bluuebunny/crossref_metadata_2025 huggingface dataset:
EVALUATION OF THE PROPOSED FUNCTIONS OF PROTEIN KINASE C IN PLATELET SIGNAL TRANSDUCTION BY THE USE OF A DIACYLGLYCEROL KINASE INHIBITOR
Authors: de Chaffov de Courcelles D , De Clerck F , Roevens P
Published: August 2018

Protein kinase C is suggested to play a major role in propagation as well as in termination of excitatory signal transduction in the platelet. Most of its properties were discovered by the use of synthetic diacylglycerol analogs or phorbol esters that directly stimulate protein kinase C. It is, however, unknown to what extent activation of the protein kinase C by these exogenously added compounds can be compared to that after receptor activation. To evaluate the role of protein kinase C in excitatory signal transduction, we transiently elevated the endogenous diacylglycerol level after receptor activation by the use of a diacylglycerol kinase inhibitor (R 59 949). On addition of the agonist vasopressin to platelets prelabeled with [32P] orthophosphate, 32P-phosphatidic acid (PA) formation was inhibited by R 59 949 in a dose-dependent manner (IC50 α 10−6 M). Vasopressin induced formation of 32P-phosphatidylinositol-4’-phosphate (PIP) and the phosphorylation of the 40 k Da protein (major substrate of the protein kinase C) were increased in the presence of the compound. In platelets prelabeled with [3H]-inositol, the agonist-induced formation of all the water-soluble inositol phosphates was inhibited in the presence of the diacylglycerol kinase inhibitor and Li+. Vasopressin induced increase in intracellular Ca2+ was lower in the presence of R 59 949. The platelet shape change induced by a threshold concentration of vasopressin was reduced by the compound. By contrast, the rate and the maximum of the first-wave aggregation was enhanced in the presence of R 59 949.

These data evidence that protein kinase C, stimulated by endogenously generated diacylglycerol after receptor activation, plays a major inhibitory role on the primary steps of signal transduction since its activation reduces i) phospholipase C activity and ii) the increase in intracellular Ca2+ and the concomitant shape change reaction. The inhibitory role of protein kinase C on signal transduction is largely independent of its stimulatory role on platelet aggregation. Our data further confirm that stimulation of protein kinase C induces the formation of PIP but questions the role of the kinase in the breakdown process of inositoltrisphosphate.
GENE STRUCTURE OF VITAMIN K-DEPENDENT PROTEIN S; A REGION HOMOLOGOUS TO SEX HORMONE BINDING GLOBULIN (SHBG) REPLACES THE SERINE PROTEASE REGION OF FACTORS IX, X AND PROTEIN C
Authors: Edenbrandt C-M , Gershagen S , Femlund P , Wydro R , Stenflo J , Lundwall Å
Published: August 2018

It has recently been shown that the similarity between coagulation factors IX, X and protein C in the protein sequence is also evident in the organization of their genes. To further elucidate the relation of protein S to the other vitamin K-dependent clotting factors, we are now characterizing the human protein S gene. The size of the gene was estimated to be more than 45 kb, by hybridization of a cDNA for human protein S with chromosomal DNA in a Southern blot.We have isolated three overlapping clones from a human genomic DNA library in bacteriophage λ Charon 4A, which cover approximately 40 kb of the gene. The clones have been mapped by single- and double restriction enzyme digestion. Genomic subclones in pUC 18 which hybridize with cDNA probes for protein S have been isolated and sequenced to establish the intron/exon structure of the gene. The 5’- part of the human protein S gene closely resembles the corresponding part of the genes for factors IX, X and protein C. However, the thrombin sensitive region (amino acids 46-75), which is unique for protein S among the vitamin K-dependent clotting factors, is coded for by a separate exon. The 3'- end of the protein S gene, coding for amino acids 247-635, is not homologous to the catalytic region of the vitamin K-dependent serine proteases but shows a significant homology to human sex hormone binding globulin (SHBG).
MEASUREMENT OF FACTOR XIII ACTIVITY IN HUMAN PLATELET HOMOGENATE BY A NEW UV-KINETIC METHOD
Authors: Polgár J , Hidasi Y , Toth A , Muszbek L
Published: August 2018

Factor XIII (FXIIl) of blood coagulation is a zymogen which is converted into an active transglutaminase during the clotting process. Earlier methods used for its determination are cumbersome, laborious, and not suitable for routine laboratory measurements.Most recently we have designed a new simple UV-kinetic assay for the determination of FXIII in the plasma (Muszbek et al., Clin. Chem., 3JL, 35, 1985). The assay is performed on def:j.b-rinated plasma in which FXIII is activated by thrombin and Ca2+. Acetylateddephosphorylated (AD)β-casein and ethylamine are used as substrates and the ammonia released during thereaction is continuously monitored by a NADPH dependent indicator reaction at 340 nm. As the enzymatically active a subunit of FXIII is also present in platelets and monocytes/macrophages we attempted to adapt the above method for the measurement of cellular FXIII activity. Experiments were carried out on Lubrol extract of washed sonicated platelets. It was found that the small amount of fibrinogen present in platelets does not need to be removed and in the blank hirudin used for preventing activation of plasma FXIII should be replaced by EGTA. The concentration of substrates and activators were optimized. The methodwas found linear at least up-to 40 U/l enzyme activity. It had a good reproducibility (optimal conditionvariance was less than 3%) and correlated well with the most commonly used fluorescent amine (dansylcadaverine) incorporation assay. The method was adapted to a centrifugal fast analyser (Baker, Centrifichem). In addition to congenital FXIII deficiency the determination of FXIII in platelets by this new methodmight have a diagnostic importance in haemopoietic diseases with diminished or accelerated platelet production.
CELLULAR DISTIBUTION OF FACTOR XIII IN HUMAN UTERUS AND PLACENTA
Authors: Muszbek L , Adány R
Published: August 2018

