bluuebunny/crossref_metadata_2025 huggingface dataset:
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.
EVIDENCE FOR PLASMIN-MEDIATED FIBRINOLYSIS AFTER /RELEASE OF TISSUE PLASMINOGEN ACTIVATOR BY DESMOPRESSIN INFUSION
Authors: Lowe G D O , Douglas J T , Small M , Kluft C , Forbes C D
Published: August 2018

A relationship between tPA activity and plasmin-mediated fibrinolysis in vivo (plasma levels of Bβ15-42-containing peptides) has been suggested by our previous studies: inverse correlations of Bβ15-42 levels with obesity and triglyceride levels (both associated with high tPA inhibition) in an epidemiological study; and increased levels of Bβ15-42 following improved control of diabetes, or treatment with oral or intramuscular stanozolol (which decrease tPA inhibition). The aim of the present study was to establish whether or not intravenous infusion of desamino-D-arginine vasopressin (DDAVP, desmopressin) is followed by increases in plasmin-mediated fibrinolysis in vivo (plasma Bβ15-42 levels). Desmopressin (0.3 μg/kg body weight) was infused intravenously over 15 mins in 22 subjects. Venous blood was obtained by separate venepuncture before and 15 mins after the end of the infusion, for assay of plasminogen activator activity of the euglobulin fraction on fibrin plates, tPA activity, and Bβ15-42 levels (RIA,IMCO). 18 subjects showed normal increases in fibrin plate lysis and in tPA activity after desmopressin (median tPA activity 120 mU/ml pre-, 5000 mU/ml post-infusion, p<0.001). In these 18 subjects, Bβ15-42 levels rose significantly (median 1.5pmol/ml pre-, 4.2 pmol/ml post-infusion, p<0.001). Four subjects showed no significant increases in fibrin plate lysis or in tPA activity after desmopressin (non-responders): all had significantly elevated levels of tPA-inhibition. In these 4 subjects no increases in Bβ15-42 levels were observed. In one nonresponder, who suffered a large myocardial infarction due to angiographic thrombosis with no atheroma at the age of 22 years, long-term treatment with stanozolol normalised the high level of tPA-inhibition, as well as the fibrin plate lysis and tPA activity responses to desmopressin: Bβ15-42 level then showed a normal response after desmopressin infusion (2.2 to 5 pmol/ml). We conclude that desmopressin infusion increases plasmin-mediated fibrinolysis in vivo, but only in the presence of normal increases in tPA activity.
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.
THE ENDOTHELIAL CELL AS THE SII$ OF THE FACTOR VIII BYPASSING ACTIVITY OF PROTHROMBIN COMPLEX CONCENTRATE (PCC)
Authors: Teitel J M , Garvey M B , Freedman J J
Published: August 2018

PCC is used empirically for its factor VIII (FVIII) bypassing activity in hemophiliacs with inhibitors to FVIII. The effect of of PCC on plasma coagulation tests is variable and is not predictive of clinical response. We are testing the hypothesis that bypass activity is expressed locally at the vessel wall, which functions as a procoagulant surface at times of hemostatic stress. Monolayer cultures of umbilical vein endothelial cells (EC) were incubated for 1 hour with commerical PCC (1 u/ml), and then extensively washed to remove fluid phase PCC components. Ellagic acid-activated PTT determinations were then done over the treated monolayers using plasma deficient in FVIII (<1 u/ml). PTTs over EC pre-treated with PCC were shorter than PTTs with control EC or EC pre-treated with commercial FVIII concentrate (control 101 s, PCC treated 57.7 s, FVIII treated 84 s, means of n=3). Similar results were obtained using non-activated PTTs and with adult saphenous vein EC. The bypass effect of PCC was dose-related:non-activated PTTs were 172 s (control),134 s (PCC 0.1 u/ml),and 107 s (PCC 1 u/ml). Pre-treatment of EC with cycloheximide shortened the PTT of FVIII deficient plasma, presumably due to impaired expression of endogenous anticoagulant activity.Subsequent incubation of cycloheximide pre-treated EC with PCC still provided FVIII bypass activity. In contrast, FVIII consistently failed to shorten the PTT using FVIII deficient plasma added to cycloheximide pre-treated EC. Thfe ability of PCC to bypass the FVIII defect was not impaired using paraformaldehyde 1fixed in place of live EC, but was reduced or abolished if the ECwere incubated with PCC at 4° instead of 37°.The putative FVIII bypassing component present in PCC may therefore express its activity only upon interacting with EC at sites of vascular interruption. Further studies using purified reagents will be necessary to identify the responsible substance(s). These observations may help explain the discrepancy between the clinical. efficacy of PCC and its inconsistent effect on plasma coagulation tests.
INTERNALIZATION OF THROMBIN-ANTITHROMBIN III BY CULTURED HUMAN ENDOTHELIAL CELLS AND ITS SUBCELLULAR LOCALIZATION
Authors: van Iwaarden F , Acton D , Sixma J J , de Groot P G , Meijers J C M , Bouma B N
Published: August 2018

