bluuebunny/crossref_metadata_2025 huggingface dataset:
PLATELET ACTIVATION BY HUMAN CANCER CELLS GROWN “IN VITRO” OR DISSOCIATED FROM TUMOUR TISSUES
Authors: Grignani G , Pacchiarini L , Zucchella M , Dezza L , Rizzo S C
Published: August 2018

The mechanisms of platelet activation by human tumour cells grown “in vitro” or freshly dissociated from tumour tissues have been investigated.

MoCCL human T-lymphoblastic cells cultured “in vitro” induced platelet aggregation through the production of ADP, as evidenced by inhibition of the effect by apyrase. The maximum of ADP production by tumour cells was reached after 1 hour and was 225 p moles/106 cells.

On the contrary, platelet aggregation induced by 5637 human bladder carcinoma cells was not inhibited by apyrase, but was abolished by hirudin, indicating the important role of thrombin in this effect.

Tumour cells dissociated from 3 breast carcinomas showed a very high platelet aggregating activity, which was not inhibited by hirudin or apyrase, but was abolished by iodoacetic acid, suggesting a role for a cystein-protease in platelet activation.

These results confirm that platelets can be activated by tumour cells through different mechanisms; they also suggest that the methods employed to obtain the tumour cells can influence the results, probably because of the different cell populations which are present in the dissociated tumour tissues.

Informations obtained with freshly dissociated cells are interesting, because this method has been used seldom so far and because it provides a more physiological approach to the study of the interactions of tumours and platelets.
ADHESION OF TUMOR CELLS TO EXTRACELLULAR MATRIX IS MEDIATED BY FIBRONECTIN
Authors: Almirall L , Aznar-Salatti J , Calopa I , Ordinas A , Bastida E
Published: August 2018

Tumor cell (TC) vessel wall adhesion is thougth to occur at specific sites of exposed extracellular matrix (ECM).To determine the role of fibronectin (FN) in TC/ECM adhesion,we measured; 1)TC adhesion to intact cultured endothelial cell monolayers (EC) or their ECMs,with or without incubation with a polyclonal antibody (Ab) to human FN, or a monoclonal antibody (Mab) to the cell binding site of FN (3E3);2)TC adhesion to EC or their exposed ECMs incubated with specific peptides against bacteria adhesion sites (I 133-79) or the peptide GRGDSP contained in the cell binding sites of several adhesive proteins.TC adhesion was measured as number of 111In-labeled A-549 adenocarcinoma cellsxlO /disc. ECMs were exposed by removing the ECs with N2 flow or EGTA treatment.There was 5.5±1x10 A-549/ EC covered disc (Table). Treatment of ECs with the Abs or peptides had no effect on TC adhesion.TC adhesion to the two ECM preparation were 42 ± 12 and 55±10 respectively.(Table).Blockage of the FN adhesive site by either Ab inhibited TC adhesion (p < 0.01). In contrast,blockage of the bacteria-adhesion site or incubation with GRGDSP had not significant effects in TC adhesion. The table shows the results (mearv±SEM (* p < 0.01).

We conclude that TC adhesion to ECM but not to ECs,is dependent upon FN.
ENOXAPARINE (LOVENOXR), DURING RENAL DIALYSIS, IN 46 PATIENTS WITH CHRONIC RENAL FAILURE (CRF) AND WITH HIGH RISK OF BLEEDING (HRB)
Authors: DECHELETTE E , POUZOL P , JURKOVITZ C , POLACK B
Published: August 2018

Standard heparin, usually used as an anticoagulant during hemodialysis, may be dangerous in patients with HRB. Low-molecular-weight-heparin (LMWH) fragments have been shown to induce less bleeding than standard heparin. We assessed the ability of Enoxaparine, a LMWH, to prevent clotting during hemodialysis, in such patients.

