Glu275 was cleared more slowly, having a 1.7 fold slower clearance rate.These results support the conclusion, that the "one chain" Glu275 rt-PA is still active in vivo and that it is slightly more potent than the wild type in these animal models. Some of this increase in potency is apparently due to a slower clearance rate for the Glu275 rt-PA.
A MONOCLONAL ANTIBODY SOLID PHASE COMPETITIVE RADIOIMMUNOASSAY (RIA) WHICH MEASURES ALL FORMS OF PROTEIN S (PS) IN PLASMA
Authors: Bovill E G , Landesman M , Mann K G , Tracy R P 
Published: August 2018

PS is bound to one (C4bp) and possibly two (protein S binding protein) other proteins in plasma, necessitating the validation of any assay, both by standard criteria and for its ability to measure the different forms of PS in plasma. Murine monoclonal antibody PS-2a was adsorbed to solid phase microtiter plate wells at 10 ug/ml, pH 9.6. For the assay, affinity purified PS standard, free of C4bp, or plasma samples were incubated with 125 I labelled PS.Serial dilutions of pooled normal plasma paralleled the standard curve. Competition studies with other vitamin K dependent coagulation proteins, anti-thrombin III, C4bp and bone Gla protein showed no significant crossreactivity. When 5.7 ug of PS was added to 10 normal plasmas the mean recovery was 5.86 ± 0.8 ug/ml. Sensitivity was 0.03 ug/ml at the working plasma dilution (1/64). Normal pooled plasma had a mean of 15 ug/ml with an intra-assay CV of 5.7%. The normal range based on 61 normal plasmas is 9.5-24 ug/ml. The mean of 24 warfarinized plasmas was 8.9 ug/ml (1 SD = 3.5)Normal pooled plasma was totally depleted of PS using anti-PS-2a coupled to sepharose 4B at 5mg/ml. Depleted and undepleted normal pooled plasmas were then compared to a standard curve of purified PS in a reduced, SDS PAGE Western Blot utilizing a rabbit polyclonal anti PS. This experiment demonstrated that the monoclonal antibody used in the assay totally depleted the pooled plasma of all forms of PS and therefore appears to recognize an epitope on PS not involved in its binding interactions with other plasma proteins.PS and C4bp were measured following HPLC using a Beckman TSK-3000 column for rapid separation. In pooled plasma 70% of PS appeared bound to C4bp.(Supported by NIH Grant HL 35058)
INDEPENDENT ISOLATION OF HUMAN PROTEIN S cDNA AND THE ASSIGNMENT OF THE GENE TO CHROMOSOME 3
Authors: Ploos van Amstel J K , van der Zanden A L , Bakker E , Reitsma P H , Bertina R M 
Published: August 2018

Protein S is a vitamin K-dependent glycoprotein, that serves as a cofactor of activated protein C. A hereditary deficiency in protein S is associated with an increased risk for the development of venous thrombosis. The deficiency is inherited as an autosomal dominant trait. We isolated a cDNA coding for protein S and assigned its gene to chromosome 3.A human liver cDNA library in phage xgtll (complexity 1.2 × 106 , D. Stafford, Chapel Hill) was screened by using immuno-purifiedpolyclonal anti-protein S IgG as a probe. Approximately 1.5 x 10 recombinants of the amplified library were screened. Out of eighteen positive clones one clone was found, after nucleotide sequence analysis, to code for a peptide with a high degree of homology with the carboxy terminal region of the already published bovine protein S. This clone pSP84 (450 bp) was used as a probe to screen a human liver cDNA library in plasmid pUC9. From this library we isolated several positive clones. Clone pSUL5 contained the largest insert (2200 base pairs). Dideoxy sequencing revealed that it codes for 330 amino acids of the carboxy terminal part of protein S. Furthermore, it contained a 1200 base pairs 3' untranslated region. The predicted amino acid sequence did not differ from the published sequence of human protein S, although at the nucleotide level some differences could be detected.Clone pSUL5 was used to localize the protein S gene to its chromosome. The assignment was done by hybridization to Pst I digested DNA from human-hamster c.q. human-mouse somatic cell hybrids. In this way we got strong indication that the protein S gene is located on chromosome 3.