bluuebunny/crossref_metadata_2025 huggingface dataset:
ON THE DOUBLING DISTRIBUTIONS OF DAY 7-MEGAKARYOCYTE (MKC) PROGENITORS
Authors: Paulus J M , Fernandez-Delgado R , Grosdent J C , Lecocq J , Prenant M
Published: August 2018

It has often been assumed that hemopoietic progenitors undergo a rather uniform, deterministic number of proliferative cycles before they generate the elements which are recognizable by pano-ptiG and cytochemical methods. We now confirm and extend previous data on the distribution of the number of doublings undergone before polyploi'dization by day 7-MKC progenitors. Cultures of mouse bone marrow were stimulated by erythropoietin, WEHI conditioned medium (CM) and/or pokeweed CM. In all cases, cumulative doubling distributions of pure MKC colonies could be precisely fitted by exponential lines, whose slope and length depended on the stimulus or combination of stimuli used. The frequency of single MKC could be fitted by the same line as that plotting the number of doublings in MKC colonies, suggesting that single MKC and MKC colonies can be generated by the same class of progenitors. Exponential shape implies that the coefficient of variation (SD/mean) of doubling numbers is one, indicating significant variability in proliferative behavior among day 7-MKC progenitors. Such exponential distributions are best explained in the framework of Renewal Theory (Cox, Methuen, 1962). Whatever the distribution of the number of progenitor renewals in fixed time, the distribution of renewals will be geometric (i.e discontinuous exponential) if the total time spent by individual colonies in the proliferation phase (1) is distributed exponentially, and (2) is independent from the renewal process itself. These results suggest that the wave of MKC progenitor mitoses is randomly arrested by a differentia-tive event which strikes progenitor clones independently from their proliferative past.
ARACHIDONIC ACID (AA) INDUCED AGGREGATION OF RAT PRO- MEGAKARYOBLASTS (RPM)
Authors: Gladwin A M , Martin J
Published: August 2018

Platelet aggregation can be induced in vitro by a variety of platelet agonists acting upon membrane receptors. Since platelets have only a limited ability to synthesise proteins, these receptors must be present in megakaryocytes. This was investigated using an eternal line line of RPMs. Cells were suspended in rat platelet free plasma (PFP) at a concentration of 105 cells ml-1 . 200μl aliquots of this were placed in a light aggregometer. For this suspension, and for an aliquot of PFP, light transmission was adjusted to zero and 100% respectively. Addition of ADP (plasma concentrations 1-100μM), thrombin (0.5-5 I.U. ml-1 ), and adrenaline (0.1-1 I.U. ml-1 ) to the suspension caused no change in transmission. However, addition of AA (1.5-6mM) increased light transmission, indicating RPM aggregation. Radioimmunoassay (RIA) on the resultant supernatant showed no thromboxane B2 was produced. Scanning and transmission electron microscopy showed the aggregate to be composed of non-lysed cells. Aggregation of RPMs was not inhibited by preincubation with PGL (1500ng ml-1 ) indomethacin (100μg ml-1 ) or fenoprofen (1002μg ml-1 ). However, preincubation with aspirin (30μM) blocked aggregation.

These results indicate that RPMs can aggregate in response to AA. Mechanism of this is unlike that observed for platelets, since PGI2, indomethacin and fenoprofen did not block aggregation. The response was only inhibited by aspirin. Indomethacin, fenoprofen and aspirin are all known inhibitors of cyclo-oxygenase. In addition, aspirin also blocks 12-lipoxygenase. Therefore, this may suggest that the effect of AA on RPMs is mediated via 12-lipoxygenase pathway. Further investigations are in progress
CHANGES IN PROTEIN SYNTHESIS PROFILES OF MEGAKARYOCYTES (MK) DURING MATURATION
Authors: Jackson C W , Hutson N K , Steward S A
Published: August 2018

Several key differentiation events occur within the recognizable MK compartment; however, little is known about the macromolecular changes responsible for these events. In this study, protein synthesis profiles of morphologically immature and mature guinea pig MK populations have been analyzed by twodimensional gel electrophoresis after in vivo labeling with 35S-methionine. MK were enriched by a bovine plasma aggregation enrichment procedure (Blood 69:173, 1987) and then fractionated into immature and mature populations based on differences in their respective buoyant densities (Brit. J. Haematol. 64:33, 1986). With this protocol, immature and mature MK populations were obtained in which MK constituted 95% of the cell mass. Ninety percent of the MK in the immature population had basophilic, immature morphology while ≥90% of those in the mature population had acidophilic, mature staining characteristics after Wright's staining. Protease inhibitors were used throughout the isolation procedure. The cells were solubilized and proteins subjected to two-dimensional electrophoresis according to O'Farrell (J. Biol. Chem. 250:4007, 1975). To examine basic proteins, proteins were electrophoresed in the first dimension under nonequilibrium conditions in a pH gradient as described by O'Farrell et al. (Cell 12:1133, 1977). Analyses of fluorograms revealed both qualitative and quantitative differences in synthesis profiles between these two MK populations. Among acidic proteins whose synthesis was readily detected in immature but not mature MK were ones whose MW and pi were respectively: 120K, 6.4; 7OK, 5.9; 70K, 6.9; 65K, 6.8; 55K, 6.2; 55K, 6.0; 53K, 5.8; 53K, 6.5; 52K, 6.7; 50K, 6.8; 41K, 5.5 and 33K, 6.7. Acidic and neutral proteins prominently synthesized in mature but not immature MK were found at MW and PI of: 110K, 5.7; 110K, 5.8 and 80K, 7.2. Basic proteins prominently synthesized in immature but not mature MK were found at MWs of: 110K; 70K; 52K; 48K; 39K and 18K. Basic proteins actively synthesized by mature but not immature MK had MWs of: 83K; 43K and 17K. These findings demonstrate that differences in protein synthesis patterns can be readily detected between immature and mature MK and provide baseline data with which to explore the role of these proteins in MK differentiation
ETHANOL INHIBITS RABBIT PLATELET RESPONSES TO COLLAGEN IN VITRO BUT DOES NOT AFFECT RABBIT PLATELET ADHERENCE TO DE-ENDOTHELIALIZED A0RTAE IN VIVO
Authors: Rand M L , Groves H M , Kinlough-Rathbone R L , Packham M A , Mustard J F
Published: August 2018