46 patients with CRF, requiring regular maintenance hemodialysis were included. 136 episodes of HRB occurred in these 46 patients : renal transplantation (17) , deep vessel catheterization (18) , biopsy (11), surgical.operation other than renal transplantation (46), obvious bleeding or HRB (44). 493 hemodialysis were performed using a single dose of Enoxaparine, as a bolus, 0.5 mg/kg for a double pass system, 0.75 mg/kg for a single pass system. Blood flow rate was maximum (350 ml/mn) in case of arterio-venous fistulas, and varied between 200 and 250 ml/mn in case of superior veina cava catheters. Air-blood exchange areas were maintained as small as possible. Bubble traps were examined every half an hour. Hemodialysis lasted 4 or 5 hours.

Extensive clotting within the extracorporeal circulation occurred only 3 times (0.6 %) ; (in 2 of the 102 hemodialysis performed with a single pass system and in 1 of the 391 hemodialysis performed with a double pass system). Efficiency of hemodialysis was always very good ; one patient had a genital hemorrhage requiring red cell transfusion. No other bleeding complication due to Enoxaparine was detected. 7 surgical operations and 3 renal biopsies were performed just after a hemodialysis without any problem.

This study shows that hemodialysis can be performed with Enoxaparine, 0.5 - 0.75 mg/kg, as a bolus in patients with HRB. It is efficient in preventing clotting, does not increase the risk of bleeding and ensures a very good quality of extrarenal filtration.

Enoxaparine (LOVENOXR) - PHARMUKA S.F.
THE ANTICOAGULANT EFFECT OF ENOXAPARINE (LOVENOXR) DURING HEMODIALYSIS
Authors: POUZOL P , DECHELETTE E , JURKOVITZ C
Published: August 2018

Enoxaparine is a low-molecular-weight-heparin obtained by partial and controlled depolymerization of a heparin of mucosal hog origin. We aimed at the evaluation of the efficacity of a unique injection of Enoxaparine in the prevention of blood clotting in the dialysis circuit in 42 patients with chronic renal failure. Three doses were tested : 0.75, 1.00, 1.25 mg/kg body weight in an open study with a crossing over of the three doses which were randomized according to a latin square design with a one week interval between each dose. None of the dialysis were impaired with clotting of the circuit and never an additional administration of anticoagulant was required in order to achieve the dialysis. Antithrombotic effectiveness was assessed by the quality of arterial and venous lines restitution and by visual inspection of the bubbles trap. No hemorrhagic event occurred. APTT didn’t show residual hypocoagulability at the end of dialysis. Anti-Xa and anti-IIa activities 4 hours after administration were :

Anti-Xa and anti-IIa activities exhibited a linear dose level
relationship. Efficiency and tolerance were excellent for the three doses.

Enoxaparine (LOVENOXR) PHARMUKA S.F.
EFFICACY AND SAFETY OF A LOW MOLECULAR WEIGHT HEPARIN (L M W H KABI 2165) IN PROPHYLAXIS OF POSTOPERATIVE DEEP VEIN THROMBOSIS (D.V.T.) AFTER ONCOLOGICAL SURGERY
Authors: Graunebaum L , Fricker J-P , Wiesel M-L , Vergnes Y , Cazenave J-P , Barbier P , Kher A
Published: August 2018

The effect of a LMWH - Kabi 2165 on thrombo-embolism prophylaxis was studied in a prospective, randomized trial of 74 patients scheduled for surgery due to malignant diseases. 38 patients received 2 500 anti-Xa units S.C. 2 hours before operation, then 12 hours after and then 5 000 anti-Xa units once a day for 10 days. 36 patients were given calcium heparin 5 000 i.u S.C. 2 hours before operation and then three time a day for 10 days. 125I-Fibrinogen uptake test was used in all patients to detect D.V.T. Positive scan was controlled by phlebography. In the Kabi 2165 group, 2 patients had positive scan not confirmed by phlebography No pulmonary embolism was reported. In the calcium heparin group, 2 patients developed non fatal pulmonary embolism. The fibrinogen test was negative in all the patients. Intra and postoperative blood loss, transfusion requirements, wound hematomas and infectious complications did not differ significantly between the two groups. The mean values of hematocrit and hemoglobin preoperatively and post operatively (day 1, and 10) were similar for the two groups :