As spontaneous abortion is a frequent finding in females with Factor XIII (FXIIl) deficiency it has been presumed that the plasmatic or cellular form of this clotting factor is essential to normal fetus development. In this context it is of special interestthat FXIII subunit a has been demonstrated in the homoge-nate of human uterus and placenta by activity measurements and immunobiochemical methods. However, no information on its cellular distribution has beenpublished, so far. In the present study first FXIII subunit a was detected in paraformaldehyde-fixed, paraffin-embedded sections by immunoperoxidase technique. FXIII containing cells were localized in the connective tissue of uterus and in the mesenchyme of placental chorionic villi. Though in a single report FXIII containing connective tissue cells were interpreted as fibroblasts (Fear et al., J.Clin.Pathoi.,37,560, 1984) the mononuclear, multipolar, stellate morphological appearance of these cells suggested that they rather belong to the monocyte/macrophage cell line. To characterize them the immuno-fluorescent detection of FXIII subunit a was combined by the visualization of different marker antigens for tissue macrophages (RFDT, Leu M3, HLA-DR) and fibroblasts (IIG10) on frozen sections. The coexpression of FXIII subunit a with RFD7 and Leu M3 macrophage markers, butnot with fibroblast membrane antigen IIG10 clearly proves that FXIII containing cells both in the uterusand in the placenta are tissue macrophages. HLA-DR was strongly expressed in cells positive for FXIII subunit a. in the uterus, but not or only very weakly in the placental mesenchyme, which might be due to the relative absence of extrinsic antigens during fetal development. The morphological findings on the presence of FXIII subunit a in placental macrophages were further strengthened by immunobiochemical experiments. FXIII subunit a, but not b content of isolated placental macrophages was also verified by immunoblotting, while fibroblasts were shown to be exemptof this factor. The results well agree with our previous findings demonstrating FXIII subunit a in humanmonocytes and peritoneal macrophages (Muszbek et al., Thrombos. Res. ,37, 401,1985; Adány et al. , Eur.J.Cell Biol.,38,171,1985).
EFFECTS OF NICOTINE AND COTININE ON THE SYNTHESIS AND RELEASE OF PLASMINOGEN ACTIVATOR FROM BOVINE AORTIC ENDOTHELIAL CELLS
Authors: Kuo Be-Sheng , Dryjski Maciej , Bjornsson Thorir D
Published: August 2018

While cigarette smoking has beenimplicated in the development of cardiovascular diseases, it has been reported to increase fibrinolytic activity in vivo. However, no data is available regarding the underlying mechanism of action. The present investigation was carried out to evaluate the effects of nicotine and its major metabolite, cotinine, on the seretion of plasminogen activator (PA) and PA inhibitor (PAI) by cultured bovine aortic endothelial cells. PA activity was determined by the fibrin plate method, and individual molecular species with PA and PAI activities were separated and visualized using SDS-PAGE with zymography and reverse fibrin autography. Both nicotine and cotinine increased PA secretion in a time- and dose-dependent manner. A maximum stimulation in PA secretion after 24-hour incubation was observed for nicotine at 10-8 M and for cotinine at 10-7> M, which corresponded to 2.5- and 2.7-foldincreases over control, respectively. These concentrations are in the range observed after cigarette smoking. The pharmacological stimulation of PA activity required both RNA and protein synthesis, as evidencedby its inhibition by cycloheximide and actinomycin D. Both control cells and cells treated with nicotine and cotinine produced multiple forms of PA and a single form of PAI. The PAI was mainly of the latent form as no quenching effect was observed on standard tissue plasminogen activator (tPA) and urokinase (UK) after they were mixed with the conditioned culture medium. The PAs werefound to consist of both tPA and UK,and the corresponding complexes with PAI, however, the UK bands were wider than the tPA bands. Both species were enhanced by nicotine and cotinine. Although activities of all species of PA were enhanced by nicotine and cotinine, these compounds had no apparent quantitative or qualitative effects on the release ofPAI. These results thus suggest that the mechanism underlying the enhanced fibrinolytic activity after cigarette smoking may be due to nicotine- and cotinine-induced stimulation of PA synthesis and subsequent release.
CYCLOOXYGENASE AND LIPOXYGENASE PRODUCTS SYNERGISTICALLY MODULATE TUMOR CELL INDUCED PLATELET AGGREGATION
Authors: Steinert Bruce W , Onoda James M , Sloane Bonnie F , Taylor John D , Honn Kenneth V
Published: August 2018

