bluuebunny/crossref_metadata_2025_split huggingface dataset:
ON THE FACTOR XIIIa-INDUCED CROSSLINKING OF HUMAN FIBRIN α-CHAINS
Authors: Henschen A , Müller E 
Published: August 2018

Factor XIIIa catalysis the formation of isopeptide bonds Between γ-carbamoyl groups of peptide-bound glutamines and ε-amino groups of lysines or lysine analogues. During fibrin crosslinking two such bonds are rapidly formed between the C-termini of two γ-chains in adjacent molecules and then several bonds are more slowly formed between several α-chains. The crosslinking sites in the γ-chain were identified already 15 years ago, those in the α-chain are still only tentatively or partially identified,, However, by determining the incorporation of lysine analogues in the α-chain it could be shown that the glutamines in positions 328, 366 and possibly also 237 may participate in crosslinking reactions. Analyses of cyanogen bromide fragments isolated from crosslinked fibrin indicated the segments 271-776 and 518-587 to contain the primary crosslinking sites.In the present study factor XHI-containing fibrinogen was incubated over night with thrombin in presence of calcium ions and cysteine or, as a control, in presence of EDTA. The fibrin material was cleaved with cyanogen bromide, mercaptolysed, pyri-dylethylated and then subjected to Sephacryl S-300 chromatography. The early protein fractions were tested by reversed-phase high-performance liquid chromatography (HPLC) using fibrinogen fragments as reference. In the control sample Aa-chain fragment 271-776 eluted first but in the crosslinked sample it was missing and instead a heterogeneous mixture of higher-molecular weight components was observed. N-Terminal sequence analysis showed the mixture to contain not only the expected fragments 241-476 and 518-584,but in fact all glutamine- or lysine-containing Aα-chain fragments between positions 208 and 611. In the corresponding 6 fragments a total of 6 glutamines and 21 lysines as potential crosslinking sites are present. Two fragments contain only one each of these residues which therefore must be true crosslinking sites. Remaining sites and the actual linkages were identified after reversed-phase HPLC of the tryptic peptide mixture by N-terminal sequence and total amino acid analyses.The linkage pattern will provide information about the localisation and conformation of the C-terminal part of the α-chain and its contribution to the fibrin polymer structure.
THE DEPENDENCE OF PLATELET ATP SECRETION IN RESPONSE TO VARIOUS AGONISTS ON Ca2+ MOBILIZATION AND AGGREGATION IN QUIN 2 LOADED PLATELETS
Authors: Powling M J , Hardisty R M 
Published: August 2018

Thrombin (0.O5 and 0.5 u/ml), ADP (10 uM), PAF. (30 nM), vasopressin (vp, 0.018 u/m1 ), arachidonate (AA, 10 uM), U46619 (0.125 ug/ml) and A23187 (2OO nM) induce aggregation ATP secretion, TxB2 generation and a rapid elevation of intracellular calcium (Ca2+i) concentration provided the medium contains 1 mM extracellular Ca2+. Phorbol ester (TPA) induces aggregation and secretion with no alteration in [Ca2+i]. Pre-incubation with Fab fragments of Ml48, a monoclonal antibody against GPIIb/lIIa, completely abolished aggregation in response to all agonists without affecting Ca2+i flux. ADP and A23187-induced secretion was completely abolished by M148 Fab while that induced by VP, PAF, U46619, AA, thrombin and TPA was inhibited by 70%, 70%, 30%, 25%, 25% and 10% respectively.In the presence of 1 mM EGTA (Ca2+o <100 nM), aggregation was again abolished and the Ca2+i fluxes were about 10-20% of those in the presence of 1 mM Ca2+ o. ADP and A23187 again induced no secretion, but 0.05 u/ml thrombin, PAF, VP, U46619, AA, 0.5 u/ml thrombin and TPA induced respectively 6%, 8%, 12%, 30%, 40%, 57% and 85% of control (1 mM Ca2+ o) levels. Addition of Ml48 Fab and EGTA together had no greater effect than EGTA alone.At the concentrations used, ADP and A23187 are thus completely dependent on aggregation for the induction of ATP secretion, while TPA and thrombin (0.5 u.ml) can actlargely independently of both aggregation and changes in[Ca2+ i]. Low concentrations of thrombin induce secretion in the absence of aggregation provided the stimulated [Ca2+i] is sufficiently high (ie greater than that seen with <100 nM Ca2+ o). VP and PAF appear to be more dependent on aggregation for their secretory responses, while U46619 and AA are more dependent on stimulated [Ca2+i].
REGULATION OF RECEPTOR-OPERATED Ca2− CHANNEL OPENING IN HUMAN PLATELETS
Authors: Moffat Katrina J , MacIntyre D Euan 
Published: August 2018