The presence of antithrombin III (ATIII) was demonstrated in cultured human endothelial cells derived from the umbilical cord using immunofluorescence, immunoelectron microscopy studies and an enzyme linked immunosorbent assay (ELISA) specific for ATIII. Immunofluorescence studies indicated the presence of ATIII in granule-like structures in endothelial cells. Immunoelectron microscopy studies performed with ultrathin cryosections of endothelial cells showed a co-localization of ATIII and a lysosomal marker protein in low electron dense organelles, indicating a lysosomal localization of ATIII. Using an ELISA, 77 ± 26 ng (n = 8) of ATIII was quantitated in 10° endothelial cells. Immunoprecipitation studies performed with metabolically labelled cultured endothelial cells indicated that ATIII was not synthesized by the cells. Endothelial cells cultured in ATIII depleted human serum did not contain ATIII as was measured by the ELISA. The lack of synthesis of ATIII, the absence of ATIII in endothelial cells cultured in medium devoid of ATIII and the lysosomal localization of ATIII strongly suggest that ATIII is internalized by cultured human endothelial cells from the culture medium. Therefore internalization studies were undertaken with radiolabelled ATIII and ATIII complexed to thrombin to determine whether ATIII and/or ATIII complexed to thrombin was internalized by endothelial cells. When labelled ATIII was added to endothelial cells, it was not internalized by the cells. However, ATIII preincubated with thrombin prior to the incubation with the cells was internalized, indicating that either residual free thrombin stimulated the endothelial cells to internalize ATIII or ATIII in a complex with thrombin is internalized. To discriminate between these possibilities, the ATIII-thrombin complex was purified and radiolabelled. The purified ATIII-thrombin complex was internalized by endothelial cells in a specific and time dependent way. Preincubation of the endothelial cells with thrombin did not enhance the internalization of the ATIII-thrombin complex nor stimulated the internalization of ATIII by endothelial cells. These results indicate that endothelial cells internalize ATIII when it is complexed to thrombin.
THROMBIN INTERACTION WITH CULTURED AORTIC AND CAPILLARY ENDOTHELIAL CELLS: BINDING, INTERNALIZATION, DEGRADATION AND RELEASE OF PROTEASE NEXINS
Authors: Savion N , Gamliel A , Farzame N
Published: August 2018

Thrombin (Th) binds specifically to confluent cultures of bovine aortic (ABAE) and brain capillary (BBC) endothelial cells. Saturation of 125I-Th binding is observed after 1 h exposure to the ligand and at an extracellular concentration of 0.5 and 1.0 µg/ml for ABAE and BBC cells, respectively. Under optimal conditions both ABAE and BBC cultures bind about 2 to 5 ng/106 cells, which represents about 20% of Th binding.to bovine corneal endothelial (BCE) cells. The cell associated 125I-Th in ABAE and BBC cells is internalized and degraded as described in BCE cells. The nature of the cell associated radioactivity is analyzed on SDS-polyacrylamide -gel electrophoresis and in ABAE and BBC cells about 30% of the I-Th appears in a complex with protease nexin I (PN I) while in BCE cells about 70% of the binding is mediated by PN I. ABAE cells possess 3 types of complexes, one which appears only on the cell surface with a molecular weight of 78 kDa, and two other complexes which appear only in the conditioned medium (CM) with molecular weights of 84 and 85 kDa. BBC and BCE cells demonstrate only one type of complex with a molecular weight of 77 kDa which appears both on the cell surface and in the CM. Preincubation of BCE cultures in the presence of Th is known to up-regulate the amount of PN I on the cell surface and in the CM, but this Th induced up-regulation effect is not observed in ABAE or BBC cells.The results described above indicate a difference between ABAE and BBC cells although both cell types growunder similar conditins and demonstrate similar morphological appearance. However, in both vascular endothelial cell types the total amount of PN I and its metabolism is relatively small compared to corneal endothelial cells. It, therefore, may indicate the lower capacity of vascular endothelial cells to control serine proteases activity at or near their cell surfaces as compared to corneal endothelial cells. This research was supported by a grant from the NationalCouncil for Research and Development, Israel and G.S.F. Munchen, Germany
DIFFERENTIAL STIMULATION OF INOSITOL TRISPHOSPHATE ACCUMULATION IN CULTURED HUMAN ENDOTHELIAL CELLS BY THROMBIN
Authors: Carter A J , Eisert W G , Mμller T H
Published: August 2018