There has been considerable controversy surrounding the ability of inhibitors of arachidonic acid metabolism to concomitantly inhibit tumor cell induced platelet aggregation (TCIPA). Reconciliation of this controversy has been difficult due to the wide variability of experimental conditions (e.g., inhibitor concentration, strength of the inducing agonist).In the present study, we examined the effects of several cyclooxygenaseand lipoxygenase inhibitors on the induction of platelet aggregation by Walker 256 carcinosarcoma (W256) cells. We have previously demonstrated that aggregation of platelet rich plasma (PRP), induced by W256 cells, was initiated via a thrombin dependent mechanism. Platelet aggregation was induced by the addition of W256 cells (75,000-J500,000 cells/cuvette) to rat PRP preincubated with inhibitor(s) or diluent. The strength of the inducing stimulus affected both the degree of aggregation and the production of thromboxane A2 (TXA2) in a dose dependent manner. A weak stimulus (low concentration of W256 cells) produced only a low level of aggregation and low TXA2 production, whereas aggregation induced by a strong stimulus (high concentration of W256 cells) resulted in significant aggregation and increased TXA2 production. Preincubation (5 min., 37°C) of rat PRP with cyclooxygenase inhibitors (e.g., aspirin, indomethacin, ibuprofen) resulted in complete inhibition of platelet aggregation at low agonist concentration (75,000 W256 cells), whereas when a high agonist concentration (500,000 W256 cells) was used to induce aggregation, the inhibitors failed to inhibit TCIPA. The addition of fewer than 50,000W256 cells failed to induce any measurable platelet aggregation in the presence or absence of inhibitors. TCIPA was not affected by lipoxygenaseinhibitors (e.g.,quercetin) alone regardless of agonist concentration. Both cyclooxygenase and lipoxygenase inhibitors, however, were required to significantly inhibit TCIPA induced by high agonist concentration. Compounds which inhibited both the cycloogygenase and lipoxygenase pathways (e.g.,hydroquinone, BW755c) inhibited TCIPA at all agonist concentrations. Nafazatrom failed to inhibit TCIPA consistant with a lack of effect on platelet cyclooxygenase and lipoxygenase. Therefore, we conclude cyclooxygenase (e.g., TXA2) and lipoxygenase (e.g., 12-HETE) products of platelet arachidonic acid metabolism and the strength of the inducingagonist are important criteria in TCIPA. This study may help to clarify the current controversy regarding the inhibition of TCIPA by inhibitors of arachidonic acid metabolism.
A MONOCLONAL ANTIBODY (PL/IM 430) THAT BLOCKS THE ACTIVE TRANSL0CATI0N OF Ca2+ INTO HUMAN PLATELET INTRACELLULAR MEMBRANE (ER) VESICLES
Authors: Hack N , Wilkinson J M , Crawford N
Published: August 2018

In earlier studies [1] we identified a number of important biological properties associated with highly purified human platelet intracellular membrane (ER), isolated by continuous flow electrophoresis. These included a high affinity Ca2+Mg2+ ATPase and protein phosghorylation both of which are involved inthe active uptake of Ca into ER vesicles. The stored Ca2+ could be released with inositol(1,4,5)trisphosphate, (IP ), (approx. 50% release in 30 s 1/2 max. for release - 0.253 μM IP3,) [2]. To probe the structure-function relationship of proteins in these ER vesicles, a panel of monoclonal antibodies (Mabs) has been raised, using the ER membrane preparation as immunogen. Four of these Mabs recognise a single 100 kDa polypeptide by immunoblotting. This protein is present in platelet membranes and can also be identified in cultured human monocyte, macrophage and endothelial cell lines. None of the M^bs showed any significant effect upon the ER membrane Ca2+ Mg2+ ATPase activity but one, PL/IM 430 (of IgGl subclass), inhibited the Ca2+sequestration by the vesicles significantly (approx. 70% inhibition at 10 μM IgG). This inhibition was independent of the ATP concentration over a range2of 0-2 mM ATP, but was2dose-dependent for external free Ca 2between 30-300 nM Ca2+, giving maximum inhibition at 300 nM Ca with 10 pM IgG2+ Binding of the antibody substantially lowers the Vmax for Ca2+for Ca2+ uptake but is without effect upon the Km. PL/IM 430 therefore appears to recognise a 100 kDa polypeptide closely involved with Ca2+ trnslocation but at a site which i, s without effect upon the Ca2+Mg− ATPase associated with the Ca pump.

We are grateful to the Wellcome Trust and the British Heart Foundation for financial support for these studies.

[1] Hack, N., Croset, M. and Crawford, N. (1986) Biochem. J. 233, 661-668.

[2] Authi, K. S. and Crawford, N. (1985) Biochem. J. Z3O, 247-253
PROGRESSION OF HIV INFECTION IN THE POPULATION OF FRENCH HEMOPHILIACS
Authors: Sultan Y
Published: August 2018

A national inquiry including 28 hemophilia centers was carried out in France in order to appreciate the epidemiology of HIV infection among hemophilic patients. Information about 1781 patients was obtained with an overall prevalence of 50% seropositive patients. This percentage of HIV seropositivity was unchanged in comparison with 1985 confirming that no seroconversion was observed since the use of heat treated products for bleeding episodes. It is to be noted that there is an important progression in the number of AIDS which increased from 16 hemophiliacs last year to 23 this year with a total of 11 deaths against 7 last year related to this affection. In the' remaining hemophilic population, twenty per cent of HIV positive patients have developed an ARC. For the biological abnormalities related to immune deficiency, it was found that patients with lymphopenia less than 1000 lymphocytes had not progressed and represent 23 patients. Patients with thrombocytopenia less than 150,000 platelets had increased from 29 to 62. Patients with a number of T4 lymphocytes subset less than 400 ha increased from 54 to 110. 22% of HIV positive hemophiliacs had a T4/T8 ratio less than 1 in 1986 in comparison with 11% in 1985.17.5% of HIV positive population showed an elevated level of gamma-globulins above 20 g/liter of plasma against 12.5% last year. The conclusion of the present study is that HIV infectionhas progressed in severity from 1985 to 1986 in the population of multitransfused HIV positive French hemophiliacs.
GENERATION OF COMPLEMENT ACTIVATION PEPTIDES DURING STORAGE OF PLATELET CONCENTRATES (PC)
Authors: Bode A P , Miller D T , Newman S
Published: August 2018

Platelets are routinely stored for transfusion at room temperature in autologous, citrated plasma. We have demonstrated previously that these conditions do not completely block activation of plasma enzyme systems, as indicated by generation of thrombin activity (Vox Sanguinis, JL1:192,1986). Here, we demonstrate the conversion of large amounts of complement factor C3 during storage of citrated PC by using radioimmunoassay quantitation of the activation peptide C3a des-Arg (Upjohn Diagnostics). Supernatant samples from stored PC and from citrated platelet-poor plasma (PPP) stored under the same conditions showed a rapid linear increase in C3a levels over time with no significant difference (paired t-test, p<0.5) between PC and PPP (see table). The values at Day 10 represent conversion of approximately 11% of the native C3. Possible effects on stored platelets of C3 conversion in the surrounding plasma include:activation of platelets by C3a des-Arg (M.Polley and R. Nachman; J.Exp.Med. 158:603, 1983) and deposition of C3b on the cell surface as "innocent bystanders" (A. Salama and C. Mueller-Eckhardt; Transfusion 25:528,1985).

