bluuebunny/crossref_metadata_2025 huggingface dataset:
TISSUE FACTOR AND FACTOR VII PRODUCTION BY MURINE BONE MARROW-DERIVED MACROPHAGES:
Authors: Shands J W , Sunnenberg T D , Lottenberg R
Published: August 2018

Monocytes and macrophages (macs) make factor VII (VII) and can be induced to make tissue factor (TF), but when during differentiation this capacity is acquired is unknown. We approached this by measuring the synthesis of these factors by bone marrow cells (BMC) during in vitro differentiation into mature macs. BMC were harvested and cultured in RPMI-1640 supplemented with 15mM HEPES buffer, gentamicin, 10% FCS, 15% L-cell conditioned medium, and 2 mM L-glutamine. At intervals, characteristics of macs were measured: size, staining for non-specific esterase, phagocytosis of IgG sensitized RBC, induction of TF by a 6 hr exposure to endotoxin (LPS), and production of VII. The cells increased in mean diameter from 8.5u on d 1 to 15u on d 7 and to 17u on d 15. On d 2-3 of culture few cells were esterase pos, but 51% were phagocytic. By d 7, 95% were esterase pos and phagocytic. After 5 d of culture, macs spontaneously produced small amts of TF as measured by a coupled amidolytic assay. This increased 3 X over the total 15 d of culture. Two d macs did not synthesize TF in response to 1 ug/ml LPS. Responsiveness to LPS began at d 5 and reached a maximum between d 7 and 10 (5X control). The lack of response of 2-3 d macs was not due to suppressor cells, since mixing these with 10 d cells failed to blunt the response of the latter. VII, also measured by a coupled amidolytic assay, was produced on d 2, maximally on d 5, and decreased thereafter. It was not influenced by a 6 hr "induction" with LPS. BMC cultured in the continuous presence of 50 ng/ml LPS were incapable of producing VII and TF. These data suggest that immature macs can synthesize factor VII but not TF and that TF and factor VII synthesis are not coordinately expressed during mac maturation. Culture of BMC in the continuous presence of 50 ng/ml of LPS shut down the production of the coagulant factors by unknown means.
APPLICATION OF SPECIFIC SERINE PROTEASE INHIBITORS IN ASSAYS FOR ACTIVATED CONTACT FACTORS.
Authors: Tans G , Janssen-Claessen T , Rosing J , Griffin J H
Published: August 2018

We developed amidolytic assays to determine human Factor Xlla, Factor XIa and plasma kallikrein in mixtures containing variable amounts of each enzyme. The commercially available chromogenic substrates pro-phe-arg-pNA (S2302 or chromozym PK), glu-pro-arg-pNA (S2366), ile-glu-(piperidyl)-gly-arg-pNA (S2337), and ile-glu-gly-arg-pNA (S2222) were tested for their suitability as substrates in these assays. 8-Factor Xlla, Factor XIa and plasma kallikrein each exhibit considerable activity towards a number of these substrates. This precludes direct quantitation of the individual enzymes when large amounts of other activated contact factors are present. Several serine protease inhibitors were tested on their ability to selectively inhibit those contact factors that may interfere with the factor tested for. Soybean trypsin inhibitor efficiently inhibits kallikrein, inhibits Factor XIa at moderate concentrations, but did not affect the amidolytic activity of Factor Xlla. Therefore, this inhibitor can be used to abolish a kallikrein and Factor XIa contribution in a Factor Xlla assay. We also report the rate constants of inhibition of contact activation factors by three different chloromethylketones. D-phe-pro-arg-CH 2 Cl was moderately active against contact factors (k - 2271 M-ls-1 at pH 8.3) but showed no differences in specif ity. D-phe-phe-arg-CH2 Cl was a very efficient inhibitor of kallikrein (k = 118,000 M-ls-1 at pH 8.3) whereas it slowly inhibited Factor Xlla (k = 1389 M-ls-1) and Factor XIa (k = 110 M-ls-1). Also dansyl-glu-gly-arg-CH2Cl was more reactive towards kallikrein (k 15,662 M-ls-1) than towards Factor Xlla (k = 462 M-ls-1) and Factor XIa (k = 63 M-ls-1). Since phe-phe-arg-CH2Cl is highly specific for kallikrein it can be used in a Factor XIa assay to selectively inhibit kallikrein. Based on the catalytic efficiencies of chromogenic substrate conversion and the inhibition characteristics of serine protease inhibitors and chloromethyl ketones we were able to develop quantitative assays for Factor Xlla, Factor XIa and kallikrein in mixtures of contact activation factors.
RAPID FORMATION OF LARGE MOLECULAR WEIGHT O-P0LYMERS IN CROSSLINKED FIBRIN INDUCED BY HIGH FACTOR XIII CONCENTRATIONS: ROLE OF PLATELET FACTOR XIII.
Authors: Francis C W , Marder V J
Published: August 2018

Following fibrin polymerization, activated factor XIII stabilizes the clot by catalyzing the formation of specific intermolecular covalent crosslinks between pairs of y chains to form dimers and also among two or more a chains to form polymers. We have identified a series of previously uncharacterized a chain polymers with a wide range of sizes, including some with apparent Mr in excess of several million. Additionally, we establish the role of high concentrations of factor XIII in the extent and rate of α-polymer formation and provide evidence that the factor XIII required can be provided by platelets. Using SDS gel electrophoresis, we find that fibrin prepared from purified fibrinogen or from platelet-deficient plasma contains a series of 21 factor XIIIa crosslinked a chain polymers with Mr from 140,000 to 770,000. The mean Mr difference between individual polymers of 32,000 is consistent with a staggered, overlapping sequential addition of monomers to the growing α-polymer chain. In plasma containing no platelets, α-polymer formation was incomplete with residual α-monomer remaining. Progressively higher platelet counts facilitated more rapid crosslinking of a chains into larger polymers. Intact platelets were not required to promote crosslinking, since platelets lysed by freezing and thawing were also effective. Enrichment of plasma with placental factor XIII in an amount equal to that contained in platelets was as effective as platelets in accelerating the rate of formation and increasing the size of α-chain polymers. We conclude that platelets are a principal source of factor XIII for maximal fibrin stabilization, providing a larger quantity than is available from plasma alone and regulating both the rate and extent of α-polymer formation in thrombi or hemostatic plugs at sites of vascular injury.
MONOCLONAL ANTIBODY IDENTIFICATION OF FIBRIN DEPOSITS IN RENAL DISEASE
Authors: Bini A , D"Agati V , Pirani C , Kudryk B , Kaplan K L
Published: August 2018