Vascular endothelial cells possess specific receptors for thrombin, and thrombin can interact with these receptors to activate the endothelial cells. However, the signal transduction mechanisms which mediate the cellular responses are not yet characterised. The aim of this study therefore, was to determine whether thrombin influenced the inositol phosphate transduction pathway in cultured human endothelial cells. Endothelial cells were isolated from both large and small vessels; these were human umbilical vein and the microvasculature of human omentum respectively. The endothelial cells stained positively with antibodies against Factor VIII antigen and another endothelial cell specific antigen (BMA 120). Pure human thrombin (0.1 - 10 units/ml) induced a dose-dependent formation of inositol phosphate, inositol biphosphate and inositol trisphosphate (IP3) in endothelial cells from large vessels prelabelled with tritiated inositol. The formation of IP3 was significantly increased after 15 sec., maximal after 1 min. and had returned almost to baseline levels after 4 min. This time course is consistent with its role as a second messenger. When the enzymic activity of thrombin was removed with phenylalanyl-prolyl-arginine chloromethyl ketone or d i i sopr opyIfluorophosphate, thrombin lost its ability to stimulate the accumulation of IP3. Thrombin at all concentrations tested was unable to stimulate the formation of IP3 in small vessel endothelial cells. However, IP3 formation could be stimulated by bradykinin (0.1-10 μM) in cells from both small and large vessels. The results demonstrate that active thrombin can induce the formation of IP3 in large vessel endothelium. But that there are differences in the way small vessel endothelium responds to thrombin.
SIMULTANEOUS PRODUCTION OF ENDOTHELIAL CELL-DERIVED PROTEIN S AND FACTOR V, AND INACTIVATION OF FACTOR Va AT THE ENDOTHELIAL CELL SURFACE
Authors: Waart P v d , Preissner K T , Delvos U , Müller-Berghaus G
Published: August 2018

Several proteins synthesized and expressed by endothelial cells are involved in the regulation of coagulation. The synthesis and expression of factor V and protein S has been demonstrated in independent studies. The present work evaluates the simultaneous synthesis and expression of bovine factor V and protein S and the effect of endothelial protein S on the inactivation of endothelial factor Va by activated protein C. The accumulation of both proteins in conditioned medium was detected by SDS-PAGE followed by immunoblotting, and their activities were tested by functional assays. The synthesis of protein S and factor V per 105 cells over 24 h amounted up to 2 ng protein S and 440 ng factor V, respectively. The addition of thrombin did not increase the yield of synthesized cofactors. Thrombin did neither proteolyse protein S on endothelial cells nor in a purified system in the presence of thrombomodulin and calcium ions. Factor V was secreted partly in its activated form as evidenced by the appearance of active intermediates with M = 220,000-280,000 on immunoblots as well as by only a three-Fold further activation of factor V/Va following addition of thrombin. The rate constant for the inactivation of factor Va by activated protein C was only two-fold higher for factor Va derived from cells cultured in the presence of vitamin K as compared in the presence of warfarin. For the inactivation of comparable factor Va concentrations in conditioned medium a 10-fold higher and on endothelial cells a 40-fold higher concentration of activated protein C was required to obtain similar inactivation rates of factor Va as compared to a purified system. These results suggest that resting endothelial cells contain a factor V activator, and that a regulatory mechanism is operative on the endothelial cell surface that suppresses the inactivation potential of activated protein C/ protein S.
ROLE OF RELEASED ADP IN THE STABILIZATION OF PLATELET AGGREGATES: STUDIES IN PATIENTS WITH DEFICIENCY IN AMINE STORAGE GRANULES CONTENTS
Authors: Cattaneo M , Canciani M T , Mustard J F
Published: August 2018