Agonist-induced elevation of the platelet intracellular free Ca2+ concentration ([Ca2−]i), as monitored using quin2, is not electrically mediated and is attenuated by removal of extracellular Ca2− and by lanthanides (e.g Gd3−).Collectively these data suggest that elevation of [Ca2−]i in platelets derives in part via influx of external Ca2−presumably through a receptor-operated Ca2− channel (ROC). Hal lam & Rink (FEBS Lett. 186: 175: 1985) showed that Mn2−also enters platelets via these ROC. To investigate the possible regulatory mechanisms that govern ROC status, we utilized quin2-labelled human platelets suspended in a Ca2+-free Hepes buffered Tyrodes solution, and monitored agonist-induced Mn2+-mediated quenching of quin2 fluorescence as an index of ROC opening.Thrombin (Th, 0.01-1 U/ml), Vasopressin (VP, 10-1000 nM) and the TxA2-mitnetic, EP171 (1-100 nM) all induced ROC opening which occurred rapidly (<30s), was maximal within 30-60s and thereafter declined. Gd3+ (≤2 mM) markedly impaired this Mn2ࢤ-mediated quenching of quin2 fluorescence induced by all 3 agonists. The adenylate cyclase stimulant PGD2 (3-3000 nM) and the guanylate cyclase stimulant sodium nitroprusside (0.01-10 μM) impaired ROC opening induced by Th (0.5 U/ml), VP (100 nM) and EP171 (25 nM) whether added to platelets ≤120sbefore or 30s after the agonists. In contrast, agents that selectively antagonize, at the receptor level, the effects of VP (e.g. d(CH2)5Tyr Me AVP, 10 ¼H) or EP171 (e.g.EP092, 250nM), or that inhibit the action of Th(e.g. Hirudin 1 U/ml)only impaired ROC opening when added to platelets simultaneously with or before the agonist.These results indicate that, although initiated by agonist-receptor interaction, maintenance of the open state of ROC in human platelets does not require continued receptor occupancy or activation by agonist. Moreover, besides acting to impair the transduction processes initiated following occupancy by agonist of platelet Vi, TP and Thrombin receptors, cAMP-and cGMP-dependent reactions also can terminate or otherwise limit opening of ROC.
ARACHIDGIIC ACID METABOLISM II PLATELETS STORED FOR FIVE DAYS
Authors: Cascar J M , Kavarro J L 
Published: August 2018

Arachidonlc acid (AA) netabolisn has been extensively studied in fresh platelets, but there Is little infomation available for stored platelets . we stored platelets in CLI bags far five days at 22+/-2°C and, on days 0, 3 and 5, six si of platelet concentrate were revved from the container and platelets were labeled with (C-14J-AA. Both incorporation and distribution of radiotracer sere studied in rest and thrcnfcln stivlated platelets.Total uptake of, (C-14)-AA dropped fron day 0 to 5 (p 0.01). Distribution oh day 0 was sinilar to fresh platelets. Incorporation of (C-14)-AA on phosphatidyl inositol (FI) decayed fron 12.4+/-1.5 on day 0, to 7.9+/-0.9 on day 3 (p 0.001), While the percentage attached on phosphatidylserine (PS),increased fron 5.3+/-0.9 to 8.8+/-1.5 (p 0.001). There were not any changes fron day 3 to 5.On day 0,17.7+/-5.2X of radiactlvity was released fron phospholipids by thronbin. This anount decreased to 7.3+/-2.5X (p 0.01) on day 5. I^xaiment in breakdown of both PI and phosphatidylcholine (PC) was detected. Generation of phosphatidic acid (PA) by thronbin, decreased fron 2.6+/-0.4X of total radiactlvity on day 0 to 1.4+/-0.3X on day 3 (p 0.001) and 0.9+/-0.2X on day 5 (p 0.01). Ve did not find changes in TxB2 and HHT, but HETE decayed fron 7.2+/-2.9X on day 0, to 2.3t/-0.9% on day 5 (p 0.01).We concluded that both activities of phospholipases A-2 and C are affected by storage.
CLINICAL EXPERIENCE WITH TRANSFUSION OF LEUKOCYTE-POOR PLATELET CONCENTRATES PREPARED BY FILTRATION WITH PROSTACYCLIN
Authors: Van Marwijk Kooy M , van Prooijen H c , Reimens T I , Akkerman J W N 
Published: August 2018