It is concluded that a single daily injection of 5 000 anti-Xa units of Kabi 2165 may provide a prophylaxis against postoperative venous thromboembolism as effective and as safe as 3 daily injections of 5 000 i.u. of calcium heparin in oncological surgery.
COMPARATIVE CLINICAL PHARMACOLOGY OF LOW MOLECULAR WEIGHT HEPARINS IN MAN
Authors: Ordu Y , Augustin J , Hodenberg E V , Bode V , Harenberg J
Published: August 2018

Low molecular weight (LMW) heparins are obtained by diffent chemical procedures from conventional pig intestinal mucosa heparin. The LMW heparins differ in their molecular weight distribution and physicochemical properties. Therefore, we report of comparative studies on the anticoagulant and lipolytic effects of low molecular weight heparins in man.

The following LMW heparins were used: BM 21-23 (Braun, Melsungen, FRG), CY 216 (Choay Laboratories, Paris, France), Heparin NM (Sandoz, Niimberg, FRG), Kabi 2165 (Kabi Vitrum AB, Stockholm, Sweden), RD Heparin (Hepar Industries, Franklin, US A), normal heparin (Braun). All heparins were administered intravenously and subcutaneously to six volunteers each.

The data show considerable differences in the anticoagulant and lipolytic effects between the different low molecular weight heparins. From the area under the activity time curves (AUC) of the clotting assays for factor Xa (heptest), aPTT and thrombin clotting time the aXa/aPTT ratio ex vivo and aXa/alla ratio ex vivo were determined (table, average values)

It can be seen that there are clear differences in the ex vivo ratios of the LMW heparins. There is a good correlation between the average molecular weight of the LMW heparins and the aXa/aPTT ratio after s.c. administration and of the aXa/alla ratio ex vivo after s.c. administration. Therefore, LMW heparins differ significantly in their clinical pharmacological properties.
DOSE FINDING STUDY OF LOW MW HEPARIN (LMWH) CY222 IN HAEMODIALYSIS FOR CHRONIC RENAL FAILURE: RELATIONSHIP BETWEEN ANTI-FACTOR XA ACTIVITY AND ANTICOAGULANT EFFECT
Authors: Ireland H , Lane D A , Flynn A M , Pegrum A C , Curtis J R
Published: August 2018

A dose finding study of the LMWH CY222 (MLT2800) in patients (n=8) has been carried out to < i ) establish an effective dose and <ii) determine the relationship between ex vivo anti-factor Xa levels in plasma and the anticoagulant effect (in vivo suppression of FPA levels). Doses of CY222 were compared to a dose <5000 iu bolus+1500 iu/hr) of unfractionated heparin (UFH) that has been shown to suppress FPA levels during prolonged <>5hr) dialysis (Ireland et al , J Lab Clin Med 103, 643, 1984). CY222 given iv in doses (a) 5000 IC (Institute Choay)u bolus+1500 ICu/hr (b) 10000 IC bolus (c) 10000 ICu bolus+ 1500 ICu/hr (d) 15000 ICu bolus+1500 ICu/hr (e) 20000 ICu bolus (f) 20000 ICu bolus+1500 ICu/hr produced a dose related increase in anti-factor Xa levels (measured with CY222 as standard) and suppression of FPA levels. Dose (f) did not alter KCCT, produced FPA levels that were statistically indistinguishable from those of the UFH regime (indicating comparable anticoagulant effect), but had anti-factor Xa Levels (ICu) that were 2-3 times those of UFH. All samples were also assayed against the proposed LMWH Standard. Plasma levels of CY222 were then found to be 2-3 times lower, so that dose (f) had very similar anti-factor Xa levels to those of UFH. Clinically, doses <c), <d), (e) and (f) were all effective, although increasing amounts of fibrin were visible in the dialyser circuit with decreasing dose. T t is concluded that CY222 has comparable anticoagulant effect to UFH when present at similar anti-factor Xa concentration (measured against the LMWH Standard). CY222 may prove to be an effective alternative to UFH for use in haemodialysis.
LOW MOLECULAR-WEIGHT HEPARIN CY216 IN THE TREATMENT OF ACUTE MASSIVE PULMONARY EMBOLISM
Authors: Zambon G , Vigo M , Scatigna M , Di Giacomo C , Prandoni P
Published: August 2018