Glomerular and vascular "fibrin" deposition has frequently been reported in human and experimental renal diseases. The biochemical form of this "fibrin" has not been well defined. We studied 16 renal biopsies (Bouin's fixed paraffin embedded) with the ABC-immunoperoxidase technique using monoclonal antibodies (MAbs); MAb I8C6 (Bβ1-42) to fibrinogen and fibrin I; MAb T2G1 (β15-42) to fibrin II; and MAb GC4 to fragment D or D-D. Polyclonal antisera to fibrinogen, albumin and IgG were used as controls. Renal biopsy specimens included 9 cases of microangiopathy (Group I: 6 hemolytic uremic syndrome (HUS), 1 sclerodero-ma, 2 acute humoral rejection) and 7 miscellaneous cases of other renal disease (Group II: 2 IgA nephropathy, 2 minimal change disease, 2 membranous GN, 1 acute interstitial nephritis). Fibrinogen and fibrin I were present on the glomerular (glom) endothelial cells in 8/9 Group I and 7/7 Group II cases, but was present in glom capillary lumens in Group I only. Staining of the endothelial aspect of interstitial capillaries, arterioles and arteries was also observed in both groups. Fibrin II was present in most glom and interstitial capillaries in both groups. However, intense staining for fibrin II was observed in arterioles and arteries in Group I only. Staining for fragments D and D-D was observed in glom capillaries in 6 Group I cases (6 HUS) and 3 Group II cases (2 IgA nephropathy, 1 membranous GN) but was most diffuse and intense in Group I. Traditional histochemical stains for fibrin (Lendrum and PTAH) were positive in 4 Group I cases only, indicating that the ABC technique is far more sensitive. Controls (14 needle biopsies of non-renal tissue) showed no vascular ractivity for fibrinogen, fibrin I, II and fragments D and D-D, suggesting that the vascular staining observed in renal biopsy tissue is not caused by the biopsy procedure itself. These findings indicate that 1) Fibrin formation and lysis occur in many renal diseases of both vascular and non—vascular origin. 2) Fibrinolytic activity is higher in the glom capillaries than in the larger vessels. 3) Damaged renal endothelium may be an active participant in these processes.
HUMAN OMENTAL TISSUE MICROVASCULAR ENDOTHELIAL CELLS (HOTMEC): ISOLATION AND NEW ASPECTS OF CHARACTERIZATION
Authors: Anders E , Alles J U , Delvos U , Pötzsch B , Preissner K T , Speiser W , Müller-Berghaus G
Published: August 2018

To study the function of microvascular endothelial cells in comparison to large vessel endothelial cells, HOTMEC were enzymatically isolated from human omental tissue and plated on petri dishes precoated with an extracellular matrix prepared from isolated fibroblasts of the same tissue or precoated with fibronectin. The culture medium was supplemented with 10% fetal calf serum; growth factors were not needed. HOTMEC were subcultured in a split ratio of 1:3 and maintained in culture for up to 3 month. Cultured HOTMEC were identified and discriminated from other non-endothelial cells by different characteristics and functions. 1. The cells demonstrated the typical polygonal shape as known for endothelial cells isolated from umbilical veins. In comparison to human umbilical vein endothelial cells (HUVEC), however, HOTMEC showed prominant nuclei with several nucleoli and presented a pronounced granulation of the perinuclear cytoplasm. 2. A monoclonal antibody specific for endothelial cells was bound to cultured HOTMEC. 3. Von Willebrand Factor (vWF) antigen was demonstrated within the cells by immunofluorescence staining; measurable amounts of vWF were only found in HUVEC in contrast to HOTMEC using an ELISA. 4. The addition of purified human protein C to HOTMEC preincubated with thrombin led to the activation of the zymogen as demonstrated by a chromogenic assay system. The kinetics of protein C activation were identical for HOTMEC and HUVEC. Tissue plasminogen activator (tPA) as well as plasminogen activator inhibitor (PAI) activity were detected in the culture supernatant of HOTMEC. After incubation period of 12 h in serum-free medium, the conditioned medium of confluent HOTMEC contained 100-fold higher levels of tPA than that of HUVEC. The data demonstrate that the cells isolated from human omental tissue have morphological as well as functional characteristics typical for endothelial cells. Furthermore, the study indicates that HOTMEC and HUVEC present quantitative differences in coagulant and fibrinolytic activities.
THE EFFECTS OF HEMORRHAGIC FEVER VIRUS INFECTION OF ENDOTHELIAL CELLS
Authors: Lewis R M , Jahrling P B , Griffin B P , Cosgriff T M
Published: August 2018