In contrast, <10 ng/mL C5a was found in all samples tested, representing less than 0.2%conversion of C5a.Nephelometricassay of native C5 levels in PC samples showed a slight but significant difference by a paired t-test (p=0.04) between fresh PC (mean=117 ug/mL±12.0, n=6)and P
stored for 10 days(nean=108 ug/mL±9.7). Nochange in C5 levels was observed in stored PPP (106 ug/mL to 107 ug/mL). Radiolabelled monoclonal antibodies to C3 fragments showed less than 600 molecules bound per platelet. This study demonstrates for the first time the extent of complement activation in stored platelet concentrates.
PLASMINOGEN ACTIVATOR INHIBITOR ACTIVITY IN SEPTICAEMIA
Authors: Páramo J A , Cuesta B , Hernrnández M , Fernrnández J , Paloma M J , Rocha E
Published: August 2018

In vitro and in vivo studies have shown that endotoxin induces a marked increase in plasma plasminogen activator inhibitor (PAI) activity. Plasma PAI and endotoxin concentration (limulus lysate chromo-genic peptide substrate) were determined in 61 patients with sepsis: temperature greater than 38° and either positive blood cultures (n= 32) or negative blood cultures in neutropenic patients. Thirty age-matched healthy subjects served as control group.

There was a marked increase in PAI in patients (7.1±10.5 U/ml) as compared to controls (0.9±0.8 U/ml) with statistical differences (p<0.002). Mean endotoxin concentrations in patients was 1779 pg/ml (Ref. = no detectable). PAI concentration was significantly higher in patients with positive blood cultures (p<0.009). Such an increase was higher in patients with Gram-negative bacteria (n= 26) than in those with Gram-positive bacteria (n= 6), but without statistical differences. The highest PAI concentration was found in 9 patients with disseminated intravascular coagulaition (DIC) as compared with those without DIC (p<0.002). No correlation was found between PAI and endotoxin concentrations.We conclude that there is a marked increase of PAI activity in septicaemia which may contribute to the pathogenesis of DIC-associated sepsis.
TWO ABNORMAL FIBRINOGENS DESIGNATED AS OSAKA II AND MORIOKA WITH A HITHERTO UNIDENTIFIED AMINO ACID SUBSTITUTION; γARG-275 BY CYS
Authors: Terukina S , Matsuda M , Yoshida N , Yamazumi K , Takeda Y , Takano T
Published: August 2018

A hitherto unidentified amino acid substitution of γ Arg-275 by Cys has been found in two abnormal fibrinogens, Osaka II and Morioka. The propositi are both asymptomatic heterozygotes for the abnormality characterized by altered polymerization of fibrin monomers. Reducing SDS-PAGE revealed that fibrinogens derived from thé propositi both consist of two populations; one with a normal and the other with an abnormal longer γ-chain by 0.5 Kd.

The γ-γ cross-linking took place nearly normally, however. Analyzing plasmic digests of fibrinogen by SDS-PAGE, we located the abnormality residing in the γ-chain remnant of fragment D. Chromatofocusing of D1 obtained by plasmic digestion in 5 mM Ca++ of purified fibrinogen separated the variant D1 (vD1) from the normal one (nD1) distinctly, as confirmed by SDS-PAGE and functional studies. As anticipated, vD1 failed to interfere with normal fibrin polymerization and thrombin clotting of normal fibrinogen, whereas nD1 inhibited these reactions significantly. After reduction and pyridylethylation, vD1 and nD1 were individually digested with lysylendopeptidase (lysEP). Analyzing the digests by reverse phase HPLC, we noted a single peak present in the digests of vD1 but missing in those of nD1, and vice versa. Analysis of N-terminal five cycles of these peptides suggested that both of them corresponded to the peptide with residues 274302 based on the known sequence data. Primary sequence and total amino acid analyses revealed that γ Arg-275 has been substituted by Cys in both of these abnormal fibrinogens. Analysis of the lysEP-digests of the isolated γ-chain also gave the same result. Since no free SH has been identified at the γ Cys-275 substitute, the variant γ-chain may be endowed with some additive by an S-S linkage. Even if so, elucidation of an apparent elongation by SDS-PAGE of the γ-chain variant must await further investigation. In any case, however, the substitution of γ Arg-275 by Cys may have induced critical alterations in the γ-chain-dependent polymerization site in the D domain in these two abnormal fibrinogens.
REMOVAL OF HUMAN ANTIBODIES TO FACTOR VIII :C FROM HEMOPHILIAC PLASMA USING NEW SYNTHETIC SORBENTS
Authors: Messaïkeh H , Belattar N , Gulino D , Jozefonvicz J , Sultan Y
Published: August 2018

Human antibodies that neutralize factor VIII procoagulant activity (Anti VIII:C) detected in polytransfused patients with hemophilia A cause serious difficulties for the affected patients as they inactivate any injected FVIII preparations.