A 68 years old women presented to our Department with signs and symptoms suggestive of acute massive pulmonary embolism twenty days after knee fracture. She was in shock: heart rate, respiratory rate and blood pressure was 140/m, 36/m and 70 mmHg respectively. Two months before she had a severe episode of cerebral haemorrhage. Clinical suspicion was confirmed by a pulmonary digital subtraction angiography (DSA) and computerized tomography (CT), which showed a large filling defect in the right main pulmonary artery and a high degree of fascular sequestration of the lung (Miller index = 18). After rejecting the use of thrombolytic drugs, it was decided to treat the patient with low molecular-weight heparin CY216, due to the possible weaker bleeding potential with respect to the unfractionated heparin, and a dose of 17,500 U anti-Xa Choay was administered subcutaneously two times daily. A significant clinical improvement (decrease of heart and respiratory rate and increase of blood pressure) was observed within the first 48 hours of treatment. Pulmonary DSA and CT scan, repeated after 7 days of treatment, showed an important reduction of the above-mentioned defects (Miller index = 7). Not any bleeding complication developed and the patient was discharged within 15 days from the Hospital on warfarin therapy.
TREATMENT OF PROXIMAL DEEP VEIN THROMBOSIS (DVT) OF THE LOWER LIMBS BY CY 216* (LMWH) VERSUS UNFRACTIONATED HEPARIN (UFH)
Authors: Duroux P
Published: August 2018

Preliminary clinical data showed CY 216 could be effective in the treatment of established DVT. Thus, we undertook an European collaborative controlled randomised study to assess the efficacy and tolerance of this new antithrombotic agent.

140 patients presenting a proximal DVT of less than 5 days confirmed by phlebogram, are randomly allocated into 2 groups, one treated by UFH with constant pump infusion, at doses daily adapted to laboratory tests, the other one by CY 216 at a mean daily dosage of 450 aXa IC u** calculated at onset of treatment on body weight, administered daily by 2 sub-cutaneous injections during 10 days, without dosage adaptation whatever the lab. tests results.

The efficacy is assessed on comparison on both phlebograms (Harder and Arnessen Indexes) and lung Scans performed before inclusion and after end of treatment. In addition, tolerance is appreciated on the incidence of bleeding complications. Furthermore, incidence of clinical DVT recurrence is assessed at 12th week.

The present study is still in progress, 130 patients are included ; the preliminary results tend to show that CY 216 at fixed doses, body weight adapted, is an effective and safe treatment of proximal deep vein thrombosis.

* CY 216 : FraxiparineR Choay

** Axa IC u : anti-factor Xa Institut Choay unit.
EFFECTS OF ORG 10172, A NOVEL LMW HEPARINOID ON PRIMARY HAEMOSTASIS IN RATS AND HUMANS. A MORPHOLOGICAL STUDY
Authors: Wester J , Van Mensvoort F W J , Meuleman D G , ten Cate H , Henny C P , ten Cate J W
Published: August 2018

Org 10172 is a novel low molecular weight heparinoid isolated from animal mucosa with a higher anti-thrombotic/bleeding risk ratio than standard heparin. The effects of Org 10172 and heparin on primary haemostasis in rats and humans have been studied by light and electron microscopy (LM&EM).

In rat ears bleeding wounds were inflicted. The wound areas were excised 5, 15 or 30 min. after bleeding induction, and processed for LM and EM. Org 10172 and heparin have been administered in doses of 300 or 600 anti-Xa U/kg i.v., 5 or 1 min. prior to bleeding induction. Bleeding wounds of drug treated animals have been compared with those of placebo animals. Heparin inhibited degranulation as well as fibrin deposition and groups of not aggregated, sometimes even discoidal platelets could be found after heparin treatment. Org 10172 inhibited degranulation to some extent but less than heparin and Org 10172 hardly inhibited fibrin deposition.