Epidemiological studies indicate that moderate consumption of alcohol is associated with a reduced risk of coronary heart disease, but it is not known whether inhibition of platelet functions by ethanol is involved. We studied the effects of ethanol on rabbit platelet responses to collagen in vitro and in vivo. Addition of ethanol (4 mg/ml) to suspensions of washed platelets prelabelled with [14c]serotonin inhibited aggregation and secretion in response to low (0.4 μg/ml) concentrations of acid soluble collagen (14% secretion without ethanol, 3% secretion with ethanol). With a higher concentration of collagen (1.25 μg/ml), 4 mg/ml ethanol had no inhibitory effect. The inhibitory effect of ethanol on collagen-induced aggregation was also observed in citrated platelet-rich plasma (c-PRP) to which ethanol was added in vitro and in c-PRP from rabbits given ethanol acutely by gavage (3.5 g/kg) 30 min before blood sampling. The accumulation of [51cr]-labeled platelets on the subendothelium of rabbit aortae de-endothelialized with balloon catheters was measured in vivo in rabbits given ethanol (blood ethanol concentration at time of vessel wall injury: 4.1 ± 0.2 mg/ml, mean ± S.E., n=6). Ten min after de-endothelialization, there was no difference between the number of platelets adherent per square mm of injured aorta of control rabbits (39,400 ± 2,600, mean ± S.E., n=6) and intoxicated rabbits (36,800 ± 3,700, mean ± S.E., n=6). Thus, although ethanol inhibits platelet aggregation and secretion in response to collagen in vitro and ex vivo, it does not alter platelet adherence to the subendothelium, including its constituent collagen, in vivo. Therefore, it is unlikely that ethanol exerts its beneficial effects against coronary heart disease by altering the initial adherence of platelets to injured vessel walls.
EFFECTS OF 12-O-TETRADECANOYL PH0RB0L-13-ACETATE (TPA) ON PLATELET ADHESION-INDUCED SHAPE CHANGE
Authors: Mattson C J , Estry W D
Published: August 2018

Adherent platelets undergo changes in shape which involve conversion of the resting discoid platelet to a sphered or dentritic/pseudopodial form followed by cytoplasmic spreading to produce a fully spread cell with centralized organelles. Platelets from donors who have taken 10 gr aspirin (ASA) 4 hr prior to venipuncture show adequate pseudopod formation but defective spreading. Addition of 0.5 uM ADP is sufficient to reconstitute normal spreading suggesting that shape change is a two step process and that formation of spread platelets may require release of dense granule ADP. Since activation of platelets in suspension by ADP is associated with degradation of phosphoinositides and phosphorylation of a 40 KD protein, the potential role of this pathway in the formation of spread platelets was examined. Citrated platelets formed large aggregates when stimulated by TPA interfering with evaluation of adhesion and contact-activated shape change. When EGTA treated platelets were stimulated with TPA, aggregation was blocked but adhesion and spreading were unaffected by calcium chelation in the presence of magnesium. Therefore ASA-inhibited, EGTA-treated platelets were stimulated with 100 ng/ml TPA to activate protein kinase C, and platelet morphology was evaluated by whole mount transmission electron microscopy. TPA stimulated platelets underwent normal shape change to the fully spread stage despite endoperoxide inhibition by ASA. This data supports a role for ADP-trigqered phosphoinositide degradation in the spreading phase of adhesion induced shape change.
CYCLIC NUCLEOTIDE PHOSPHODIESTERASE INHIBITORS PREVENT AGGREGATION AND SECRETION OF HUMAN PLATELETS BY RAISING CYCLIC AMP AND REDUCING CYTOPLASMIC FREE CALCIUM MOBILIZATION
Authors: Beretz A , Lanza F , Stierlé A , Cazenave J-P
Published: August 2018

Drugs that raise platelet cyclic AMP (cAMP) are potent inhibitors of platelet activation. We have studied the effects of 5 inhibitors of cyclic nucleotide phosphodiesterase (PDE) of different chemical structures (quercetin, Ro 15-2041, HL-725, cilostamide and MY-5445), which are all potent inhibitors of platelet function. The concentrations that inhibit by 50 % crude cAMP-PDE activity (IC50) from human platelets are: 0.06 μM(HL-725), 0.15 μM(Ro 15-2041 ), 0.23 μM(cilostamide), 6.9 μM(MY-5445) and 44.4 μM(quercetin). We measured on the same preparation of washed human platelets loaded with quin2, the aggregation and the increase in intracellular Ca2+ ([Ca2+]i) induced by 5 μM ADP alone or in the presence of PDE inhibitors.