Human platelets aggregated by thrombin (T) under conditions in which the release reaction (RR) occurs to only a small extent can be deaggregated by agents that dissociate 125I-fibrinogen bound to platelets. In contrast, when platelets undergo the RR, they cannot be reading deaggregated even though combinations of inhibitors cause 125I-fibrinogen to dissociate. Therefore, material released from platelet granules seems to stabilize aggregates. T-induced aggregates of washed platelets deficient in fibrinogen or von Willebrand factor cannot be deaggregated readily by deaggregating agents, implying that released fibrinogen or von Willebrand factor do not have a major role in stabilizing aggregates. To examine the role of platelet (δ-granule contents in stabilizing platelet aggregates, aggregation and deaggregation were studied with platelets from patients with (δ- Storage Pool Deficiency (δ- SPD). Platelet aggregation and the release ofβ-TG in response to T (1 U/ml) were similar for platelets from patients and controls. Platelets from patients (but not from controls) could be deaggregated by PGE1 (10 uM) plus chymotrypsin (10 U/ml), with hirudin (5 U/ml) added to block further effects of T. Addition of ADP (20 uM) to the (5-SPD platelets 5 sec after T abolished the ability of this combination of inhibitors to deaggregate the platelets. The addition of serotonin (2 uM) 5 sec after T did not prevent inhibitors from deaggregating δ-SPD platelets. When apyrase was added to normal platelets immediately before they were aggregated by T, the combination of inhibitors readily deaggregated the platelets. Therefore, released ADP may stabilize platelet aggregates through a mechanism that could be independent of released fibrinogen and von Willebrand factor.
VIRUS INACTIVATION AND COAGULATION FACTOR PREPARATIONS
Authors: Evatt B L
Published: August 2018

Nonheat-treated factor concentrates were used for the therapy of congenital and acquired coagulation deficiencies until 1984. These unheated factor crticentrates, which are manufactured from pooled plasma obtained from between 2500 and 25,000 blood or plasma donors, have been epidemiologically implicated in exposure of large numbers of hemophilia patients to several viral infections Including human immunodeficiency virus (HIV), hepatitis B, and non-A non-B hepatitis. Of these, HIV has been fdund to be very heat labile. After the introduction in 1984-85 of heat treatment of concentrates to reduce the risk of! hepatitis to recipients, several studies documented a lack of HIV serconversion in patients treated with clotting-fadtor concentrates. However, subsequent reports described a few hemophilia patients who had seroconverted to HIV! after receiving heat-treated concentrate from unscreened donors. To determine the significance of these seroconvers(ions, an international survey was conducted on 11 hemophilia treatment centers in Europe, Canada, and the United Kingdcpn whose total patient population comprised more than 2300 hemophilia A patients and 400 hemophilia B patients. Only three patients were found who seroconverted beyond a 6-month period after switching to heat-treated material, a(nd no seroconversions have occurred in these centers between November 1985 and February 1987. In addition no cases of seroconversion on donor screened heat-treated concentrate have been reported since its widespread introduction to the hemophilia patients during 1985-1986. Other modes of viral inactivation are currently being tested, and they appeiar to be effective in inactivating HIV and hepatitis B virus. Some of these methods have shown some promise for the inactivation of non-A and non-B hepatitis, but more data are needed for final assessment of these methods.
IDENTIFICATION OF ANTIPLATELET ANTIBODY IDIOTYPlSS ASSOCIATED WITH GLYCOPROTEIN Ib SPECIFICITY, PRESENT IN ITP PLASMA AND PRODUCED BY HUMAN HYBRIDOMAS FROM ITP SPLEEN CELL FUSIONS
Authors: Nugent Diane
Published: August 2018