Repeated transfusions with platelets from randomly selected donors lead to HLA alloimmunization in about 50% of thepatients. This is caused by lymphocytes that contaminate the platelet concentrates. Attempts to remove the leukocytes from the platelet concentrates by additional centrifugation steps lead to substantial loss of platelets.We report here a new procedure for removal of almost all leukocytes with excellent platelet recoveries. Single donor concentrates are treated with 50 ng/ml prostacyclin in order to inactivate the platelets transiently. The concentrates are then passed through a cellulose acetate filter to remove the leukocytes. In 30 fresh concentrates this treatment reduced the contamination by leukocytes to less than 0.1 million per concentrate with a platelet recovery of 89± 1% (mean ±SEM). In concentrates stored for 3 days the contamination was reduced to about 5 million per concentrate. Thirty filtered platelet concentrates, obained from single donors by platelet apheresis using a Haemonetics U 50, were transfused to ten thrombocytopenic patients within 1 hour after filtration and were well tolerated. No signs of hypotension or other side effects were observed. The transfusions led to corrected count increments of (22.0±1.1) x 10 per ml blood after one hour and normal survival thereafter. In four out of five patients these concentrates reduced the bleeding time.We conclude that transient inactivation of platelets by prostacyclin enables optimal removal of leukocytes and may help to reduce alloimmunization during frequent transfusions with platelet concentrates.Supported by the Praeventionfund (grant 28953), The Hague.
PROSTACYCLIN IMPROVES SURVIVAL AND REDUCES MICROCLOT FORMATION IN RABBIT ENDOTOXEMIA
Authors: Ditter H , Matthias Fr R , Voss R , Rottger P 
Published: August 2018

Arachidonic acid metabolites seem to play a pivotal role in the pathophysiology of endotoxin (ET) shock. Therefore, attempts to intervene into the balance of eicosanoids may affect the course of ET shock. Several studies had shown a reduction of ET-induced mortality by non-steroidal antiinflammatory drugs in various animal species.We investigated whether an infusion of prostacyclin (PGI2) has an effect on survival rates and on the incidence of renal microclots in a rabbit shock model, which is based on an intravenous infusion of ET over 4 hours. Thirty animals being exposed to 75 μg/kg x h of lipopolysaccharide B, were allocated to three groups (E, El, EA; n=10 each), either receiving ET only (E), or PGI2 (500 ng/kg x min) simultaneously to ET (El), or aspirin (20 mg/kg) before ET (EA).A control group (C; saline infusion) consisted of 8 animals.At the end of the observation period (8 hours), the mortality of the treated animals (El and EA: 4/10 each) was significantly lower than in group E (8/10). However, only in the PGI2-treated group El a significant reduction of ET-induced glomerular fibrin deposition (GFD) was observed. Indices of GFD after semi-quantitative evaluation of renal slices were 10/27 (E), 1/24 (El), 3/21 (EA), and 0/24 (C). PGI2 exerted a platelet protective effect as shown by higher blood platelet counts (El 61.3 % vs. E 33.4 % of initial values), and a better preserved aggregation (El 60.5 % vs.E 31.7 %) and thromboxane formation capacity (El 52.0 ng/ml vs.E 23.4 ng/ml) of platelet rich plasma stimulated by 5 μg/ml collagen (all values at six hours after the start of ET infusion).ET caused a profound granulocytopenia which was not prevented by PGI2. Furthermore, PGI2 did not affect the ET-induced metabolic acidosis.These data confirm a beneficial effect of prostacyclin during a prolonged endotoxemia in rabbits, which may be a consequence of the known vasodilating, platelet inhibiting and cytoprotective properties of the substance.
FORMER STUDIES OF FIBRINOGEN NEW YORK I : ANALYSIS OF HE GENUINIC C DISORDER for the deletion OF MINO ACID SEQUENCE 9-72 OF THE Bβ CHAIN
Authors: Liu Chung Y , Chung Dominic , Davie Earl , Chess Leonard 
Published: August 2018