Pichindé viral infection of strain 13 guinea pigs is a model for Lassa fever virus in humans. Infected animals show impaired platelet function and altered coagulation parameters. Human endothelial cells and the human endothe1ia1-1 ike cell line, EA926, were infected with Pichinde virus. Following infection, cultures were monitored by phase contract microscopy for cytopathic effect (CPE). Assays of supernatant were used to document viral growth and to measure those endothelial-produced components that might affect hemostasis. In addition, the cells were stimulated with phorbol ester (PMA), which stimulates the production of prostacyclin. Infection showed no noticeable effect on the endothelial cells or EA926 cells which were untreated with PMA. PHA-treated EA926 cells were subject to CPE. Factor VIII antigen was not significantly affected by viral infection, PMA treatment, or endotoxin exposure. The production of PGFl, measured as an estimate of prostacyclin synthesis, was dependent on the concentration of stimulating PMA. Infected cultures showed decreased responsiveness to PMA stimulation when infected by increasing concentrations of Pichindé. The most noticeable effect was noted when cultures were infected with a multiplicity of infection of 0.1 and 100 ng/ml PMA. Thromboxane B2 an estimate of thromboxane A2, showed no significant change. No detectable leukotriene C4 was produced and no significant change in leukotriene B4 was measured. The decreased prostacyclin production by the infected endothelial cells may indicate a role for the endothelium in the hemorrhagic syndrome that accompanies some viral diseases.
EFFECTS OF HYPER0XIC AND HYPOXIC CONDITIONS ON ALBUMIN TRANSPORT ACROSS CULTURED ENDOTHELIAL MONOLAYERS
Authors: Bizios R , Holleran L A , Ladd T P , Iveson R D
Published: August 2018

Transport of albumin across cultured bovine pulmonary artery endothelial monolayers was studied under control (ambient air), hyperoxic (95% O2, 5% CO2) and hypoxic (95% N2, 5% CO2) conditions at 37°C over 3 hours. The experimental system consisted of two compartments separated by the endothelial monolayer grown to confluence on a gelatinized polycarbonate membrane (0.8 μm pore diameter). Albumin (4 gm% in PBS) solution and PBS buffer were placed on the luminal and abluminal sides of the confluent endothelial monolayer respectively. Albumin concentrations in the abluminal compartment at various time intervals were determined (Lowry assay) and the results are shown in the table.

Compared to control, exposure to hyperoxia resulted in increased albumin transendothelial transport by one order of magnitude for time intervals up to 1 hr, and by over 50% for later times. In contrast albumin transendothelial transport under hypoxic conditions was comparable to the results obtained under control conditions. The results of this study indicate that hyperoxia greatly affects the transport properties of endothelium. (Supported by the Whitaker Foundation.)
ALTERATIONS IN VASCULAR ENDOTHELIAL CELL FUNCTION BY OXYGEN-FREE RADICALS
Authors: Shatos M , Doherty J , Allen D , Hoak J
Published: August 2018

The vascular endothelium is a target for oxidant-induced damage in many disease states including hyperoxia, inflammation, ischemia and reperfusion injury. However, little is known concerning oxidant injury to endothelial cells and its relationship to hemostasis. Our studies have focused on the ability of oxygen free radicals to injure and/or alter selected vascular endothelial cell functions pertinent to the regulation of hemostasis. Xanthine and xanthine oxidase, a well-characterized generating system for the production of the superoxide anion radical (O−
2) was used to sublethally injure human umbilical vein endothelial cells (HUVE) in vitro. We examined the effects of a 15 min exposure of HUVE cells to xanthine (50μM), and xanthine oxidase (2.5-100mU) (previously determined to be non-toxic using trypan blue dye exclusion) on platelet adherence, and prostacyclin release using established assays. The antioxidant enzymes superoxide dismutase (SOD) 200μg/ml and catalase 50μg/ml were added to endothelium incubation systems to evaluate any protective effects upon O−
2-induced alterations. All experiments were conducted in a serum-free HEPES-Tyrode's buffer, pH 7.4 incubation system. Our results show that exposure of HUVE cells to sublethal concentrations of oxygen free radical generating systems causes significant enhancement of platelet adherence (65%) to injured endothelium. A 12-fold increase in prostacyclin release resulted after a 15 min treatment with xanthine and xanthine oxidase. The addition of exogenous PGI2 (150nM) to platelet-endothelial systems did not completely prevent the enhanced platelet adherence suggesting that lack of prostacyclin was not completely responsible for the adherence of platelets to O−
2 injured cells. When SOD and catalase, scavengers of O−
2 and H2O−
2, were added to treated cells, platelet adherence decreased by 42-77% and prostacyclin release approached that of control cultures. These data implicate an active participation of activated metabolites of molecular oxygen in the alteration of vascular endothelial cell function.
MODULATION of PROSTACYCLIN PRODUCTION BY HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS WITH ECGF/HEPARIN MEDIUM : ROLE OF CELLULAR DENSITY AT CONFLUENCE
Authors: BOUTHERIN-FALSON O , BLAES N
Published: August 2018

Prostacyclin (PGI2) is a major product of arachidonic acid metabolism in vascular endothelial cells. In addition to the role of exogenous agents, its production could be modulated by culture conditions : proliferative state, medium renewal, subcultivation... The use of endothelial cell growth factor (ECGF) associated with heparin has been shown to improve human endothelial cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) grown in that medium produce less prostacyclin than without growth factor.

HUVEC were cultured in RPMI-199 1:1 + 20% fetal calf serum, added or not with ECGF (Bovine hypothalamus extract BTI Cambridge, 24 ug/ml) and heparin (from porcine intestinal mucosa, Signa, 90 ug/ml). After 4 days in culture, medium was removed and replaced by Tyrode Hepes buffer and basal production was measured after 20 min. Cells were then submitted to 5 min thrombin to assess PGI2 production in stimulated conditions. PGI2 production was estimated by specific radioimmunoassay for 6 keto PGFjalpha. For each point, cell number in the culture was counted after Trypsin EDTA treatment. In the present study, cells grown in ECGF-heparin medium produce lower amount of PGI2, compared to heparin or control medium. This result was observed in both basal and stimulated conditions. For each medium (ECGF-heparin, heparin, control), correlations between PGI2 production per cell and log cell density were shown to be significantly negative.