In six human volunteers SimplateR bleeding time wounds were excised by punch biopsy, 20 min. after bleeding induction and processed for LM and EM. Org 10172 was administered as single bolus injection of either 3200 or 6400 anti-Xa U i.v., 10 min. prior to bleeding induction. Heparin was given in a dosis of 10.000 anti-Xa U i.v. All biopsies from post-drug bleeding time wounds were compared with biopsies taken from pre-drug bleeding time wounds in the same volunteer. As in the rat studies, heparin inhibited degranulation and fibrin deposition whereas after Org 10172 treatment these effects were hardly detectable. In general the effects of both drugs on haemostasis in the human volunteer study were less distinct than in the rat study. This may be attributed to the lower dose levels used.

In conclusion, primary haemostasis after Org 10172 treatment is somewhat retarded, but essentially normal, whereas haemostasis after heparin treatment is more severely disturbed.
CLINICAL PHARMACOLOGY OF DERMATAN SULFATE: EFFECT ON HAEMOSTATIC PARAMETERS
Authors: Piovella F , Pogliani E M , Custodi P , Antonioli R , Gianese F
Published: August 2018

In experimental studies dermatan sulfate (DS) has been characte riZed as an effective antithrombotic agent. This property is not associated with modifications of standard coagulation tests or in creased blood loss “in vivo”.(*)

In view of a possible clinical use of DS, we performed a prelirmi nary study on 10 consenting hospitalized patients without eviden ce of haemostatic abnormalities.

Each subject was given i.m. single increasing doses (50, 100, 200 mg) of DS (MF 701, Mediolanum Farmaceutici), separated by 3 days wash-out intervals. Prothrombin Time (PT), Partial Thrombo plastin Time (PTT), Antithrombin III (ATIII), Heparin Cofactor II activity (HCII, measured by amidolytic method) were determined before each administration and after 30, 60, 120 and 240 minutes. Bleeding time (BT) and platelet count (PIC) were determined before and after 120 or 240 minutes respectively.

No systemic or local adverse reactions were observed. Analysis of variance showed no significant modifications of PT, PTT, ATIII, BT and PIC following DS administrations nor significant differen ces between the three doses. A trend towards reduction was registered for HCII activity at 100 and 200 mg doses.

Our results suggest that DS can be safely considered for further clinical investigation

(*) F. Fernandez et al, Br J. Hematol, 64, 309, 1986
EVALUATION OF A FULLY AUTOMATED CENTRIFUGAL ANALYSER FOR ITS PERFORMANCE OF HEMOSTASIS TESTS
Authors: Chantarangkul V , Tripodi A , Mannucci P M
Published: August 2018

A fully automated centrifugal analyser (ACL, Instrumentation Laboratory, Milan, Italy) specifically designed for hemostasis tests was evaluated for its performance of methods based on clot detection by light scattering measurement or on splitting of chromogenic substrates.In general ACL performed at least as precisely as the comparative methods.PT,APTT,factor VIII and factor VII values obtained by ACL using reagents recommended by the manufacturer were correlated with those obtained by a semiautomated coagulometer (Coag A Mate X2, General Diagnostics, Morris Plains, N.J.) using reagents routinely employed in our laboratory.ACL was compatible with a widely used commercial APTT reagent (Automated APTT,General Diagnostics) both for APTT and factor VIII,whereas it was not so with Manchester Comparative Reagent,being fully compatible for factor VII but not for PT.Fibrinogen results obtained during the performance of PT by ACL,were correlated with those obtained by the standard clotting method performed with an electromechanical coagulometer,however a discrepant result was obtained in plasma from a patient with dysfibrinogenemia.Antithrombin and plasminogen results obtained by ACL using reagents supplied by the manufacturer were correlated with those obtained with other commercial reagents and standard photometric methods. In conclusion ACL presents features such as high output, easy operability, high precision and large panel of clotting and chromogenic assays which makes it suitable to cope with the large work load of the routine coagulation laboratory.Its incompatibility to accept some reagents together with the need to follow strictly the specified operational parameters (e.g. quantity of plasma and reagents and incubation time) makes it rather rigid for adaptation to research specifications.
RESULTS OF THE MULTICENTRIC EVALUATION OF A NEW SYSTEM FOR PHOTOMETRIC DETERMINATION OF COAGULATION PARAMETERS
Authors: Dati F , Becker U , Keller J , Huber J , Schmitz-Huebner U , Ostermann H , Gressner A , Zimmermann R
Published: August 2018

The classic coagulation analyses based on the clot formation present basic disadvantages which make a standardization of reagents difficult. The use of photometry for coagulation methods represents nowadays an important step towards test optimization.