Platelet membrane glycoproteins (GP) express anumber of antigenic determinants important in theetiology of autoimmune thrombocytopenia. Sensitization to GPIb, although not the most frequent cause of ITP, leads to a particularly severe form ofthe disease. We have identified a number of casesof ITP in which GPIb bears the relevant immunogen. Using GPIb-specific autoantibodies isolated from the plasma of one such patient, we have produced a number of rabbit polyclonal and murine monoclonal anti-idiotypic antibodies. These antibodies recognize an idiotype expressed on the IgM antibody of this patient as well as IgG or IgM antibodies from several other patients with ITP, all of which can be shown to bind specifically to GPIb. Statistical analysis of a series of plasmas from normal individuals and thrombocytopenic patients demonstrated that there is a very strong correlation between the presence of the idiotype and GPIb reactivity, (p < 0.00001). These anti-idiotypic antibodies are useful for the detection and characterization of GPIb-specific antibodies in the sera of patients with a clinically severe form of ITP. The classification of patients bearing this idiotype in their plasma may be useful in predicting disease outcome, thus identifying a group of ITP patients in whom more aggressive therapeutic regimens may be indicated. The use of these reagents and the development of human B lymphoblastoid cell lines producing monoclonal anti-GPIb antibodies will serve to elucidate the clonal origin and cellular regulation of autoantibody production in this disease
A SURVEY OF THE CLINICAL EFFICACY OF RETICULOENDOTHELIAL BLOCKADE EMPHASIZING INTRAVENOUS IgG AND ANTI-RH(D) GLOBULIN
Authors: Mueller-Eckhardt C
Published: August 2018

"Blockade" of the RES is the maj0or therapeutic principle in autoimmune thrombocytopenic purpura (AITP) and related conditions. It can be accomplished by a number of possibilities which include, first, removal of the main sequestration site of antibody-sensitized platelets (i.e. splenectomy), or, second, functional "modulation" of the phagocytic capacity of the RES. The latter is achieved by either "unspecific" or "specific" means. "Unspecific" means are defined as being operative via a mechanism in which apparantly no immunological reactants are involved, while "specific" means comprise the direct or indirect participation of antibodies and/or immune complexes. "Unspecific" means are the injection of red blood cell (RBC) stroma (SHULMAN et al, 1965) or heat-treated RBC (PREIS et al, 1980). "Specific" means seem to be effective during the course of some patients with acute viral infections as mirrored in spontaneous remissions of thrombocytopenia, and by treatment with high doses of human IgG (HDIgG) (IMBACH et al, 1981) or with anti-D (SALAMA et al, 1983). Until now more than 30 studies on the efficacy of HDIgG have appeared describing now over 600 patients. Most IgG preparations for intravenous use on the market have been used. Although they differ in their content of di- and polymers and the integrity of their Fc portion they all seem to be effective. Pepsin-treated IgG, however, is not. A response rate to HDIgG of approximately 60-70% of AITP patients is usually seen with the platelet peak between day 5-10 post infusions; the effect is always transitory. The mechanism(s) of HDIgG is (are) still obscure. Since pepsin-treated IgG is ineffective the Fc portion of IgG seems indispensable. Interference with Fc-IgG receptors of the RES by HDIgG via Fc receptor blocking antibodies or via immune complexes is at present the most likely explanation. Coating of autologous RBC by anti-D antibodies is an alternative way for RES "blockade". It is similarly effective in AITP as HDIgG, but, unlike HDIgG, much more in unsplenectomized than in splenectomized patients. Its mechanism is dependent on antibody-coating of RBC, since Rh (D)-negative recipients are unresponsive to anti-D, but responsive to anti-c. Comparative studies of RES clearance of racdio-actively labelled aggregated albumin have shown that the clearance of liver uptake is significantly increased by HDIgG, but not by anti-D, although their influence on the platelet count is similar. This speaks in favor of a more general effect of HDIgG on the RES, whereas anti-D is Fc-receptor dependent. The clinical efficacy and conceivable mechanisms of these recent treatment modalities will be discussed.
CALCIUM IN PLATELET ACTIVATION
Authors: Rink T J
Published: August 2018