These observations suggest that ECGF effect on PGI2 production could be a consequence of its growth factor activity, notably by the fact that it leads to an endothelial monolayer made of more numerous cells. Since it is now suggested by a number of clinical observations that PGI2 is rather produced in pathological conditions, culture models showing a weak production of PGI2 appear in that connection doser to the physiological conditions.
DUAL EFFECT OF FLUORIDE ON ENDOTHELIAL PROSTACYCLIN PRODUCTION
Authors: Kienast J , Arnout J , Pfliegler G , Deckmyn H , Van Houtte E , Vermylen J
Published: August 2018

The effects of fluoride at mM concentrations on prostacyclin (PGI2) production by human umbilical vein endothelial cells in culture were investigated. At lower concentrations, with a peak activity around 20-30 mM, sodium fluoride (NaF) caused a slow rise in PGI2 release reaching a maximum of 6 to 8 × control values after 1 hour. With increasing concentrations, the NaF-induced PGI2 production progressively decreased to almost control levels at 100 mM. Preincubation of the cells with 100 mM NaF inhibited thrombin— and A23187-stimulated PGI2 production by 46 % and 64 % respectively. The response to NaF was not significantly enhanced by AlCl3 nor was it inhibited by pertussis toxin.

The dual, stimulatory and inhibitory, effect of NaF on endothelial PGI2 production as well as its slow mode of action resemble the stimulus-response pattern observed in platelets upon exposure to NaF as it has previously been described by our grcup. Therefore, NaF might be an interesting tool for the investigation of intracellular regulatory processes triggering endothelial PGI2 production, particularly in view of the known capacity of fluoride to activate guanine nucleotide—binding regulatory proteins.
TICLOPIDINE AND PREVENTION OF PLATELET ACCUMULATION IN CAROTID ENDARTERECTOMY. EVALUATION WITH INDIUM-111 LABELED PLATELETS
Authors: Catani L , Vitacchiano G , Scatigna M , Monetti N , Pedrini N , Gugliotta L
Published: August 2018

In endoarterectomy (EA) the vascular endothelium is removed, thus promoting platelet adhesion and aggregation. This situation may cause microembolic phenomena that manifest at the carotid level with the appearance of TIA in the postoperative period. An interesting method of evaluating platelet behavior “in vivo” consists of the use of indium-111 labeled autologous platelets and the subsequent evaluation of scintigraphic images.

We used this method to perform a pilot study on the efficacy of ticlopidine, an antithrombotic drug with platelet antiaggregant activity, in the prevention of platelet accumulation after carotid EA.

A total of 20 patients undergoing carotid EA were randomly allocated to receive ticlopidine 500 mg/day (10 patients, T group) or placebo (10 patients, P group) in double-blind conditions. Drug administration was started 5 days before the operation and continued during the postoperative period; infusion of the labeled platelets was performed on the first and scintigraphic evaluation on the second postoperative day.

Analysis of the two groups, which were comparable for age, sex, risk factors and associated patologies, showed 3 scintigraphic-positive cases in the T group and 6 in the P group. The fixation index was 1.12 ± 0.18 in the T group and 1.20 ± 0.16 in the P group. One patient in the P group presented an episode of amauros is fugax in the first postoperative day.

The results of this pilot study suggest that ticlopidine is effective in preventing platelet accumulation after carotid EA.
PROTECTIVE EFFECT OF A NEW SYNTHETIC COMPOUND: PCA-4230, ON SEVERAL In vivo THROMBOSIS MODELS.
Authors: Ortega M P , Sunkel C , Priego J G
Published: August 2018

PCA-4230 is a new anti-thrombotic compound which inhibits pla. telet aggregation In vltn.0 and ex. vivo in several species, including man, prolongs the bleeding time and has potent protective ac tivity in several thrombosis models. Phase I trials with different dosage schedules have recently been initiated.

In the present study, the effects of PCA-4230 on bleeding time and on several In vivo thrombosis models were studied in mice. Mice were treated with one single oral dose of PCA-4230 (1-10 mg/ kg). One hour after treatment, mice were injected intravenously with four thrombotic challengers {arachidonic acid (AA), thromboxane agonist (U46619), Platelet Activating Factor (PAF) or collagen/epinephrine combination (C/E)} at a dose which induced 80-90% of mortality. The thrombotic agents were prepared in saline. The appropiate doses were dissolved in a volume of 100 μl/mouse. Bleeding time was measured in non-anesthetized mice by the tail transection technique.

Effects of compound were recorded from1 to 4 hours after dosage. Acute pre-treatment with PCA-4230 showed a significant dose-depen dent protective effect.

Results of each series of experiments are given in the next table.

The compound inhibited thrombotic sudden death induced by U46619, PAF or C/E combination, reduced the duration of respiratory distress induced by AA and prolonged bleeding time. Thus, PCA-4230 is protective against a variety of thrombotic stimuli.