Fibrinogens New York I and la (NY-I and NY-la) have been purified from blood plasma samples of a^ sister and a brother in a white family with thrombotic tendency. Both are heterozygous and contain both thrombin-clottable fibrinogen with two normal Bβ-chains and thrombin-nonclottable fibrinogen with two abnormal Bβ chains. The abnormal β-chains result- fran deletions of ammo acid residues 9-72, which are encoded exactly by exon II of the gene. To study the genomic disorder for this deletion, gencmic DNAs were isolated respectively from leukocytes of NY-la, NY-Ib (a nonaffected brother), and four normal individuals outside the NY-I family, and analysed in Southern blotting experiments with a human gencmic DNA probe containing exons I-V. Digestion of various DNAs were performed with two different restriction enzymes, and these digestions were analyzed respectively by agrose electroptoresis.Digestion with Hind III reveals 3 cleavage sites (one site in intron A near exon II)witn formation of two fragments of equal size (2 bands : 3.1kb and 3.1 kb) in normal, NY-la and NY-Id, but an extra fragment (one band = 6.0 kb) in NY-Ia.Digestion with Pvu II reveals 3 cleavage sites (one site in exonII) with formation of two fragments (2 bands : 7.5 kb and 2.9kb)m normal, NY-Ia and NY-Ib, but an extra fragment (one band - 5.7 kb) in NY-Ia. These results show that one Hind III and one Pvu II cleavage sites which are present in the normal allele are absent in the abnormal allele of NY-Ia. Thus, these studies indicate that generic disorder is associated with the patient (NY-Ia) with a thrombotic tendency, and further suggest that the genomic defect in the aonormal allele is near the junction of intron A and exon II. A possible mechanism for this genomic disorder is due to that an inverse double crossover have taken place in a region covering this junction, resulting in m abnormal RNA-splicing site in this junction. Thus, exon II is eliminated with intron A during RNA processing and absent in the abnormal rnRNA. Accordingly, the β(9-72) amino acid sequence disappears from one abnromal β-chains.
STRUCTURE-FUNCTION RELATIONSHIPS IN ABNORMAL FIBRINOGEN WITH Bβ14 ARG→CYS SUBSTITUTION: FIBRINOGENS SEATTLE I AND CHRISTCHURCH II
Authors: Kaudewitz H , Henschen A , Pirkle H , Heaton D , Soria J , Soria C 
Published: August 2018

Genetically abnormal, dysfunctional fibrinogen variants may be used as unique models for studies of structure-function relationships both in vitro and in vivo. Out of the over kO so far structurally elucidated abnormal fibrinogens only 4 have an amino acid substitution in the Bg-chain. These variants are named Fibrinogen Pontoise, Nev York I, Christchurch II and Seattle I.Fibrinogens Seattle I and Christchurch II are slow-clotting fibrinogens which on thrombin-treatment release only half the normal amount of fibrinopeptide B and therefore were expected to contain an amino acid substitution close to the thrombin cleavage site in the Bβ-chain. In order to sequence the abnormal Bg-chains the fibrinogens were cleaved with thrombin and cyanogen bromide. The abnormal Bβ-chain components were isolated from the mercapto-lysed-pyridylethylated N-terminal disulfide knots. After pyroglu-tamyl-peptidase digestion the Bβ 14 Arg→Cys substitutions could be demonstrated for both variants by direct N-terminal sequence analysis. The form of the cyst(e)ine residue was determined by amino acid analysis of the alkylated native fibrinogen. As no alkylated cysteine was detected it was concluded that Bβ 14 Cys participates in a disulfide bridge.Fibrinogens Seattle I and Christchurch II are the first two elucidated fibrinogens with substitutions at the Bβ-chain thrombin cleavage site. Surprisingly, both the thrombin and Reptilase times are prolonged. It may be assumed that the half-cystine residues in position 14 of the two Bβ-chains within one fibrinogen molecule are disulfide-linked to each other, in an analogous way to that already established for the half-cystine residues in position 16 of the Aα-chains of several abnormal fibrinogens.This additional disulfide bridge might change the conformation and charge in the N-terminal region sufficiently to explain the prolonged clotting times. Furthermore, this bridge would provide the evidence for the parallel arrangement of the Bβ-chains at the fibrinogen N-terminus in a similar way as previously shown for the Aα-chains.
A NEW DDAVP-PREPARATION FOR IMPROVEMENT OF SUBCUTANEOUS (S.C.) INJECTION
Authors: Köhler M , Hellstern P , Harris A , Hammer M , Wenzel E 
Published: August 2018