We have evaluated a new analytical system (ChromoTimeSystem, Behringwerke AG, Marburg/FRG) based on a special instrument and reagents for photometric tests for coagulation and fibrinolysis. The instrument is a microprocessor-controlled 4-channel-photometer operating at 37°C and connected to a microcomputer. Photometric methods for prothrombin time (PT: Chromoquick®) and activated partial thromboplastin time (aPTT: Partochrom®) have been developed using a new chromogenic substrate (Tos-Gly-Pro-Arg-5-amino-2-nitro-benzoic-acid-isopropy-lamide) specific for thrombin. These tests are based on the time necessary to attain a fixed increase of absorbance (0.1 A). Tests for thrombin time (TT), batroxobin time and fibrinogen rely on turbidimetric techniques. In the screening tests PT, aPTT and fibrinogen 25 μl sample (TT: 50 μl) and 250 μl reagent are used. Single coagulation factors are assayed by mixing an undiluted sample (10 μl) with the correspondent factor deficient plasma (50 μl) in connection with the chromogenic PT or aPTT reagent (500 μl). The evaluated specific chromogenic substrate methods are: antithrombin III, α2-antiplasmin and plasminogen.

Tne characteristics of the ChromoTimeSystem are: fibrinogen-independent PT and PTT, small sample volumes (10-50 μl), no predilution or preincubation, PT standardization according to WHO recommendations, coefficients of variation between 1 and 4 %, good correlation between photometric and coagulometric tests, the reference values for photom. PTT being 90-120 sec.
THE IMPLEMENTATION OF THE WHO INTERNATIONAL NORMALISED RATIO (INR) SYSTEM OF PROTHROMBIN TIME STANDARDISATION
Authors: Poller L , Thomson J M , Taberner D A
Published: August 2018

The implementation of the INR system has been assessed in a group of laboratories from over fifty countries. These centres participate in the World Health Organisation International Quality Control Programme in Blood Coagulation. Prothrombin times are performed on various lyophilised human coumarinised plasmas using both the local thromboplastin reagent and British Comparative Thromboplastin (BCT) reference preparation. During 1985 and 1986 a programme of education has been undertaken whereby instructions in the derivation of INR values have been given. The calculation of INR has been shown by means of a worked example and a chart for the conversion of the prothrombin ratio with BCT to INR was included. Prior to this, prothrombin results were expressed as British Corrected Ratios (BCR) with a minority of centres using INR. In 1984 the CV varied between 23.7% - 36.8% for results with local reagents, but between 8.2% - 14.3% with BCT. This suggested that conversion into INR/ BCR was unsatisfactory. The table shows the impact of the continuing educational programme.

The number of laboratories expressing results as INR has steadily increased and the CV is now approaching that obtained with BCT. The improvement in CV suggests that the general application of the INR system will help improve inter-laboratory standardisation of oral anticoagulant control and should be encouraged.
ACUTE INTERRUPTION OF ORAL ANTICOAGULANT THERAPY: A THROMBOTIC RISK?
Authors: Rouvier J , Vidal H , Gallino J , Boccia M , Scazziota A , Altman R
Published: August 2018

It is still on discussion how oral anticoagulant therapy must be interrupted. A progressive diminution of drug intake have been proposed in order to avoid a MreboundM of vitamin K-dependent procoagulant factors. At the present, it is well known that coumarin drugs affect not only the biologic activity of factors II, VII, IX and X but also Protein C (PC), an inhibitor of coagulation kinetics, and their cofactor Protein S. With the aim to determine the recovery level of PC in relation with the others vitamin K-dependent factors, the effect of suppression of anticoagulant therapy in patients under chronic treatment with acenocoumarin was studied.