Studies with calcium ionophores, permeabilised platelets, and platelets containing fluorescent calcium indicators quin2 and fura-2 have shown that elevation [Ca2+]i is an effective trigger for shape-change, aggregation, secretion and release of TxA2; and that elevation of [Ca2+]i is an important part of a complex “activation cascade” set up by natural agonists combining with their surface receptors. We have used calcium ionophores to impose [Ca2+]i changes, monitored by indicator dyes, to construct [Ca2+]i/function relationships for shape-change, secretion, aggregation, arachidonic acid release, TxA2 production, and myosin phosphorylation in intact platelets(e.g.1,2). Some of these functions can also be studied by analagous experiments using Ca2+-buffers to set known [Ca2+]i in permeabilised platelets. Our ability to monitor and modulate [Ca2+]i with fluorescent indicators has also allowed us to see what happens when [Ca2+]i changes are greately reduced or even absent and to investigate other pathways of intracellular activation. We think that formation of diacyl glycerol and activation of protein kinase-C can explain, some, but not all, of the cell activation that some agonists can apparently evoke at or near resting [Ca2+]i, and that combined or synergistic actions of Ca2+ and other intracellular mediators is the usual basis for physiological activation(3). Most agonists seem to promote both Ca2+ entry across the plasma membrahe and discharge from intracellular organelles, presumably the dense tubular system. The available evidence fits with the prevailing idea that Ins 1,4,5 P3 formed by agonist evoked hydrolysis of PIP2, is the internal messenger for Ca2+ release. Our kinetic measurements of [Ca2+]i transients require that optimal concentrations of InsP3 are formed within 250 milliseconds(4,5). The question of whether ADP receptors in human platelets are directly coupled with PIP2 breakdown remains contentious. Probably they do, weakly, and the differences from most other receptors are quantitative rather than qualitative. We do not understand the mechanisms of agonist-evoked Ca2+-entry; there is now plenty of evidence that argues against a role for membrane depolarisation and voltage-gated Ca channels, including some recent work with ionic substitution(5). Stopped-flow fluorescence analysis of [Ca2+]i rises in fura-2-loaded human platelets reveals some intriguing new insights(4,5). With thrombin, vasopressin and PAF at optimal concentrations, there is a highly reproducible delay before the signal starts to rise, which is approximately 250msec in the absence of external calcium compared to 190msec in the presence of external calcium. This suggests that Ca entry leads internal release, and gives ample time for complex coupling mechanisms for both processes.

The delay with ADP, in the presence of external calcium, is much smaller suggesting a different coupling mechanism for Ca entry.
ACTIVATION OF FACTOR VII IS RELATED TO BLEEDING TENDENCY IN LIVER CIRRHOSIS
Authors: De Angelis V , Zambon M , Toffolo L , Donada C , Molaro G L , Zuin R
Published: August 2018

Coagulation abnormalities are among the number of potential risk factors toinitiate the bleeding episodes from gastrcr-esophageal varices in liver cirrhosis. The impairment of liver clearance of activated coagulation factors, the release of thromboplastin-like activity from the necrotic liver cells and the hemodynamic changes due to expanded bollaterals may all contribute to activate the coagulation cascade.

However, little is known about the mechanisms leading to this activation. Activated Factor VII (FVIIa) is known totrigger both intrinsec and extrinsec coagulation pathway. Therefore, we measured FVIIa in a group of 33 cirrhotic patients in order to see if a difference between bleeders and non-bleeders patients would correlate with Factor VII activation. The patients were divided in two groups according to the presence or the absence of major bleeding from gastroesophageal varices; haemorragic episodes were confirmed by a gastroscopic examination performed during or immediately after bleeding episodes. Factor VII coagulant assay (VII:C - one stage clotting method) and Factor VII coupled amidolytic assay (VII:CHR) were performed and a factor VII activity ratio (VIIa) was calculated as VII:C/VII:CHR. The results (mean ± S.E.) are summarized in this table:

No difference in Vila distribution was seen when the patients were divided on the base of liver impairment (according to Child’s criteria) .Our study shows that FVII activation is related to bleeding from esophageal varices but not to the degree of liver impairment and strongly suggests the existence of an hypercoagulable state in liver cirrhosis, probably related to major bleeding from gastroesophageal varices in cirrhotic patients.
IMMUN0BL0TTING STUDIES OF THE INTERACTION OF PLASMA-FACT0R XI WITH KAOLIN
Authors: Mannhalter C , Deutsch E
Published: August 2018

An immunoblotting technique with a commercially available, polyclonal Factor XI antibody was developed for Factor XI in plasma samples.Aliquots containing equivalents of 6 ul plasma were treated with appropriate amounts of SDS, electrophoresed on 7.5 % SDS-PAGE and immunoblotted onto nitrocellulose membranes. The blots were quenched with low fat milk and incubated with 2 % Factor Xl-antiserum, which was preadsorbed with Factor XI deficient plasma. The bound Factor XI antibody was identified by incubation with 125I-labelled Factor XI followed by autoradiography. Using this method, the interaction of Factor XI with kaolin was studied in plasma. Normal plasma was incubated with kaolin for various time periods. Aliquots were removed, centrifuged and the plasma supernatant and the kaolin pellets were examined after treatment with SDS on 7.5 % SDS-PAGE followed by immunoblotting.