PGE1 (2 nM) potentiates significantly (1.6 to 3.3 fold) the inhibitory effects of PDE inhibitors on [Ca2+]i rises and platelet aggregation. Adrenaline, an inhibitor of adenylate cylase, prevents the effect of PDE inhibitors on ADP-induced [Ca2+]i rise and platelet aggregation. These results suggest that these compounds inhibit [Ca2+]i mobilization and subsequent ADP-induced aggregation through a rise in cAMP, because both effects are potentiated by PGE1 and inhibited by adrenaline. The inhibitor concentrations which potentiate the action of PGE1, on [ Ca2+]i levels also potentiate the rise in platelet cAMP induced by PGE-<. These results suggest that PDE inhibitors inhibit platelet aggregation Ly raising cAMP levels and subsequently inhibiting [Ca2+]i mobilization.
SYNTHETIC RGD-CONTAINING PEPTIDES OF VON WILLEBRAND FACTOR INHIBIT PLATELET ADHESION TO COLLAGEN
Authors: Fresslnaud E , Sadler J E , Girma J P , Baumgartner H R , Meyer D
Published: August 2018

The Arg-Gly-Asp (RGD) sequence is common to fibrinogen (Fg), fibronectin (Fn) and von Willebrand Factor (vWF). RGD-containing peptides compete for binding of these adhesive proteins to platelet membrane GPIIb/IIIa and inhibit thrombin-induced platelet aggregation as does an unrelated dodecapeptide from the γ Fg COOH terminus (γFg 400-411). We compared in flowing blood the effect of γ Fg 400-411 and of 3 synthetic peptides derived from the sequence of human vWF upon platelet adhesion to collagen. The 3 vWF peptides (13 or 18 aminoacids) contained an RGD sequence in the NH2 (peptide 03), central (peptide 07) or COOH (peptide 02) portions. Collagen was coated onto plastic coverslips and exposed in a parallel-plate perfusion chamber to reconstituted human blood at a shear rate of 2,600 s™1 for 3 min at 37°C. Perfusates contained physiological concentrations of 51 Cr-platelets and red cells in either citrated autologous plasma or modified Tyrode buffer containing 4% human albumin ; in the latter case, the collagen-coated coverslips were preincubated with normal plasma or purified human vWF prior to perfusion. Platelet-collagen interactions were estimated by radioactivity counting and quantitative morphometry. RGD peptides 02, 03 and 07 inhibited platelet-collagen interactions in a dose-dependent manner. With peptide 07, deposition of 51 Cr-platelets decreased from 283.8 ± 32.5 × 105/cm2 (mean ± SEM, n = 3) with buffer to 169.6 ± 33.0 in the presence of 50 μM peptide (p < 0.05), 133.7 ± 26.4 with 150 uM (p <0.012) and 101.8 ± 27.1 with 300 uM (p <0.005). The inhibitory effect of γ Fg 400-411 upon platelet deposition was less significant than that of the RGD peptides at 50 and 150 uM concentrations (224.4 ± 39.8, N.S. and 139.5 ± 55.3, p < 0.05, respectively). RGD peptide 07 also inhibited in a dose-dependent way both platelet adhesion to collagen and thrombus growth. Similar results were observed with peptides 02 and 03, indicating that the position of the RGD sequence is not critical. No synergetic effect between RGD and γFg 400-411 peptides was observed. These results with vWF peptides confirm that GPIIb/IIIa is involved not only In platelet aggregation (thrombus growth) but also in vWF-mediated platelet adhesion to collagen.
EFFECTS OF CELL ADHESION PEPTIDE, Arg-Gly-Asp-Ser (RGDS), ON RESPONSES OF WASHED PLATELETS FROM HUMANS, RABBITS AND RATS
Authors: Harfenist E J , Packham M A , Mustard J F
Published: August 2018

Fibrinogen (Fbg) is a cofactor in the aggregation of human platelets, and washed platelets do not aggregate to a significant extent in response to ADP unless Fbg is added to the suspension; however, exogenous Fbg is not required for ADP-induced aggregation of washed platelets from rabbits or rats. Since, with human platelets, Arg-Gly-Asp-Ser (RGDS) inhibits aggregation and the binding of 125I-Fbg to ADP-stimulated platelets, its effects on the responses of rabbit and rat platelets were studied in an attempt to elucidate the differences in Fbg requirements of platelets from the three species. Aggregation and Fbg binding were studied using washed platelets suspended in Tyrode solution containing albumin, apyrase and 2 mM Ca2+. 50 μM RGDS caused over 80% inhibition of the aggregation of human platelets stimulated with 9 yM ADP in the presence of 0.2 yM Fbg, but only 3-9% inhibition of the ADP-induced aggregation of rabbit or rat platelets regardless of whether exogenous Fbg was added. 50 yM RGDS also inhibited the aggregation of human platelets stimulated with thrombin (0.9 U/mL), but produced no more than 3% inhibition with rabbit or rat platelets. The binding of 125I-Fbg to ADP-stimulated human platelets was inhibited by 80-90% by 30 yM RGDS, but even at 50 μM, RGDS inhibited Fbg binding to rabbit or rat platelets by only 15-27%. The differences were due to the species of platelets, since, with both human and rabbit platelets, human Fbg could be replaced by rabbit Fbg without significantly changing the results. RGDS, added to human platelets that had been aggregated with thrombin, did not cause deaggregation, but did partially inhibit aggregation when added within 1 min; this inhibitory effect was less than when RGDS was added before thrombin, and decreased progressively as the length of time before the addition of RGDS was increased. These observations indicate a difference in aggregation mechanism between human platelets and those from rabbits and rats, and are consistent with a Fbg-independent component to the aggregation of rabbit and rat platelets.
FREVENTION OF MYOCARDIAL REINFARCTION BY LOW DOSE HEPARIN
Authors: Neri Semeri G G , Rovelli F , Gensini G F , Pirelli S , Carnovali M , Fortini A
Published: August 2018