Quick time, functional factors II, VII, X (one stage methods), functional PC (Francis method) and immunological Factor II and Protein C (Laurell) were determined before and 36 hours after suspension of acenocoumarin administration.

Results showed that: 1) Recovery levels of functional Protein C (increased from 28.55% ±2.57 to 72.64% ±5.9) were significantly higer than functional Factor II (22.09% ±2.34 to 30.73% ±8.64), Factor VII (22.55% ±2.01 to 40.73% ±4.85) and Factor X (23.27% ±2.66 to 39.18% ±3.19). Statistical analysis (Newmann-Keuls test) showed at least a p<0.01 between PC increase and factors II, VII or X increment.

2) No significant differences were seen between immunological levels of Factor II before and after suspension of acenocoumarin.

3) Levels of immunological PC in patients under anticoagulant therapy were higer than functional PC. After acenocoumarin suppression, not correlation was seen between immunological and functional Protein C recovery.

It is concluded that acute suppression of acenocoumarin does not induce a thrombotic tendency because the recuperation of functional Protein C is more important than factors II, VII and X recovery.
SALICYLATE-WARFARIN INTERACTION: EFFECTS ON SYSTEMIC ANTICOAGULATION, BLEEDING TIME, AND EXPERIMENTAL VENOUS THROMBOSIS
Authors: Roncaglioni M C , Reyers I , Bolognese Dolessandro A P , Cerletti C , Donati M B , de Gaetano G
Published: August 2018

The potential benefit of the aspirin/warfarin association as an antithrombotic treatment has been matter of debate in view of the major haemorrhagic effect reported with this drug combination. We have tested the effect of such association in a model of venous thrombosis already shown to be prevented by a fully anticoagulant schedule of warfarin. CD-COBS male rats were treated for three days with either warfarin (0.1 mg/kg i.v. once a day) or salicylate (175 mg/kg i.p. twice a day) or their combination (W+S). Systemic anticoagulation (thrombotest), template bleeaing time and occurrence of experimental venous thrombosis (ligature of inferior vena cava) were followed. Treatment with W or S alone did not affect template bleeding time, whereas the association (W+S) did (320+35 sec versus 120± 10 sec in the control group, p<0.01). Thrombotest was only slightly prolonged by single drug treatment (W= 43%, S=48% versus 90% of controls) but strongly prolonged in the association group (S+W=5%; p<0.001). The mechanism of this combined effect may be multifaceted; competition of both drugs for protein binding and the anticoagulant effect of salicylate itself could contribute. In any case, neither the incidence nor the weight of the thrombus were reduced by any drug treatment. Thus, W+S, in contrast to W alone (<5% thrombotest), was unable to prevent or reduce venous thrombosis, while prolonging bleeding time. Bleeding complications reported in clinical trials by the association of W and aspirin might not be solely due to the antiplatelet effect of aspirin.
COAGULATION, FIBRINOLYSIS AND PLATELET FUNCTION DURING COUMARIN THERAPY
Authors: Taberner D A , Thomson J M , Poller L
Published: August 2018

The inactivation of factors VIII:C, V:C and fast acting TPA inhibitor by activated Protein C indicates that oral anticoagulation is more than simple reduction of prothrombin complex activity. To investigate these changes, six patients were studied after stopping oral anticoagulant treatment. Protein C activity and C antigen, Factors VIII:C, VIII:vWFAg, V:C, V:Ag, X:C, VII:C, fibrinogen and TPA activity were measured during long-term nicoumalone therapy (duration of therapy 8-96 months, mean 28 months), and after discontinuation on days 2, 4, 8, 10, 15, 30 and 42.