These results suggest that PCA-4230 may be a promising anti-throm botic drug.
PHARMACOLOGICAL STUDIES OF PLATELET ANTIAGGREGANTS USING AN IN VITRO MODEL OF PRIMARY HEMOSTASIS.
Authors: Bellucci S , Cambau E , Candalot B , Caen J P
Published: August 2018

We used a new device simulating in vitro primary haemostasis : more precisely the reactivity of blood to collagen and ADP. Thus an artificial vessel was created consisting of two main parts : a glass capillary (ID 140 um, length 16 mm, siliconized) simulating the haemodynamic resistance of an arteriole and an aperture (ID 150 um) reflecting the injured part of a cut arteriole. This aperture was performed in a cellulose acetate filter covered with collagen type I (3 mg/ml) to provide a defined surface for the adhesion of platelets and soaked with ADP in a concentration similar to that of injured endothelial cells (2 x 10-2 M). The mean - sd control values were 110 ± 24 s, 156 -± 40 ul (n = 25) and correlated well with in vivo bleeding time values (p< 0.01). We studied the effect on this test of classical antiaggregant drugs which act on primary hemostasis by different mechanisms of action. Acetylsalycilic acid (Egic laboratories) prolonged this test for concentrations above 10−5 M, ticlopidine (Millot-Solac laboratories) above 3 × 10−4 M, prostacyclin (Wellcome laboratories) above 5 Õ 10−9 M, the synthetic octapeptide LYS-PRO-GLY-GLU-PRO-GLY-PR0-LYS derived from type III collagen (gift from Y. Legrand) above 5 × 10−4 M. We evidenced a synergistic action between collagen octapeptide and ticlopidine. Thus this device permits the screening of new drugs for their effects on primary hemostasis and the study of ex vivo repeated measurements for the monitoring of antiaggregant therapy.
PLATELET DERIVED GROWTH FACTOR (PDGF) BINDS TO HUMAN BONE MARROW FIBROBLASTS AND STIMULATES THEIR PROLIFERATION.
Authors: Bryckaert M C , Wasteson A , Lindroth M , Tobelem G C
Published: August 2018

A role for the Platelet Derived Growth Factor (PDGF) has been suggested in the abnormal proliferation of bone marrow fibroblasts occuring during myelofibrosis. To investigate this hypothesis, human bone marrow fibroblasts were isolated, and the cultures were characterized by immunofluorescent staining and electron microscopy. Electron microscopy eliminated the presence of endothelial cells by the absence of Weibel-Palade-Bodies. A positive intra and extra cellular antifibro-nectin staining was observed by immunofluorescent staining. The cultured cells didn’t show any labeling with specific antibodies for factor VIII von Willebrand factor, desmin or macrophage. Following the characterization of the bone marrow fibroblasts, using human pure 125I-PDGF, a specific binding of 125I-PDGF was demonstrated. The binding reached a plateau after 3 hours at 20°C, and after 4 hours at 4°C. Addition of unlabeled PDGF decreased this binding until 25 %.Saturation curve and scatchard analysis indicated two classes of sites with respectively 21,000 sites/aall and 37.000 sites/cell with an apparent Kd of 0.3 X 10-10 M and 0.5 X 10-9 M. Normal human serum at a concentration of 20 % induced a maximal DNA synthesis measured by-3H thymidine incorporation. When PDGF was added alone to the cultured fibroblasts at a concentration of 15 ng/ml, it induced a maximal DNA synthesis of 400 %.In the presence of 5 % of Platelet Poor Plasma (PPP), the same concentration of PDGF (15ng/ml) increased the incorporation of 3H thymidine up to 900%.

In conclusion i) PDGF binds to human bone marrow fibroblasts, ii) PDGF stimulates their proliferation. These results are in favour of a role of PDGF in the proliferation of bone marrow fibroblasts associated with the development of myelofibrosis.
THE FUNCTIONAL DOMAIN OF PLATELET MEMBRANE GLYCOPROTEIN lb FOR VON WILLEBRAND FACTOR AND THROMBIN-BINDING
Authors: Taki M , Sato K , Ikeda Y , Yamamoto M , Watanabe K
Published: August 2018

In this paper, we have examined the functional domain of platelet membrane glycoprotein lb (GPIb) by using elastase and a monoclonal antibody against GPIb which specific inhibits both von Willebrand factor (vWF) and thrombin interaction with platelets. Elastase was purified from human granulocytes by using affinity column chromatography according to the method of Okada et al.. A monoclonal antibody against platelet membrane GPIb (56-2) which inhibits both vWF and thrombin-binding to platelets was used for this study. Platelet surface glycoproteins were labelled with 3H by the method of Nurden et al.. Purified GPIb was obtained by a modification of the method of Coller et al. and labelled with 125I using chloramine-T method. Either 3H-labelled platelets or 125I-labelled GPIb was treated with elastase for various time periods. Elastase-treated l25I-GPIb was subjected to immunoaffinity chromatography using 56-2 antibody to determine the functional site of GPIb. Elastase inhibited platelet aggregation or 5-HT release by thrombin, ristocetin-induced platelet agglutination and vWF-binding to platelets in the presence of ristocetin in a dose- and time dependent manner. A fluorogram of SDS-PAGE of 3H-labelled platelets treated with elastase revealed that GPIb band was reduced gradually, and fragments with MW of 97, 70, 60, 47, 44, 37, 25 and 15 KD were released from the platelets. The 47 KD fragment was initially cleaved from the platelets, and subsequently other fragments were digested. Similar results were obtained when purified 125I-GPIb was digested by elastase. When the fragments from purified 125I-GPIb were reacted with 56-2 antibody, only three fragments with MW of 47, 44 and 25 KD were immunoisolated. The electrophoretic mobility of all these three bands was altered under reduced conditions, indicating that all these fragments contain disulfide bonds in their molecules. The 25 KD band showed a much fainter in 3H-labelling than in 125I-labelling.