We hypothesized that FVIII:C might possess active sequences composed of amino acids able to bind Anti VIII:C antibodies. Consequently, completely synthetic resins with suitable chemical substituents mimicking these sequences might interact with Anti VIII:C antibodies. Based upon this hypothesis, crosslinked polystyrene was substituted by various amino acids or their derivatives in order to obtain completely synthetic adsorbents able to remove Anti VIII:C antibodies from hemophiliac plasmas. To establish the relationship between chemical composition of the resins and their affinity towards Anti VIII:C antibodies, the "in vitro" removal of these inhibitors from hemophiliac1s immunoglobulins G was tested by measuring simultaneous adsorptions of either IgG or Anti VIII:C antibodies. Specific and accurate methods veil adapted for studying the adsorption of these two kinds of proteins were used for evaluating either the IgG concentrations (rocket iirrnunoelectrophoresis) or the Anti VIII:C concentrations (immunoradicmetric method). To determine the most suitable chemical groups able to develop a specific adsorption, a first screening on amino acid substituents was undertaken and allowed the selection of glutamic acid and hydro -xyproline. In fact, among the twenty resins tested, the most interesting one is obtained by linking glutamic dimethyl ester derivative onto the polystyrene matrix. This resin possesses a pseudo-specificity towards Anti VIII :C antibodies as it is possible to remove 12 % of Anti VIII :C antibodies and only 3 % of IgG on 5 mg of this resin. Furthermore, the hydrophobic sites on the surface seems to be involved in the Anti VIII :C adsorption as demonstrated by comparing selectivity obtained with mono and diester derivatives either in aspartic or glutamic series.
TOLERANCE INDUCTION IN HIGH-RESPONDING HEMOPHILIACS WITH F VIII ANTIBODIES BY MEANS OF COMBINED TREATMENT WITH IgG, CYCLOPHOSPHAMIDE AND F VIII
Authors: Nilsson I M , Berntorp E , Zettervall O
Published: August 2018

Of 10 patients with hemophilia A and antibodies, 7 have been rendered tolerant by means of combined treatment with high-dose IgG i.v., cyclophosphamide and F VIII. When the initial antibody concentration exceeded 10 Bethesda inhibitor units per ml, the treatment was preceded by antibody adsorption to protein A. Six of the tolerant patients were originally classified as high-responders. After one week of the combined treatment, VI11:C dropped and the inhibitor reappeared in low titer. The F VIII infusions being continued alone, the inhibitor disappeared in the following week and VIII:C increased satisfactorily after infusion, while VIII:Ag (assayed immunoradiometrically) reached very high concentrations. In one patient the treatment had to be repeated once. Except for transient leukopenia, no side effects occurred. Earlier treatments with F VIII in combination with cyclophosphamide gave high anamnestic response. In two of the remaining three non-tolerant patients, anamnestic response decreased dramatically after two courses of the combined treatment. The tolerant state seems to be stable, as the tolerant patients have now been on regular prophylaxis with F VIII concentrate for periods varying from four months to four years. The half-life of infused F VIII is normal, while that of VIII:Ag is prolonged. On the basis of similar findings in hemophilia B patients, we believe the VIII:Ag to have become modi Tied and complexed to a 'new' antibody which lacks VIII:C inhibitory activity. It is known that modified antigen may act as a tolerogen. The tolerant state may thus be sustained by maintaining consistent concentrations of the modified antigen by means of the F VIII treatment. We conclude that the combined treatment described here is a safe and effective method of tolerance induction in hemophilia A patients.
FIBRIN STIMULATION OF ENDOTHELIAL CELL (EC) PRODUCTION OF PGI AND TISSUE PLASMINOGEN ACTIVATOR (t-PA)
Authors: Kaplan L K , Mather T , DeMarco L , Solomon S
Published: August 2018

Many substances are known to stimulate EC production of PGI2 and t-PA. Additionally, it has been reported that fibrin can Be formed on the EC surface. In this study, the possibility that fibrin generated on the surface of cells can stimulate production of PGI2 and t-PA was examined. Human umbilical vein ECs were incubated for various time intervals with citrated human plasma clotted on the cells by the addition of CaCl2 . Control dishes contained plasma without Ca++ or serum. Time-dependent generation of PGI2 and t-PA was seen over 22-24 hours. Maximal production of PGI2 occurred when fibrin on the cells was formed from 10 to 50% plasma, with serum comprising the remainder of the incubation volume, while maximal t-PA production occurred with clots formed from 100% plasma. Fibrin I formed by addition of batroboxin to citrated plasma stimulated less synthesis of t-PA than did fibrin formed by thrombin action, and it did not stimulate PGI2 production. Thrombin clots were significantly more adherent to the cells than were batroboxin clots. PG^ synthesis induced by fibrin was fully inhibited by indomethacin, approximately 50% inhibited by actinomycin D and cycloheximide, and 20% inhibited by trifluoperazine, but was unaffected by cytochalasin D and vinblastine. Stimulation of t-PA synthesis by fibrin was unaffected by indomethacin, completely inhibited by actinomycin D and cycloheximide, and 60%, 80% and 40% blocked by cytochalasin D, vinblastine, and trifluoperazine, respectively. Thus, thrombin-induced fibrin clots stimulated PGI2 synthesis, and both thrombin and batroboxin clots stimulated t-PA synthesis. Protein and RNA synthesis were essential to stimulation of t-PA synthesis but inhibition of these processes only partially inhibited stimulation of PGI2 synthesis. Integrity of the cytoskeleton was necessary for full stimulation of t-PA synthesis, but not for stimulation of PGI2 synthesis. Thus the mechanisms of stimulation of these two cellular products were different. Increased PGI2 production could serve to limit further fibrin formation by preventing platelets from contributing to the coagulation process and increased t-PA could stimulate lysis of existing fibrin.
DIAGNOSIS AND MANAGEMENT OF ITP DURING THE PERINATAL PERIOD
Authors: Christiaens G C M L
Published: August 2018