Factor XI was adsorbed onto kaolin and, after five minutes no Factor XI antigen was present in the plasma supernatant.

However, after twenty minutes a band corresponding to Factor XI became again visible in the plasma, indicating a release of Factor XI from the kaolin surface. The electrophoretic results obtained with the SDS-eluates of the kaolin pellets confirm these observations. After thirty seconds a significant amount of Factor XI was present on the kaolin, and it exhibited the same electrophoretic mobility as Factor XI in the starting material. After ten minutes incubation, the protein concentration adsorbed onto the kaolin had increased. However, after twenty minutes clearly less Factor XI was present on the kaolin than after ten minutes.

Thus, our results indicate that Factor XI is quickly adsorbed from plasma onto kaolin. However, Factor XI or XIa do not remain surface-bound but are released into solution.
SULPHINPYRAZONE (SP) AS A CAUSE OF ACUTE RENAL FAILURE (ARF): A REVIEW
Authors: Boelaert J , Lijnen P , Daneels R , Schurgers M , Amery A
Published: August 2018

A total of 41 patients (all but one since 1980, 10 from our own group) have been reported with ARF due to SP. Their mean age was 58 years. 84 % of them received SP for coronary or cerebrovascular disease. Signs of pre-existing renal disease were absent in 91%of cases. Daily dosage of SP was < 400 mg in 21 %, 600 mg in 21 % and 800 mg in 58 % of the cases. ARF appeared within the first day of R/ with SP in31 % of cases(median delay of 4 days). Oliguria was present in 41 % and lumbar or abdominal pain in 26 %. Urinalysis showed uric acid crystals in 13 %. Serum creatinine peaked at a mean of 7.2 mg%. Extrarenal signs (fever, rash) were rare (10 %). 3 patients (8 %) died; renal function recovered in the others, acute dialysis being needed in 6 cases (15 %). Renal histology (11 cases) showed either no lesions (2), minimal tubulo-interstitial lesions (3), discrete interstitial infiltration (2), acute interstitial nephritis (3) or acute tubular necrosis (1).SP can cause ARF by 3 mechanisms which are not mutually exclusive : acute urate nephropathy, acute (immunological) interstitial nephritis or acute ischaemia due to inhibition of the renal synthesis of kallikrein-kinin and/or vasodilatory prostaglandins. We suggest that the latter mechanism is the most prevalent. The effect of SP on the renal prostaglandin synthesis is not settled; thurinary excretion of kallikrein is significantly depressed by SP

In conclusion : since 1980, R/ with SP is a frequent cause of ARF
Practically:
1) the indications for sp should be taken with care;
2) a progressive increase in the dosage of SP may decreses the incidence of ARF.
DN.9693 COMPARED WITH PROSTACYCLIN AND PROSTAGLANDIN E1 IN SEVEN PLATELET TESTS
Authors: O'Brien J R , Etherington M D , Salmon G P
Published: August 2018

A new drug, DN.9693, has low Km phosphodiesterase inhibitory properties. Its effect on seven broad spectrum platelet "function" tests has been compared with the effects of prostacyclin E1 (PGI1) and prostaglandin E (PGE ). The tests were (1) platelet aggregation induced by ADP, collagen, adrenaline, thrombin, arachidonic acid and ristocetin; (2) a new test in which platelets aggregate after adding distilled water to cause osmotic stress; (3) the loss of platelets washed in buffered saline; (4) clot retraction; (5) the glass bead column platelet retention test; (6) the in vitro filter "bleeding time" (see two other submitted abstracts); (7) the amount of platelet factor 4 (PF4) which "leaks" from platelets at room temperature.PGI2 inhibited all seven tests, 50% inhibition of the various tests required from 0.1 to 44ng/ml of PGI2. PGE1 also inhibited in all tests but on average required 18 times higher concentrations. Thus an increase in cAMP may be relevant to all these tests, but an understanding of the mechanisms involved is incomplete. DN.9693 inhibited only the first four tests; the equi-active concentration was about 600 times that of PGI2. DN.9693, 2.5μg/ml, caused 50% inhibition of ristocetin induced aggregation and at 4μg/ml had a minor effect on the filter bleeding time. Thus DN.9693 may affect the platelet membrane glycoproteins. In conclusion it is confirmed that PGE1 is less active than PGI^ but has similar activities. DN.9693 whenstudied in these tests has many, but notall, of the prostaglandin-like properties.