The effectiveness of low dose heparin in the prevention of myocardial reinfarction was investigated in a multi centric randomized controlled study. After having given their, informed consent to undergo daily subcutaneous heparin adninistratian, 728 patients of both sex aged 50-75 years, who had suffered frcm a transmural myocardial infarction 6-18 months before the enrollment and were in the I or II NYHA class were randomized. 365 patients (control group) were an the therapy usually performed by the 21 experimental canters participating in the study and 363 (heparin group) were treated with subcutaneous calcium heparin (Calciparina®) 12,500 IU daily in addition to the usual therapy of the centers. Curing enrollment the balancement of the two grcups was periodically checked for age, sex, serum cholesterol, cigarette smoking, blood pressure, site of infarction, arrhythmias and drug regimen. The prospect!vely established end-points were: transmural reinfarctioi as primary end-point; general mortality and mortality for cardiovascular events as accessory end-points over a mean follow-up period of 24 nxnths. Statistical analysis was foreseen both on drug efficacy (EE) and intention to treat (IT) basis. Patients of both groups underwent periodical examinations during the study. Acherence to the therapy and bone mineral content (bone density by double isotope technique) were also checked. At the end of the study the balancement for the factors considered was satisfactory and the drop-outs were 7.7% in heparin group and 6.3% in control group (ns). In heparin group the re infarction rate was lcwer by 62.92% than in control group. At life table analysis the difference was statistically significant (p<0.05 DE and p=0.05 IT). Mortality rate was reduced by 47.61% (DE) in heparin group (p<0.05 at life table analysis). Cardiovascular mortality was not significantly reduced (−33.06%), but the mortality attributable to thromboembolism was reduced in heparin group (p<0.05). Sixty patients (16.5%) discontinued heparin treatment, but only in 23 patients (6.3%) suspension was due to side effects.
PLASMA AND LIVER LEVELS OF VITAMIN K IN THE NEWBORN
Authors: McCarthy P , Gau G , Shearer M
Published: August 2018

Few measurements have been made of vitamin K in neonatal tissues. Using an assay based on HPLC with dual-electrode electrochemical detection we have investigated the vitamin K status of the newborn from analyses of paired cord-maternal plasma samples and liver samples obtained at post-mortem. For vitamin K1 (K1) the median value in cord plasma (16 pg/ml, range 4-45 pg/ml) in 20 babies was some 30 fold lower than that in maternal plasma (median 0.47 ng/ml , range 0.14-2.42 ng/ml). This is the highest maternal-cord gradient of all the fat-soluble vitamins and together with the lack of correlation between cord and maternal values suggests that K1 does not rapidly equilibrate across the placenta. Hepatic neonatal-adult differences in K1 levels were less marked being about 5 fold lower at birth (median 1.2 ng/g, range 0.1-8.8 ng/g, n = 22) than in adults (median 5.4 ng/g, range 1.1-21.3 ng/g, n = 32). No relationship was found between hepatic K1 and gestational age and relatively high levels (1-2 ng/g) were detected at 10-12 weeks gestation. Post mortem livers obtained after intramuscular K1 prophylaxis at birth (0.5-1.0 mg) had K1 levels which were raised dramatically (1000 to 5000 fold after 24-48 h) and which remained raised for at least one week. A preliminary assessment of the contribution of vitamins K1 (menaquinones, MKs) to vitamin K1 status revealed undetectable levels in fetal or neonatal livers until about 14 days post-partum. This was in marked contrast to adults in whom MKs 7-10 accounted for the majority of liver vitamin K (75-97% on a molar basis). In adult plasma MKs were present at much lower levels than K1; the low circulating levels and poor1placental transport would explain our inability to detect MKs in newborn livers. When expressed as total vitamin K (K1 and MKs) we concluded that the newborn may have only about 2% of adult hepatic concentrations; this relative deficit of MKs may be responsible for the high susceptibility of the newborn to vitamin K deficiency.
VON WILLEBRAND FACTOR PROTECTS FACTOR VIII FROM INACTIVATION BY ACTIVATED PROTEIN C AND PROTEIN S
Authors: Joost S , Koedam A , Meijers Joost C M , Sixma Jan J , Bouma Bonno N
Published: August 2018

Activated protein C (APC) inactivated the cofactors factor V (FV) and factor VIII (FVIII). In the case of FV, this reaction and the respective roles of Ca2+ , phospholipids and protein S have been well documented. We investigated the role of protein S and von Willebrand factor (VWF) on the inactivation of FVIII.

Purified human factor VIII (3 units/ml) was incubated with protein C (0.2 μg/ml) in the presence of 8 μg/ml phospholipid, 5 mM CaCl, and 1 unit/ml hirudin. Factor VIII coagulant activity decreased with a pseudo first-order rate constant of 0.09 min . The reaction rate increased linearly with the concentration of prot^ig S in the incubation mixture.

12I-FVIII was incubated under the same conditions. SDS-polyacrylamide gel electrophoresis showed cleavage products of Mr 43 and 22 kDa. High Mr bands (FVIII-heavy chain) ranging fromMr 108 to208 kDa disappeared while the Mr 80 kDa FVIII-lightchain remained unchanged. The degradationpattern was not changed by addition of protein S.