These results suggest that the functional domains of GPIb for both vWF and thrombin-binding may be located in a less glycosylated fragment with a MW of 25 KD on the distal portion of the GPIb molecule, which should contain at least one intramolecular disulfide bond.
FACTORS AFFECTING PLATELET VOLUME ANALYSIS
Authors: Wehmeier A , Schneider W
Published: August 2018

Parameters of platelet volume have become widely available with the introduction of automated platelet counters. However, variant sample processing and in vitro platelet activation have prevented standardization of platelet volume analysis. We investigated the influence of anticoagulation, storage, temperature, and the presence or absence of RBC on platelet volume. Mean platelet volume (MPV) and the mode of the distribution were calculated from the platelet volume distribution curve recorded with the impedance method and plotted in 27 classes between 1.2 and 22 fl. The effects of EDTA (.335%), citrate (.38%), citrate (.38%)/glutaraldehyde (.125%) and a cocktail containing citrate, forskolin, indomethacin and theophyllin were determined 10, 30, 60, 120 and 240 min after blood collection. Tests were made at 4, 20° and 37°C in whole blood and platelet-rich plasma (PRP)from 6 healthy subjects. Platelet volume was strongly dependent on the anticoagulant in a time-dependent manner. MPV was lowest with citrate/glutaraldehyde and highest with EDTA. The maximum difference was 30% at 60 min both in whole blood and PRP. However, this was true only at 4 and 20°C. At 37°C, there was a constant rise in MPV using citrate/glutaraldehyde exceeding volume changes seen with the other anticoagulants. Platelet volume was higher in whole blood as compared to PRP. The difference was dependent on the anticoagulant used and the incubation time (1.8 fl for EDTA and 1.35 fl for citrate/glutaraldehyde, at 60 min). To determine the influence of platelet loss due to PRP preparation on this effect, we determined platelet volume in parallel from whole blood, PRP and the platelet population separated from whole blood by a linear Percoll gradient (n=5, recovery 94%). MPV was 7.6 fl in whole blood, 5.4 fl in PRP and 6.8 fl from the gradient (anticoagulant: citrate/glutaraldehyde). Platelet volume parameters highly depend on anticoagulation, incubation time and temperature. For clinical studies we recommend anticoagulation with citrate/glutaraldehyde and measurement within 2 hr at room temperature in whole blood.
ABNORMALITIES OF MEGAKARYOCYTE (MK) SIZE AND MORPHOLOGY IN PRIMARY AND SECONDARY THROMBOCYTOSIS
Authors: Gladwin A W , Trowbridge E A , Slater D N , Adams J M , Martin J
Published: August 2018

Platelet count elevations are observed in various clinical situations. In essential thrombocythaemia (ET), this is believed to reflect a primary disorder of megakaryocytes (MK). In contrast, thrombocytosis in malignant lymphoma (ML) is thought to arise via a secondary effect on these cells. In this study, MK parameters of whole cell, cytoplasmic and nuclear volumes were evaluated in these disorders. A trephine biopsy was obtained from the iliac crest of 10 subjects with ET, and from 10 subjects with ML. All exhibited a thrombocy^osip (considered to be a platelet count in excess of 400×109 L−1 ). In addition, a biopsy was obtained from 5 volunteer, haematologically normal subjects. Platelet counts in all subjects were measured using Coulter S+. Biopsies were processed as described elsewhere( 1). MK and MK nuclear planimetric areas were measured and converted to associated volumes(l). Mean MK, MK cytoplasmic and nuclear volumes were significantly larger in ET(p<0.05)and ML(p<0.05) when compared to those in controls. In addition, MKs in ET showed morphological abnormalities which were not exhibited in ML. These results suggest that platelet count elevations in ML and ET arise from large MKs with increased amounts of cytoplasm. The increased nuclear size observed further suggests that this may be mediated by an increased DNA content.
INCREASED SPONTANEOUS NUMBER OF MEGAKARYOCYTE COLONIES IN ESSENTIAL THROMBOCYTHEMIA (ET)
Authors: Deschamps J F , Bodevin E , Caen J P
Published: August 2018

Megakaryocytes were grown from medullary progenitor cells using the plasma clot technique, with 5 % human serum and with or without 2,5 % PHA-LCM. Using this technique megakaryocyte cultures were done in 5 patients essential thrombocythemia (ET) ( platelet count :0,6 x 106 to 1,4 x 106 /μi) before chemotherapy and antiplatelet agents and in 2 patients with secondary thrombocytosis (ST) (platelet count : 0,65 x lO6 and 0,8 x 106 /μi) corrected after effective anti-bacterial or iron therapy. The results were as follows

x number of colonies

mean number (in brackets) of cells per colony

It appears therefore that in ET, megakaryocyte progenitors grow without PHA-LCM and show however a better proliferation in its presence. On the contrary in ST, PHA-LCM is required for obtaining a 6 times increase of MK colonies. The effect of MK growth under chemotherapy is reported in some of the patients studied before treatment and results analyzed and compared to platelet function involvement
DOES THE MEGAKARYOCYTE CYTOSKELETON REGULATE IRROMBOPOIESIS?
Authors: Heynen M J , Verwilghen R L , Vermylen J
Published: August 2018

A widely held view of thrombopoiesis is that platelets arise from fragmentation of the periphery of mature megakaryocytes (MK’s). Evidence against this concept was provided by Zucker-Franklin, who showed that platelet plasma membrane is different from that of the MK (freeze-fracture, membrane antigens). Several authors have described contractile processes in MK’s

We have performed detailed electronmicroscopic studies of the numerous small MK's of a subject with congenital macrothrombocytopenia. In the young granular MK’s a central zone with organelles and a thick peripheral zone without organelles can be observed. The absence of elements of the demarcation system in the peripheral zone argues against derivation of the demarcation system from the megakaryocyte plasma membrane. In the mature granular MK’s the heart of the central zone is not occupied by the nucleus, but by an area, free of organelles and membranes, containing fibrillar structures and the centrioles. Elements of the demarcation system, delineating platelet territories, radiate from this fibril-rich area, but do not extend into the very thin peripheral zone. In the platelet producing MK’s the peripheral zone is thicker and the central fibril-rich area is surrounded by separated platelet territories. The peripheral zone shows several openings. In the old MK’s the nucleus is only surrounded by a markedly thickened peripheral zone, which seems to result from contraction of the more extended peripheral zone of the platelet producing MK's.