Although maternal and perinatal mortality and morbidity in pregnant patients with ITP are lower than previously assumed, they are not negligable. Significant postpartum hemorrhage occurs in 7% of the mothers with ITP. Thrombocytopenia is found in 51% of the newborns born from mothers with ITP and 6% of these have serious bleeding problems. Tests which predict which fetuses are at risk, are not yet available. Thrombocyte counts in a fetal blood sample are falsely low in 40% of cases.A prospective controlled randomized study done in the Netherlands failed to show an effect of antenatal corticosteroid treatment on neonatal platelet counts. Elective caesarean section has not been shown to protect against intracranial bleeding in thrombocytopenic newborns. The choice between vaginal delivery and caesarean section in ITP patients should be made on obstetric grounds with one exception: no other assisted vaginal delivery than the easy outlet forceps should be done. All cases of slow progress of the second stage of labour with insufficient descent should be terminated by caesarean section as well as breech delivery with suboptimal progress. Newborn thrombocyte counts should be done daily during the first week of life, since lowest platelet counts are often found between the 3rd and 5th postpartum day. Newborn thrombocytopenia is transient and does not warrant splenectomy, but can necessitate treatment with corticosteroids and/or high doses of immunoglobulin 6. Current data do not justify to dissuade breastfeeding.

The recurrence of neonatal thrombocytopenia in subsequent patients is unknown.
FOLLOW UP OF PREKALLIKREIN AND FA3T0R VII IN PATIENTS WITH LIVER FAILURE (LF)
Authors: Ferro D , Violi F , Alessandri C , Quintarelli C , Saliola M , Cordova C
Published: August 2018

Life expectancy of patients with liver cirrhosis (LC) is dependent upon thepresence of haemodinamic and metabolic complications.Patients with ascites, hepatic encephalopathy and bleeding have lower survival than patients in compensated state. Neverthless the clinical signs of decompensated state are not useful in the individual assessment of LF. Recently we showed that some coagulation factors such as factor VII and prekallikrein (prekk) are good indexes of LF.In fact they clearly differentiate decompensated LC from compensated one and, in a short term follow up. survivors from non survivors. In order to evaluate the relation between factor VII (Mariani1s method) and prekk (Chromogenicsubstrate S-2302) activities and the survivaltime of patients with LF , we studied 23 patients with severe decompensated state and followed them up to 2 years from the hospitalization. The clinical evaluation of LC could be followed in all but 2 patients. Of 21 patients 15 (71%) died,8 for LF and 7 for bleeding, and6survived. The survival of patients was highly related to the activities of prekk and factor VII;patients with higher activities survived more than those with lower ones. There was a significant correlation between prekk (r:0,89 p<0,001), factor VII (r:0,8 p<.0,001) and survival time. The study suggests that prekk and factor VII could be useful test for the identification of patients for liver transplantation.
PROPHYLACTIC EFFECT OF ARGIPIDINE (MD-805) ON DEVELOPMENT OF LESIONS IN RAT PERIPHERAL ARTERIAL OCCLUSION MODEL
Authors: Hara T , Iwamoto M , Ogawa H , Tamikawa M
Published: August 2018

Argipidine ((2R,4R)-4-methyl-1-(N2-[(RS)-3-methyl-l,2,3,4-tetrahydroquinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylie acid hydrate) is a potent and specific inhibitor of thrombin(Ki=0.039μM). We tested the effect of argipidine on a new animal model of peripheral arterial occlusion (PA0).

PA0 was prepared by injection of 5% lactic acid into rat femoral artery. Macroscopic changes of legs progressed asfollows; change to violet color around whole paw (3-24hr), edema (3-24hr), gangrene of fingers (after 24hr) and mummification of whole paw (after 7 days). Pathological examination demonstrated partial deletion of arterial endotherial cells and subsequent occlusive arterial thrombosis. Coagulo-parameters in circulating blood did not change 3 hours after lactic acid injection. To evaluate the effect of tested antithrombotic drugs, sum of lesion grade of five fingers is used as an index of severity of. PA0.

Single subcutaneous injection of argipidine 30 minutes before lactic acid injection, inhibited significantly development of lesions of the lower limb at doses lOmg/kg and 30mg/kg in a dose-dependent manner. The number of rats which showed moderate or severe lesion on their legs at 14 days after lactic acid injection was decreased to 2/8 in argipidine treated (30mg/kg) rats from 7/8 in control rats. But argipidine injected simultaneously with lactic acid showed weak inhibitory effect (not significant) and argipidine injected three hours after lactic acid injection did not showany inhibitory activity. Slight prophylactic effect was also found by subcutaneous injection of heparin at a dose of 900U/kg, but its effect were not significant.

In a separate experiment, PT and APTT were assayed after subcutaneous injection of argipidine or heparin. Heparin (900U/kg) prolonged APTT much greater than argipidine (30mg/kg), but as for prolongation of PT, argipidine was more effective than heparin.

These results suggest that extrinsic coagulation system plays an important role in initiation of lesions of this model.
TISSUE FIBRINOLYTIC ACTIVITY:DIFFERENCES BETWEEN NORMAL AND NEOPLASTIC PULMONARY TISSUE
Authors: Doni A , Crisci C , Bartoloni Saint Omer F , Di Lollo S , Leoncini G , Rosi E
Published: August 2018

In order to verify differences between the fibrinolytic activity in normal and neoplastic pulmonary tissue, 16 subjects underwent infra-operatory biopsy.

Samples from normal lung and neoplastic tissue areas were obtained from each subject and then examined using Todd's histoehemical method (Doni A. et al.: VII International Congress on Fibrinolysis, Venezia 1984)

The results were evaluated using Wilcoxon’s test, and were expressed by an Index of the Potentiality of Fibrinolysis (I.P.F.) = % Lysis area/% tissue area and by the Ratio between I.P.F. at 30’ and I.P.F. at 45’ of incubation.