The s.c. administration of DDAVP was currently limited by the large volumes, which had to be injected. Thus, a new preparation containing 40 pg DDAVP per ml was investigated using a standard dose of 0.4 μg/kg body weight by s.c. injection. The pharmacokinetics of s.c. DDAVP and the resulting effects on the haemostatic system were assessed in 10 healthy male subjects in a placebo-controlled study. Peak levels of DDAVP, ranging from 480 to 638 pg/ml (median 505 pg/ml ) were found one hour after injection. DDAVP declined with a median (m) half-life of 3.1 h (range: 2.9- 3.6 h) . Maximum FV III levels were measured 1 or 2 h after DDAVP, the m increases were 2.7 and 3. Ox basal levels for FVIII:C and FVIII:Ag, respectively. A 2.1-fold increase of t-PA antigen was observed. The number of leucocytes significantly increased (absulute mean increment of 3.3x109 /I granulocytes) 4 h after DDAVP. These results were confirmed in plasmapheresis donors (N=13). Two bags of plasma were obtained after s.c. DDAVP with a mean content of 1.7 U/ml FVI11 :C and 1.9 U/ml ristocetin cofactor. The effect of s.c. DDAVP was was tested in 10 haemophilia A patients and 2 carriers of hemophilia A (m FVIII:C: 0.17 U/ml). The mean increase of FVIII:C was 2.3-fold (FVIII:Ag 2.5-fold) 1 h post injection. Eight bleeding episodes or operations were successfully treated with s.c. DDAVP, in one case postoperative haematoma occurred. In eight patients with uraemic bleeding the influence of s.c. DDAVP was investigated in the steady state. Bleeding time (BT) significantly shortened in 7 patients (before m BT: >15 min; 90 min after DDAVP BT: 6 min). Additionally, platelet count decreased while platelet retention increased.DDAVP, s.c. injected, was shown to be a safe and effective measure in bleeding disorders and may become a useful tool for conditioning of plasma or granulocyte donors.
A NEW DELIVERY SYSTEM FOR DDAVP: CLINICAL EXPERIENCE OF DDAVP SPRAY PUMP IN MILD HAEMOPHILIA A AND vWD TYPE I
Authors: Lethagen S , Harris A S , Nilsson I M 
Published: August 2018

Until recently, the reliability and predectibility of the intranasal route of administration of DDAVP has been too poor to recommend it in routine practice.With the development of a new delivery system in the form of a spray puip we have previously shown that absorption and biological effect of DDAVP in volunteers is enhanced(1).In this patient study we conpared intranasal administration of 300 pg DDAVP using a newly developed pre-corrpression spray pinp with intravenous administration of 0.3 - 0.4 pg/kg. We studied 24 patients with mild haemophilia A, 23 with mild vWD Type IA and 8 with Type IB. We measured VIII:C (one-stage assay), vW:Ag (IRMA) and bleeding time (Simplate II) before and 30 - 60 min and 1 hour post treatment after i.v. and i.n. administration respectively.The spray resulted in a 2 to 4 times increase in basal levels of VIII:C and vW:Ag and was comparable in effect to intravenous administration. Moreover, bleeding time was significantly reduced to within the normal range in most patients. Several patients reported good clinical effects of the spray after tooth extraction, menorrhagia and epistaxis.We conclude that the DDAVP spray is a clear improvement over previous atterpts at intranasal administration. The spray deposits well controlled and reproducible doses in the nasal cavity resulting in a clear enhancement in absorption with a magnitude and reliability of its biological effect which is corparable to the intravenous delivery. Furthermore, the spray offers a convenient and practical means of self-treatment to haemophilia patients without delay.1. Harris A.S., Nilsson I.M., Wagier Z.-G., Alkner U. Intranasal Administration of Peptides: Nasal Deposition, Biological Response, and Absorption of Desmopressin. J Pharm Sci (in press, December 1986).
SUBCUTANEOUS AND INTRAVENOUS ADMINISTRATION OF DESMOPRESSIN (DDAVP) TO HEMOPHILIACS: PLASMA PHARMACOKINETICS AND FACTOR VIII (VIII:C) RESPONSES
Authors: Mannucci P M , Vicente V , Alberca I , Sacchi E , Harris A S , Lindqvist A 
Published: August 2018