From these observations we conclude that, at least in this patient, platelets are formed inside the MK and are extruded through openings of the peripheral zone. Further studies with cytoskeleton markers are required to confirm that (!) the fibril-rich heart governs the organisation of platelet territories and (2) that platelet extrusion results from contraction of the peripheral zone, the latter however not giving rise to platelets.
FACTOR XIII IS NOT INVOLVED IN THE PLATELET ADHESION TO COLLAGEN
Authors: Kariya K , Sawada Y , Ueno K , Kudo I , Aihara M , Yoshida Y
Published: August 2018

We have reported that the adhesion of human formalin-fixed washed platelets (FWP) to collagen was enhanced by von Willebrand factor but inhibited by plasma fibronectin (Thromb. Res. 44, 1986). Recently, platelet factor XIII is reported to be a receptor for collagen (Saito, JBC, 261, 1986). To investigate the role of factor XIII in the interaction between platelets and collagen, effect of purified XIII or rabbit anti-XIIIa on the adhesion of FWP to collagen or on the in vitro bleeding time was studies. FWP adhesion was measured by either collagen-coated glass beads column or aggregometric method using bovine type I collagen (Ethicon Inc., Dr. Kronenthal). In vitro bleeding time was measured with Thrombostat-4000 (VDG-VONDERGOLTZ), in which citrated whole blood as a sample and ADP, CaCl2 and rat type I collagen as the reagents were used. Platelet adhesion to the collagen immobilized column (1,300 ug collagen, flow rate 5 ml/min) was not changed by the addition of purified XIII (Fibrogammin, Hoechst); the adhesion were 42.7 ± 1.7% in the presence of 1% human serum albumin, 42,0 ± 0.3%, 43,0 ± 1.4% in the presence of 1 or 2 u/ml factor XIII. Furthermore, the adhesion of FWP which was added by 1:100 rabbit anti-XIIIa was 42.3 ± 1.4% and not different from that of control rabbit serum (46.1 ± 1.3%). Similar results were also obtained with different technique using aggregometer. No significant change on in vitro bleeding time was observed after the addition of 1:100 rabbit anti-XIIIa to citrated normal blood. When the binding of factor XIII to the collagen was investigated by batch method, 17%, 23% and 54% of factor XHIa in normal plasma bound to 250, 500 and 1000 ug/ml collagen, respectively. These data suggest that factor XIII is not involved in the platelet adhesion to the type I fibrillar collagen, while factor XHIa in normal plasma binds to the collaeen.
SECONDARY FIBRINOLYSIS, PROTEIN C ACTIVATION, PLATELET DECREASE, BUT NOT CONTACT ACTIVATION CAN BE CORRELATED TO FREE THROMBIN ACTION IN EXPERIMENTAL DIC
Authors: Marbet G A , Satiropas P , Pantaleoni C , Duckert F
Published: August 2018

We have studied the influence of activated coagulation on antithrombotic defence mechanisms in vivo. Conventional coagulation variables, platelets (Tcy), plasminogen (Pig), α2-antiplasmin (AP) , protein C (PC), factor VIII C, factor XII and Cl-inhibitor have been measured before and during reversible tissue thromboplastin-induced DIC in the dog. Free thrombin action as derived from fibrinogen (Fbg) decrease has been expressed as integral of active thrombin concentration over time (φ). Protection by heparin H, pentosan polysulfate PPS or dermatan sulfate DS was studied. DIC had no consistent effect on the behaviour of factor XII and Cl-inhibitor, but led to the consumption (Δ) of the following variables:

The Spearman correlation coefficients between and φ in the Δ whole group were all statistically significant and ranged from rs=0.51 (ΔPlg) to rs =0.94 (ΔFbg). The response of major defense mechanisms in vivo quantitatively depends on active thrombin.
THE ARG-GLY-ASP(SER) SEQUENCE OF FIBRONECTIN, AND THE GLYCOPROTEIN IIB-IIIA COMPLEX ARE NOT INVOLVED IN FIBRONECTIN DEPENDENT PLATELET ADHESION IN FLOW
Authors: Nievelstein P F E M , Ottenhof-Rovers M , Pierschbacher M D , Sixma J J
Published: August 2018

Activated blood platelets interact with fibronectin through it to the glycoprotein IIb-IIIa(GPIIb-IIIa)-complex. The cell attachment site of fibronectin with its crucial arg-gly-asp-(-ser) (RGD(S))sequence is involved in this binding. We have studied the importance of this interaction for the fibronectin dependence of platelet adhesion under flow conditions. An RGDS-containing hexapeptide (GRGDSP) was compared with a non-reactive control peptide (GRGESP). The GRGDSP-peptide inhibited thrombin induced aggregation and adhesion under static conditions at 0.1 mM. This concentration had no effect on platelet adhesion to nonfibrillar collagen type I in flow. GRGDSP at 1 mM had a significant inhibitory effect at 1500 s™1 (8.8 ± 1.4 111In platelets* 105 /cm2, versus 19.8 ± 0.5 for the control). At lower shear rates of 800 and 300 s™1 , where platelet adhesion is also fibronectin dependent, no significant differences were obtained (respectively 11.7 ± 1.1 versus 15.2 ± 2.1, and 11.4 ± 1.0 versus 13.1 ± 0.7).