A statistically significant difference between I.P.F. of normal tissue and the neoplastic one at 30’ and 45’ of incubation was found while no difference between the Ratio of normal and neoplastic tissue was noted.

These data suggest that differences in fibrinolytic activity exist as regards normal and neoplastic pulmonary tissue and that these differences may be related to the different amounts of plasmonogen activators (perhaps tPA) present in normal and neoplastic tissue (Doni A. et al.: VIII International Congress on Fibrinolysis, Vienna 1986).

Kinetics of fibrinolysis did not show statistically significant differences
EFFECT OF DIPYRIDAMOLE ON SPONTANEOUS PLATELET AGGREGATION IN WHOLE BLOOD DECREASES WITH THE TIME AFTER VENEPUNCTURE:EVIDENCE FOR THE ROLE OF ADP
Authors: Saniabadi A R , Lowe G D O , Forbes C D
Published: August 2018

Spontaneous platelet aggregation (SPA) was studied in human whole blood at 3,5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter (Ultra Flo 100), SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of blood at 37°C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10-5M at each time interval; (b) 0.5-100 x 10-6M at 3 and 30 minutes, and (c) 15 x 10-6M in combination with 2 x 10-4M adenosine (Ad), 8 x 10-6M 2-chloradenosine (2ClAd, a specific ADP receptor blocker) and 5 x 10-5M aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy withAd, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when an effective concentration of Dipy and an ineffective concentration of Ad (10-4M) were addedtogether, the inhibitory effect of Dipy was not increased, suggesting that Dipy inhibits platelet aggregation independent of Ad.

The increase in SPA with the time after venepuncture was abolished when bloodwas taken directly into the anticoagulant containing 2ClAd (5 x 10-6M). We conclude that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture, and makes a serious contribution to the artifacts ofin vitro platelet function studies. Furthermore, the decrease in the inhibitory action of Dipy with the time after venepuncture may explain why previously, it has not been possible to observe inhibition of platelet aggregation by Dipy in platelet rich plasma which requires time to prepare.
REVERSIBLE BLOCKADE OF PLATELET ACTIVATION DURING CARDIO-PUIMONAR BYPASS IN DOGS AFTER IV ADMINISTRATION OF AJOENE
Authors: Apitz Castro R , Ledezma E , Jorquera A , Jain M K
Published: August 2018

Surgery with extracorporeal circulation (ECC) is associated with platelet activation, which greatly contribute to prolonged postoperative bleeding and increased blood loss after surgery. Antiplatelet ccnpounds which induce rapid and reversible inhibition of platelet function, without affecting platelet adhesiveness would be potentially useful in the management of the platelet-dependent hemostatic disorder observed in ECC. Ajoene, an organosulfur originally obtained from garlic, inhibits platelet release and aggregation induced ex vivo by all know agonists. It does not affect shape change or adhesion to collagen nor interfere with metabolic pathways relevant to the platelet reaction. Ajoene action is related to its direct interaction with the fibrinogen receptor on the platelet surface which irrpairs fibrinogen binding to stimulated or chymotrypsin treated platelets. IV administration of ajoene (15 mg/Kg) to mongrel dogs, inhibits platelet aggregation induced ex vivo by collagen (2-5 g/ml) or ADP (10 M). Ccnplete inhibition is attained after 20-35min and recuperation of platelet reactivity is obtained after 2.5-3.5 hours. To study the potential benefit of ajoene for the prevention of platelet activation during cardio-pulnonary bypass, ajoene was administered to anesthesized (heparin-anticoagulated) dogs, as described above, 40min before establishing the ECC. Circulation was mantained at 1.5L/min for a period of lOOmin Platelet count, and aggregation induced esc vivo by ADP or collagen were meassured immediately before ajoene administration, lOmin after the end of ECC and thereafter, hourly. Platelet count lOmin after end of ECC, in ncn-treated dogs fell to about 57% of prepump values, while in ajoene-treated animals circulating platelets represented 80% of pre-ECC values. Recovery of platelet function in ajoene-treated dogs started 2 hr after end of ECC (about 4 hr. after ajoene administration) reaching 70% 4 hr after end of ECC. Surgical bleeding in treated-dogs was not different frrm controls. Moderate bradicardia and hypotension, which in atrqpi-nized dogs returned to normal values within 3 min was observed. Although detailed pharmacological studies are still needed, our results suggest that ajoene is a potentially useful drug for the prevention of platelet activation induced by ECC.
CYCLOOXYGENASE INDIPENDENT PLATELET AGGREGATION:RELATION WITH ASPI RIN CONCENTRATION
Authors: Cordova C , Violi F , Praticò D , Ghiselli A , Alessandri C , Balsano F
Published: August 2018

Low doses of aspirin (20 mg/day) were previously reported to be uneffective in preventing platelet aggregation (PA) induced by pairs of aggregating agents such as PAF and adrenalin.This was in part attributed to the inability of such treatment to inhibit lipo oxygenase-dependent PA.The latter can be observed in vitro in"aspl rinated"platelets stimulated with high quantities of aggregating -agents.The aim of this study was to evaluate if the lipooxygenase-dependent PA was influenced by aspirin in a dose-dependent fashion. PA was studied in platelet rich plasma (PRP)(Born's method) by using threshold doses of aggregating agents (TDA) such as PAF(4-75 nM),epinephrine(0.6-2 μM) and collagen(2-4 μg/ml).PA performed in PRP pretrated with 100μM aspirin was fully prevented;in the same samples thromboxane (Tx) A2 evaluated by its metabolite Tx B2 was almost absent.Increasing amount of PAF(20 fold TDA),epinephrine(20 fold TDA) and collagen (36 fold TDA) do aggregate"aspirinated"pla telets;similarly"aspirinated"platelets aggregate when stimulated-with a pair of aggregating agents (TDA of PAF+epinephrine).This phenomenon was not detected if platelets were incubated with higher amounts of aspirin (250-500 μM).The study suggests that aspirin could influence lipooxygenase-dependent PA.This hypothesis is sup ported by a research showing the aspirin inhibits dose-dependently platelet HETE formation.A further study is now in progress to eva luate the influence of high doses of aspirin on cyclooxygenase-i"n dependent PA in vivo.
DEFECTIVE FIBRINOLYSIS IN SICKLE CELL DISEASE
Authors: Glas-Greenwalt P , Palascak J , Gruppo R , Stroop D , Pollak V
Published: August 2018