The FVIII-VWF complex was reconstitutedby mixing the two components (±2 units VWF/units FVIII) and lowering the calcium concentration to 2 mM. The inactivation of the FVIII-VWF complex by APC proceeded at a 15- to 20-fold slower rate as compared to the isolated FVIII, indicating a protection of FVIII by VWF. Protein S exhibited no cofactor activity on the inactivation of FVIII-VWF by APC. The protective effect of VWF was lost completely after activation of the FVIII-VWF complexwith thrombin (0.05 units/ml).

When FVIII (0.1 units/ml) was added toplasma of a patient with severe von Willebrand's disease, 96% of its activitywas lost in 20 min after the addition of APC. All of the FVIII activity was retained when haemophilic plasma was used. Mixing experiments showed that one unit ofVWF unit FVIII is needed to fully protec FVIII against APC. These results may explain the observed lability of FVIII in von Willebrand's disease patients.
TOPOLOGY OF PLATELET GPIb INVESTIGATED BY LOCATION OF MONOCLONAL ANTIBODY EPITOPES
Authors: Clemetson K J , Weber R , McGregor J L
Published: August 2018

A large number of monoclonal antibodies to platelet membrane glycoprotein lb (GPIb) have been described but for most of these the position of the epitope is not known. Since many of these influence platelet function, a better understanding of struc-ture-function relationships requires this knowledge. The position of the epitopes for the monoclonal antibodies API (Dr. T.J. Kunicki), AN51 and SZ-2 (Dr. C-G. Ruan), WM23 (Dr. M.C. Berndt) and PI were determined by analysis of proteolytic cleavage fragments of glycocalicin via affinity chromatography on the monoclonal antibodies coupled to Sepharose, elution with diethyl ami ne solution, separation on SDS-gel electrophoresis and detection by silver-staining. First, intact glycocalicin was examined and was found to bind to all monoclonals with the exception of PI. All monoclonals bound intact GPIb. WM23 bound a 70 kDa glycopeptide from the highly-glycosylated 90 kDa tryptic fragment of glycocalicin. API, AN51 and SZ-2 all bound to 45 kDa and 40 kDa, poorly glycosylated tryptic fragments. The 40 kDa fragment is derived from the 45 kDa fragment and has been shown to be the N-terminal region of GPIb. All these monoclonals have been shown to inhibit von Willebrand factor induced platelet agglutination. Platelets were treated with either elastase or calcium activated protease and monoclonal binding checked by immunofluorescence. The immunofluorescence with API, AN51 and SZ-2 was minimal compared to control platelets whereas that of PI remained as strong as the controls. This indicates that the epitope for PI lies on GPIb in a region other than glycocalicin and its absence from glycocalicin is not simply due to conformational changes in that fragment. Since PI inhibits platelet activation by thrombin and ADP it must act via conformational effects and not by blocking the thrombin receptor which lies on the 45 kDa region of glycocalicin. These results support a more complex role for GPIb in platelet activation.
KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS
Authors: van Breugel Hans H F I , de Groot Philip G , Sixma Jan J
Published: August 2018

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.

In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.

Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.

When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.

From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.
PREFERENTIAL RECOGNITION OF VITRONECTIN (S-PR0TEIN) BY A MONOCLONAL ANTIBODY UPON INTERACTION WITH THROMBIN, ANTITHROMBIN AND GLYCOSAMINOGLYCANS
Authors: Tomaslni B R , Mosher D F
Published: August 2018

V1tronect1n/S-Prote1n (VN/SP) is a glycoprotein present at a concentration of 200-400 ug/ml 1n plasma and serum. It has been shown to promote cel 1-substratum adhesion and to act as an Inhibitor of the membrane attack complex of complement and of the inactivation of thrombin by antithrombin III in the presence of low levels of heparin. We have previously shown that VN/SP binds more avidly to heparln-agarose and to a monoclonal antibody (MaVN/SP)-Sepharose column when present 1n serum rather than 1n plasma. In order to examine the possibility of a serum-induced conformational change, we utilized, 1n this study, an Indirect enzyme-linked Immunosorbent system to test for the exposure of new antigenic determinants. When MaVN/SP was Incubated with plasma or serum, recognition of VN/SP 1n serum was approximately 50 fold greater than recognition of VN/SP in plasma. Since VN/SP has been shown to Interact strongly with the thromb1n-ant1thrombin complex, we examined the antigenicity of VN/SP when Incubated with thrombin and antithrombin 1n the presence and absence of heparin. Incubation of VN/SP with heparin promoted a 2.5-fold Increase 1n recognition by MaVN/SP. When MaVN/SP was Incubated with thromb1n-ant1thrombin but not thrombin or antithrombin alone, recognition was Increased by 7-fold 1n the absence of heparin and by 32-fold 1n the presence of heparin. This differential recognition of VN/SP was not observed with a second monoclonal antibody raised originally against S-Prote1n. Treatment of VN/SP with various glycosaminoglycans and polysaccharides demonstrated the following relative potencies for Induction of the partial antigenic change: dextran sulfate>fucoidan>heparin> dermatan suIfate>hyaluronic acid. No effect was detected upon Incubation of VN/SP with keratan sulfate, heparan sulfate or chondroltln sulfate. These data suggest a conformational change Induced by thrombin-antlthrombin which may allow VN/SP to Interact more avidly with other molecules such as heparin. The physiological role of this putative conformational change is under investigation.
ASSOCIATION OF LIPID WTTH THE CYTOSKELETON (CS) OF AGGREGATING PLATELETS PRELABELED WITH 3H-PALMITTC ACID
Authors: Livne A E , Packham A M , Guccione A M , Mustard F J
Published: August 2018