The relation between GPIIb-IIIa and fibronectin dependence was investigated with platelets of a patient with Glanzmann’s thrombasthenia and monoclonal antibodies to GPIIb-IIIa, using endothelial cell matrix (ECM) as a surface. Platelets of normal controls or a patient with Glanzmann’s thrombasthenia showed a inhibition of adhesion in fibronectin free plasma, after the ECM had been preincubated with anti-fibronectin F(ab’)2, of respectively _J5 and 30 percent at 300 s™1 , and 43 and 65 percent at 1300 s™1 . Incubation of platelets with anti GPIIb-IIIa showed inhibition of platelet adhesion at high shear rates. Dependence on fibronectin for platelet adhesion was still observed, even though separate experiments had shown that these anti GPIIb-IIIa antibodies could block binding of radiolabeled fibronectin to thrombin activated platelets. These data suggest the existence of a second binding system from the RGD/GPIIb-IIIa system separate for the interaction of platelets with fibronectin, which may only function when fibronectin is present on a surface.
CHARACTERIZATION OF RECOMBINANT HUMAN SINGLE-CHAIN LOW MOLECULAR WEIGHT UROKINASE (RE-SC-LUK)
Authors: Günzler W A , Wolf B , Flohé L
Published: August 2018

RE-SC-LUK obtained from recomoinant b. con Bacteria showed a molecular mass similar to that of recombinant two-chain LUK (RE-TC-LUK) as judged from SDS-PAGE. By “Western“ blot analysis immunoreactivity of RE-SC-LUK was observed with monoclonal antibodies directed against the B chain but not with those against the A1 chain of urokinase. N-terminal sequence analysis c RE-SC-LUK showed identity to the A, chain of RE-TC_LUK and provided evidence for its single-chain nature, i.e. integrity of the Lys-Ile bond which is split in TC-UK. In all other respects structural identity of RE-SC-LUK and RE-TC-LUK was demonstrated by fingerprinting of fragments. Similar to recombinant pro-urokinase (RE-SCU-PA), RE-SC-LUK exhibits only marginal amidolytic activity, which is greatly enhanced by treatment with plasmin, but considerable fibrinolytic activity in a fibrin agar plate test.

Thus, RE-SC-LUK is characterized as a fragment (residues 136 -411) of RE-SCU-PA, which lacks the “growth factor” and “kringle” domains. Moreover further evidence is provided that a free N-terminus of the B chain is essential for amidolytic but not for fibrinolytic activity of urokinase in more complex systems.
IN VITRO STUDIES ON THE BINDING OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (t-PA) AND UROKINASE (u-PA) TO LIVER MEMBRANES
Authors: Kalyan N K , Lee S G , Hum W-T , Hartzell R , Levner M , Hung P P
Published: August 2018

The plasminogen activators, t-PA and u-PA, are glycoproteins known to be involved in homeostasis of the blood clotting system, and thus are of potential clinical use in the treatment of thrombosis. Several in vivo studies have shown that both t-PA and u-PA are quickly removed from the blood circulation, predominantly by the liver. The mechanism by which the liver removes these proteins is not understood. To delineate this, we conducted in vitro studies of binding of PAs or their derivatives to isolated mouse liver membranes utilizing a functional assay developed in our laboratory. The assay consisted of initial binding of t-PA to liver membranes followed by centrifugation to pellet the membranes and the assay of the activity of the membrane-bound t-PA by a fibrin-agar plate method. The bound t-PA, which retained complete enzymic activity, could be dissociated by SDS treatment in an undegraded form as shown by SDS-PAGE. The binding of t-PA as well as u-PA was very fast and did not compete with glycoproteins or sugars containing the terminal galactose, mannose and N-acetylglucosamine residues. Furthermore, the treatment of t-PA with neuraminidase and/or periodate oxidation did not affect its binding characteristics. These data suggest that the carbohydrate moieties of t-PA and u-PA, unlike many glycoproteins, do not mediate their binding to the liver. This raised the possibility of the liver binding sequence being located in the protein backbone, especially the non-protease domains which are known to determine the biological specificities of PAs. The relative binding of u-PA and its low molecular weight (LMW) derivative containing only the protease domain, to the liver membranes was studied. Unlike u-PA and t-PA, LMW-urokinase did not bind significantly. This suggests that the protein sequence containing the non-protease domains, rather than the carbohydrate moieties of PAs contain the information necessary for binding to the liver and possibly their clearance from the blood circulation.
APSAC VERSUS PLACEBO: THE FIRST PLACEBO CONTROLLED CORONARY RECANALISATION STUDY WITH PRE AND POST TREATMENTCORONARY ARTERIOGRAPHY
Authors: Timmis A D , Griffin B , Crick J C P , Flax J S , Sowton E , Cool D E
Published: August 2018

40 patients entered the first double-blind placebo controlled invasive arteriographic study of intravenous anisoylated plasminogen streptokinase activator complex (APSAC) for coronary recanalisation in acute myocardial infarction. Coronary arteriography was performed before and 90 minutes after a single intravenous injection of APSAC or matched placebo given over 2-5 minutes. Pre-treatment coronary arteriography was performed in all 40 patients 3.1 ± 1.2 hours after the onset of major symptoms of myocardial infarction. All coronary arteriograms were read blindly and scored according to the TIMI criteria by an independent cardiologist. Following randomisation occlusion of the infarct related coronary artery was demonstrable in 29 of the 40 cases at pre-treatment coronary arteriography. Patients were given either APSAC 30 units (n=l6) or matched placebo (n=13),3.3 ± 1.3 hours after the onset ofsymptoms. Repeat coronary arteriography was performed 90 minutes after administration of study drug. 9 of the 16 patients who received APSAC had demonstrable coronary recanalisation of the infarct related vessel compared with only one patient inthe placebo group (P 0.05).A third full diagnostic coronary arteriogram was performed three days after treatment and this showed persistent patency of all the recanalised coronary arteries except one in the APSAC group as well as late recanalisation in a further 4 cases, 3 of whom had received APSAC.

Of the 11 patients who had patent infarctrelated arteries on initial arteriography, 4 received APSAC and 7 placebo. Patency was maintained in all these throughout the study period.There were haemorrhagic complications related to APSAC therapy. This data confirms the thrombolytic efficacy of APSAC in acute myocardial infarction, and indicates that the drug may be used safely by the intravenous route with a 56 per cent recanalisation rate at 90 minutes. Re-occlusion of the recanalised vessel was rare.