Vasocclusive crises (VOC) cause significant morbidity and mortality in sickle cell disease (SCD). Although sickling is thought to be the predominant factor in VOC, investigators have examined the possible role of the hemostatic mechanism in the process. The data are, however, inconsistent. We studied, functionally with the fibrin plate method, the fibrinolytic system in 36 adults in the steady state and in 8 children, 7 of whom suffered from painful crises. Values in 240 normal blood donors were: tissue-type plasminogen activator activity (t-PA); 3-25 activator units/ml, corresponding to 0.04 to 0.4 ng/ml; plasminogen activator inhibitor (PA-I): 649-885 inhibitor units/ml; and α2-antiplasmin (α2-AP) : 718-970 inhibitor units/ml. In patients with sickle cell disease t-PA levels were below the normal range in 24/44. PA-I and α2-AP levels were elevated in 35/44 and 23/44 respectively. The prevalence was similar in patients in crises and quiescent. Functionally impaired t-PA was associated with either low, normal or high t-PA antigen levels measured immunologically with an ELISA technique (normal range: 3-6 ng/ml). This indicates various degrees of endothelial dysfunction, ranging from impaired synthesis to functionally defective protein to complex formation with PA-I. Fibrin has been implicated in the intravascular sludging of sickle cells. It is suggested that a defective fibrin-clearing system contributes to the syndrome of SCD.
A METHOD FOR DETERMINATION OF FUNCTIONAL α-2 ANTIPLASMIN LEVELS IN PLASMA: THE CIRCULAR LYSIS INHIBITION OF AGAROSE-FIBRIN PLATE
Authors: Avello A Garcia , Fernandez Trejo R A , Garcia Frade L J , Vigo A Pardo , Cesar J , Navarro J L
Published: August 2018

A new, simple and reliable technique .has been developed for the determination of functional α-2 Antiplasmin in plasma; It is based on the circular inhibition of lysis produced by a-2 Antiplasmin from a hole punched out in an agarose-fibrin plate in which Urokinase has been previously dissolved.

Briefly: An Agarose-Fibrin plate is made with Agarose>(l%), Fibrinogen (0.5%) and Urokinase (0.5 U/ml). The components are mixed at 422 C and thrombinCO.25 U/ml is used to coagulate Fibrinogen. The mixture is poured on a horizontal glass, plate and left at room temperature. 30 min later a series of holes are punched out in the plate and the samples and controls are seeded in the holes.

The plate is incubated during 18 hours at 37° C in a humid chamber. After that time, all the plate has been lysed by plasmin produced by Urokinase except a circular area around the holes where α-2 Antiplasmin has migrated from the border: A round opaque circle can be seen around the holes and the diameters are proportional in a log-log scale with the aα-2 Antiplasmin levels of the sample.

A standard curve is constructed with a pool of plasmas and the diameters of each sample are referred to it in a log-log scale, determining in this way the levels of functional α-2 Antiplasmin as a percentage.

The lysis inhibition circle is entirely due_ to α-2 Antiplasmin and not to any other Fibrinolysis inhibitor: This has been demonstrated incubating previosusly the samples with antibodies directed to α-2 Macroglobulin, α-1 antitrypsin and C-l Esterase Inhibitor with no change in the circle. But when samples were incubated with an antibody to α-2 Antiplasmin, the opaque lysis inhibition circle disapeared.

This method presents a very good correlation with other functional methods (cromogenic substrate s-2251 Kabi:r=0.92 p(0.01) and is comparatively simpler.

the sensibility of the method is 0.5 % and the reproductibility is 3% day by day and 1% in a same day.
A NEW ONE STAGE CLOTTING ASSAY FOR HEPARIN AND LOW MOLECULAR WEIGHT HEPARINS
Authors: Giese Ch , Knodler A , Zimmermann R , Harenberg J
Published: August 2018

Heparin and its low molecular weight (LMW) derivatives are usually measured by chromogenic or fluorogenic synthetic substrate assays and by coagulation tests. Since the activated partial thromboplastin time (aPTT) and thrombin clotting time (TCT) are insensitive to LMW heparins, we report here of data obtained with heptest, a new one stage modification of the original heparin in plasma assay of Yin.
The assay was compared with the antifactor Xa chromogenic substrate S2222 method, the TCT and aPTT tests in 100 patients receiving unfractionated pig intestinal mucosa heparin and 100 patients treated with low molecular weight heparin Kabi 2165. The results indicate a high correlation between the heptest and the anti Xa chromogenic substrate method, whereas the correlations were lower for the aPTT and TCT.
correlations with LMW heparin

The lowest detection limit of the heptest is 0,005 heparin units per ml plasma. The test is very sensitive, simple, highly reproducable and reliable clotting assay for unfractionated and low molecular weight heparins in human plasma. The test detects with high preference the inhibition on factor Xa but also the other anticoagulant effects on die coagulation tractors.