To investigate the association of lipid with the CS of platelets during aggregation, rabbit and human platelets were isolated and labeled with 3H-palmitic acid; lipid extraction showed about 80% in phospholipid. Limited aggregation was induced with ADP (undpr conditions in which release of granule contents did not occur) or thrombin, and CS isolated after lysis with 1% Triton X-1DD, 5 mM EGTA. CS from unactivated platelets had approx. 0.03% of the total platelet label, but after aggregation with ADP (2 µM) or thrombin (0.l U/ml) for 20-30 sec, 3 to 5% of the label was with the CS. Stirring was necessary and added fibrinogen enhanced aggregation and association of label with the CS; incorporation of label increased exponentially as aggregation proceeded and decreased exponentially during deaggregation. The amount of label with the CS was related more directly to the number of sites of contact than to the numbor of platelets in the aggregates. To determine how much label was bound to CS, some experiments were done in which the CS was disrupted and loss of label was assessed. In these experiments, DNase I and Ca2+ were added to the Triton X-100 lysis medium to cause actin depolymerisation, under conditions in which the Ca2+-dependent protease activity was inhibited. This treatment greatly reduced the association of label with the CS in samples taken from early time points on the aggregation curves: when aggregation proceeded further, a large proportion of label was not dissociated by this treatment. These findings, electron microscopy, and enrichment of the CS of aggregated platelets with only some membrane proteins labeled by the 125lactopproxidase method, indicated that under the conditions of limited aggregation used, the 3H-labeled lipid was mainly associated with the CS and not with trapped membrane fragments resulting from incomplete lysis. Si nee the pattern of CS 1abeli ng (3H-palmit at el and selective association of some of the membrane proteins with the cytoskeleton/1ipid complex was the same with ADP and thrombin, the reactions must be dependent on aggregation and not on events associated with the release of granule contents.
The Effect of Changes in the Leader Sequence of Human Protein C on Biosynthetic Processing and Gamma-Carboxylation
Authors: Foster D , Schach B , Rudinsky M , Berkner K , Kumar A , Sprecher C , Hagen F , bavie E W
Published: August 2018

Protein C is the precursor to a serine protease in plasma which contains gamma-carboxy glutamic acid and functions as a potent anticoagulant. Protein C shows considerable structural homology with the other vitamin K-dependent coagulation factors including prothrombin, factor VII, factor IX and factor X. This homology includes the putative pro-peptide region of the prepro leader sequences for these proteins, as well as the leader sequences for gamma-carboxylated proteins from bone. Deletion mutants have been constructed in the cDNA for human protein C in order to test the possibility that the pro-peptide portion of the 42 amino acid leader sequence serves as a molecular signal for gamma-carboxylation. Accordingly, these mutants contain the pre-peptide (hydrophobic leader) plus portions of the pro-peptide at the amino terminus of the light chain. The mutant proteins were expressed in carboxylation-competent mammalian cells and analyzed by barium citrate precipitation and N-terminal amino acid sequencing. These studies have shown that deletions in the pro-peptide region interfere with gamma-carboxylation and removal of the pro-peptide. Deletion of residues −1 through −12 had little effect on the carboxylation or secretion. Deletion of −1 through −17 completely abolished gamma-carboxylation, but had no measurable effect on secretion. Amino terminal sequence analysis of the latter mutant showed that the light chain began with Thr-Pro-Ala-Pro... This corresponds to a sequence in the prepro leader starting at −24. This indicates that the signal peptidase cleavage site for human protein C is between residues −25 and −24 and removal of the pro-peptide had been blocked by the deletion.
DDE COMPLEX DETERMINATION AS A SPECIFIC MARKER FOR FIBRINOLYSIS
Authors: Soria J , Soria C , Mirshahi Me , Mirshahi S , Mirshahi M , Pujade E , Boucheix C , Samama M , Bernadou A , Caen J P
Published: August 2018

Monoclonal antibodies (McAb) reacting with fibrin degradation products (FbDP), but not with fibrinogen have been produced in order to determine specifically FbDP directly in plasma. Most of the McAb available however do also react with fragment D. Our anti D neo McAb reacts about 10 times less with fragment D than with FbDP but does not react with fibrinogen, fragment X or Y.

In clinical investigation, even in pathological conditions in which there is a great release of tissue-type plasminogen activator (tpA), we have shown that fragment D is not generated in patients plasma. Therefore, the reactivity of our McAb with fragment D did not alter the specificity of FbDP assay.

On the contrary, using polyacrylamide gel electrophoresis in the presence of SDS followed by immunorevelation, we have evidenced that fragment D is generated in patients undergoing thrombolytic therapy even with rtpA. Therefore, using conventional Elisa procedure (capture of FbDP on polystyrene-immobilized anti D neo antibody and detection by peroxidase-labelled anti fragment D immunoglobulins), the presence of fragment D in patients plasma leads to an overevaluation of FbDP. To avoid this overestimation we have modified the Elisa procedure. The structure of FbDP was taken into acount in order to render the technique specific of FbDP. In FbDP fragment D coming from one fibrin monomer is always associated with fragment E from another fibrin monomer, as DDE complex for example. Therefore after capture of fragment D by the polystyrene-bound anti D neo McAb, FbDP were specifically revealed by peroxidase-labelled anti E antibody (polyclonal or monoclonal anti E may be used). For this reason, this test was named DDE determination and DDE determination can be used in any circumstances to evaluate fibrin degradation.
ORAL ANTICOAGULANTS IN BREAST CANCER PATIENTS
Authors: Sagripant A , Carpi A , Baicchi U , Nicolini A , Ferdeghint M
Published: August 2018