Reported studies dealing with the clinical use of DDAVP in mild and moderate hemophilia A patients show a very large between-patient variability for the maximum increase of VIII:C after the drug given intravenously (i.v.) or subcutaneously (s.c.). By measuring DDAVP plasma levels with a sensitive and specific RIA method, we elected to evaluate whether or not between-patient response variability was related to the variability of DDAVP levels achieved in their plasma. To this purpose 14 moderate or mild hemophilic volunteers (baseline VIII : C 4 to 31 U/dL) were randomly given 0.3 pg/Kg of i.v. or s.c. DDAVP with a between-treatment interval of 15 - 30 days. Plasma DDAVP pharmacokinetics in relation to the routes of administration are shown in the table.Pack levels (Cmax) were higher after i.v. DDAVP (p < 0.02). Time to peak levels (tmax) was shorter for i.v. DDAVP (p < 0.001). There was no difference between i.v. and s.c. DDAVP for plasma time curve (AUC) and half-life (t½).The bioavailability of the s.c. route relative to the i.v. route was 85 ° 32%. Of further interest, was the greater variability of the i.v. pharmacokinetics compared to the s.c. data. These differences were reflected in the VIII:C response. Maximum VIII:C increase over baseline levels was 3.2 ° 2.4 fold (i.v.) and 3.2 ° 1.3 fold (s.c.) (n.s.).Thus the i.v. route gave a marginally greater response but the effect was more variable than the s.c. route. Finally, no significant correlation was found between the VIII:C response and plasma DDAVP levels for either route of administration (i.v. route r = 0.03, s.c. route r = 0.23).These findings establish the subcutaneous route to be bioequivalent in effect to the intravenous route with less variation. This study also demonstrates that the VIII:C response to DDAVP is neither a function of the rate of absorption of the corrpound into the body nor the magnitude of the plasma concentration.
THE RESPONSE OF PROTEIN S, TISSUE PLASMINOGEN ACTIVATOR AND TISSUE PLASMINOGEN ACTIVATOR INHIBITOR TO DESMOPRESSIN (DDAVP) INFUSION
Authors: Wadhwa N K , Kim S , Glas-Greenwalt P , Kant K S , Pollak V E 
Published: August 2018

Protein S (PS) is a cofactor for activated protein C (PC) in the neutralization of tissue plasminogen activator inhibitor (PA-I) and in its profibrinolytic effect (Blood 69:231, 1987). Increased fibrinolytic activity and tissue plasminogen activator (t-PA) antigen in response to venous occlusion are dissociated because of t-PA and PA-I complex formation (Br. J. Haematol. 61:169, 1985). We hypothesized that DDAVP infusion stimulates t-PA and PS release from endothelium, thereby decreasing PA-I. These responses to intravenous DDAVP infusion (0.4 μg/kg) were studied in 1.0 healthy volunteers. t-PA activity and PA-I were measured by standard fibrin plates. t-PA antigen was assayed by ELISA. PS and PC were measured by electroimmunodiffusion. The baseline values were expressed as 100%. Mean values expressed as percent of baseline at 10, 30, 60 min after DDAVP infusion were:These data support that both t-PA and PS were released, during DDAVP infusion, thereby resulting in decreased PA-I.