As the fibrin clot may play a role in the intravascular metastatic spread of breast cancer, we have treated women with advanced breast cancer with oral anticoagulants. After mastectomy, 11 consentient women with postsurgical stage Nl (6 patients), N2 (3 patients) and N3 (2 patients) and without evidence of distantmetastasis have been treated with acenocoumarol (INR 2-4,5) for 4-22 months (mean 12 months). All the patients received adjuvant therapy (radiotherapy, polichemiotherapy and/or tamoxifen) in conformity with theclassic indications. FibrinopeptideA plasmatic level, checked in everyone on stable anticoagulation, was always lower than 2 ng/ml (mean 0,78 ng/ml). No major bleeding occurred, except a copious hematuria caused by overdosage. Until now all the 11 women are alive. Two of them stopped anticoagulant, one because of hemorrhagic cystitis, the other because of awareness of visceral metastases; all the other 9 women arebeing treated now. Three patients(2N2 and 1 N3) have evidence of visceral metastases; no sign of relapsehas been observed by serial instrumental and laboratory examinations inthe other 8 women. A control group of 13 patients (9 N2 and 4 N3)was compared with the group of anticoagulated patients in more advanced stages (2 N2 and 3 N3):

The follow-up of the anticoagulatedNl patients is too brief as yet forcomparative evaluation. Our preliminary data seem to indicate an useful role of oral anticoagulants inbreast cancer and oblige us to prolong investigation.
MONOCLONAL ANTIBODIES AGAINST THROHBIN-ANTITHROMBIN III COMPLEX: EPITOPE SPECIFICITY AND EFFECT ON THROMBIN-ANTITHROMBIN III
INTERACTION
Authors: Asakura S , Yoshida N , Matsuda M
Published: August 2018

Among monoclonal antibodies (MCA´s) raised against human thrombin (T)-antithrombin m (AT) complex (TAT), two MCA´s designated as JITAT-16 and 17 with high affinity, Kd = 4.6nMand 4.1 nfi, respectively, were selected and characterized for specificity and functions. Their respective immunoglobulin subclasses are IgGi and IgG2a, and epitopes were found to be different from each Dther as shown by crisscross inhibition experiments. Immuno-alotting of normal plasma and serum electrophoresed on non-SDS aolyacrylamide gel showed that these antibodies reacted with normal serum but not with plasma. This was verified by an anzyme-linked differential antibody immunosorbent assay using aither one of the MCA´s as the first antibody and the other MCA labeled with peroxidase as the second one. By immunoblotting after SDS-PAGE, we found that both antibodies reacted with TAT, nut not with its respective nascent constituent, AT or T.
However, they reacted with reactive site-cleaved AT (or thrombin-nodified AT, ATM) and also a complex of AT with activated factor K (Xa-AT). These results indicate that both of these antibodies recognize enzyme-treated forms of AT, including AT molecules :omplexed with enzymes reversibly or irreversibly as well as ATM. Jpon incubation of T with AT in the presence of JITAT-16, T activity remained nearly unchanged and formation of irreversible rAT did not proceed as expected. Moreover, AT was preferentially :onverted to ATM. When JITAT-16 was added after completion of FAT formation, however, neither recovery of T activity nor generation of ATM was observed. These findings were not obtained vhen JITAT-17 had been substituted for JITAT-16. These data suggest that JITAT-16 may have converted AT from an inhibitor to a substrate for T after having recognized a possible intermediate reversible complex of AT with T. Undoubtedly, in the presence of a polyclonal antibody against AT, neither TAT formation nor ATM neneration was observed at all. The mechanism of the unique Function of JITAT-16 has not been fully clarified as yet, but this antibody seems to give us new information on the kinetic study of TAT formation and ATM generation when AT was allowed to react with enzymes.
ANTITHROMBIN III (AT III) REPLACEMENT FOR THE PREVENTION OF REOCCLUSION AFTER INTRACORONARY THROMBOLYSIS
Authors: Egbring R , Bethge C , Behling A , Seitz R A
Published: August 2018

Within 4 hours of acute myocardial infarction thrombolysis of an intracoronary thrombus leads to reperfusion in 40-707. of all cases, when streptokinase (SK), urokinase (UK) andtissue plasminogen activato(TPA) areused as thrombolytic agent.Although reperfusion could be demonstrated after thrombolysis, reocclusion occurred to different extents. There are several reasons for reocclusion: 1. Reocclusion occurs at the site of theprevious thrombus, as the vessel wall is deprived of endothelium and thethrombogenic surface is exposed; 2. The post-thrombolytic anticoagulant treatment (ACT) is not sufficient oris not performed correctly. When ACT was carried out with high doses of heparin, bleeding complications could be observed. Therefore we have tried in an open pilot study to increase the anticoagulant effect by additional replacement of AT III concentrates for 5 days as follows. The first group of patients (30) was given 1000 U AT III concentrate daily with an average dose of 24000 U heparin daily for 5 days. The second group (9)received more than 30000 U heparin daily. In the first group of 30 patients neither hemorrhage nor reocclusion of the infarction vessel was observed, the AT III levels rose gradually. In 3 of 9 patients of the secondgroup, hemorrhage occurred, necessitating reduction of the heparin dose.Subsequent reocclusion of the infarction arteries occurred in all 3 cases. The mean AT III level decreased in these patients to the lower limit.

Conclusion: After reperfusion of coronary arteries following thrombolytic therapy reocclusion may occur for several reasons. The present studies show that reocclusion is rare ifanticoagulant treatment is performedwith AT III concentrate replacement in combination with ACT with heparin.Although the initial AT III levelsare not decreased beneath the critical level of 707o, the substitution leading to a further gradual increase seems to prevent reocclusive thrombosis. According to Reeve (1982) the amount of circulating thrombin is dependent on the equation 1:(AT III)2.
EXPRESSION OF BIOLOGICALLY ACTIVE HUMAN ANTITHROMBIN III IN CHINESE HAMSTER OVARY CELLS
Authors: Zettlmeiβl G , Ragg H , Karges H
Published: August 2018

Expression of human antithrombin III (AT III) at high levels has been achieved in Chinese hamster ovary (CH0) cells by cotransfection and subsequent coamplification of the transfected sequences. Expression vectors containing the AT III cDNA gene and a dihydrofolate reductase (DHFR) cDNA gene were transfected into CH0 DHFR-deficient cells. About 20% of the DHFR+ transformants secreted recombinant human AT III into the medium. Stepwise selection of the AT III producing DHFR+ -transformants in increasing concentrations of methotrexate generated cells which had amplified the AT III géne. We determined the copy number of the AT III cDNA and the relative amounts of AT III specific mRNA at different stages of the amplification process. Transfected CH0 cell lines expressed elevated immunreactive levels of human AT III.

AT III secreted from these cell lines had the same molecular weight (60 kDa), immunological properties and biological activities as AT III obtained from human plasma. In vivo data concerning the inhibition of glycosylation by different drugs suggest that recombinant AT III from CHO cells is glycosylated according to a complex type pattern.
PURIFICATION AND CHARACTERIZATION OF GENTECHNOLOGICALLY PREPARED ANTITHROMBIN III
Authors: Karges H E , Zettlemeiβl G , Naumann H , Eberhard U , Bröker M
Published: August 2018

Isolation and purification of antithrombin III (AT III) by affinity chromatography on immobilized heparin is a standard method for the large scale preparation of this protein from human or animal plasma. Hence, after AT III became available by gentechnological methods, we tried to adapt this procedure for the isolation of AT III from supernatants of mammalian- and yeast-cells. Indeed, it was possible to use this method also for the isolation of the recombinant gene products. Since, however, the cell growth media contain heterologous protein or peptide mixtures like fetal calf serum, the method had to be improved to avoid the adsorption of non human proteins or peptides. We are now able to purify AT III from CHO-cell-superna-tants to more than 95 % purity. The characterization of this AT III-product by double immuno diffusion revealed that it is immunologically totally identical with the authentic material from plasma. AT III antigen content, progressive inhibitor activity and heparin cofactor activity compare very well in the final product; hence, it is totally active compared to AT III from plasma.

In polyacrylamidegel electrophoresis most of the material migrated differently to the authentic material showing 9 bands in equal distance to each other, instead four in the At III from plasma. After degradation with sialinidase from both AT III preparations identical cleavage products were obtained migrating predominantly as a single band. Hence, the electrophoretic heterogeneity seems to be due to a different degree of sialinyla-tion of the products.
ANCROD PROPHYLAXIS AFTER SURGERY FOR FRACTURED NECK OF FEMUR: A STUDYOF FATAL PULMONARY EMBOLISM
Authors: Pretice C R M , Townsend H A
Published: August 2018

A pilot multi-centre randomized controlledtrial was carried out to compare ancrod (Arvin, Knoll) versus no medical treatment in 453 patients having surgery for fractured neck of femur to assess prevention of fatal post-operative pulmonary embolism (PE).Ancrod was given subcutaneously by 5 daily injections starting immediately post-operatively; initially 4u/kg bw were given followed by 4 injections of 1u/kg bw, to reduce fibrinogen levels to 80mg/dl. The primary objective of the study was to record mortality due to PE, as shown by the DeathCertificate, within 3 months after surgery.Death Certificates were analysed by 2 medical assessors, unaware of the patient treatment group. Of 239 control patients, not given ancrod, there were 5 deaths due to PE and 2 deaths where PE may have been contributory. Total deaths were 30 (12.5%). In 214 ancrod treated patients there were 2 deaths due to PE and a further 3 where it may have been contributory. Total deaths were 31 (14.5%), not significantly different from the control group. Deaths from PE occurred between16 and 66 days after surgery. Although in this study there was abeneficial tendency for ancrod to reduce fatal PE it is likely that at least 6,000 patients would be needed to demonstrate that any drug significantly reduces by 50% the incidence of fatal PE compared to the control group. Wound infection was recorded in 16 patients in both groups. Wound haematomas were seen in 26 ancrod patients compared to 6 controls (p<0.02) but were not sufficiently serious to warrant re-exploration or prolonged hospital stay. The low mortality due to PE in our patients with fractured neck of femur (2%) is contrasted with the figures of Sevitt & Gallagher, 1959(8%). The low incidence of fatal PE in thehigh risk group studied here should be taken into account when assessing future antithrombotic prophylaxis after surgery. Advances in anaesthetics, surgery and rehabilitation may have contributed to the decline in fatal post-operative PE. Effective assessment of drugs for prophylaxis against post-operative venous thrombosis is best carried out by large scale simple controlled trials using fatal PE a the primary end point. Collaborative Centres were located in Portsmouth, Cape Town, Glasgow, Belfast and Leeds.