The INR on the last day of therapy ranged between 2.0 - 3.3, (mean 2.6). Protein C activity and antigen and factor X became normal by day 8; factor II by day 10. Factor VII activity peaked on day 8, falling to resting levels by day 30. Factor VIII parameters remained high throughout, whereas Factor V antigen showed no significant change. Factor V activity was not quantifiable untill day 8 because of non-parallelism (? PIVKA effect), but was higher on day 8 than day 42 (p < 0.002 paired “t” test) . The higher levels of factor V activity could be protein C dependent, but the high factor VIII appears unrelated. Fibrinogen levels were higher on coumarin treatment (p < 0.05 paired “t” test) and took 30 days to fall to resting level. The effect of Protein C on TPA inhibitor would be expected to increase the activity of TPA, but this activity remained unchanged. Raised fibrinogen levels did not, therefore, appear to be mediated by the effect of protein C on fibrinolysis. Fibrinogen levels in plasma influence ADP induced platelet aggregation which is known to be increased in patients receiving coumarin drugs. In conclusion, patients on coumarin treatment, in addition to showing a reduction in protein C activity, also have higher fibrinogen levels and increased platelet aggregability all of which may be undesirable.
STOPPED-FLOW KINETICS OF PROTHROMBINASE COMPLEX ASSEMBLY
Authors: Krishnaswamy S , Jones K , Mann K
Published: August 2018

Prothrombinase is the multi -component enzyme composed of factor Xa and the cofactor, factor Va, that assemble on a phospholipid surface in the presence of calcium ion. Fluoresence stopped-flow kinetic studies of prothrombinase assembly in the absence of the substrate, prothrombin were undertaken at saturating concentrations of calcium ion, using phospholipid vesicles (PCPS), factor Va and factor Xa modified at the active-site with the fluorophore dansyl-glutamylglycinylarginylchloro methylketone (DEGR-Xa). The substantial fluorescence enhancement of DEGR-Xa in the presence of factor Va, PCPS and calcium ion was used as a continuous measure of prothrombinase complex formation. The rate of complex formation studied under pseudo-first order conditions was most rapid when factor Va was reacted with the DEGR-Xa-PCPS binary complex. Under these conditions, the rate increased linearly with increasing PCPS concentrations and was independent of the concentration of factor Va. The reaction between Va and PCPS assessed by stopped-flow light scattering produced identical rates. The data indicate that the rate-limiting step in the reaction between Va and PCPS-bound DEGR-Xa is the initial formation of a Va-PCPS binary complex (8.4×107M−1s−1) followed by the very rapid reaction (>3×109M−1s−1) between PCPS-bound DEGR-Xa and Va. When complex formation was initiated by mixing DEGR-Xa and Va with PCPS or by mixing DEGR-Xa with Va-PCPS, the pseudo-first order rates were substantially lower. Under these conditions, increasing concentrations of factor Va inhibited the rate of prothrombinase complex formation which could be overcome by increasing PCPS concentrations. The data are consistent with the interpretation that the initial formation of the DEGR-Xa-PCPS complex is a prerequisite for prothrombinase assembly. The second order rate constant for the reaction between DEGR-Xa and PCPS (9.5×109M−1s−1 ) was independently determined by stopped-flow light scattering. Collectively, the data indicate that prothrombinase assembly proceeds via the initial formation of DEGR-Xa-PCPS and Va-PCPS binary complexes. The lipid-bound constituents then rapidly react via diffusion over the vesicle surface to form prothrombinase.

(Supported by NIH grants HL-35058 and HL-34575)
REGULATION OF FACTOR V (FV) EXPRESSION IN HUMAN MEGAKARYOCYTES
Authors: Gewirtz A , shepiro C L , Bovd R , Colman R W
Published: August 2018

We have suggested that synthesis of FY by human megakaryocytes (MEGS), is a maturation relrted event. This is based on our finding that when MEGS are cloned from progenitor cells in vitro in FY depleted medium, FY is immunochemically detectable only in morphologically recognizable (mature) cells (Blood 67:1639, '86). The stimuli which induce FY synthesis in MEGS are unknown, but if they are related or linked to factors which regulate terminal maturation processes cellular FY levels might increase as the maturation routine proceeds. To test this hypothesis, mature human MEGS were isolated from normal bone marrow by counterflow centrifugel elutrietion, deposited onto gloss slides by cytocentri-fugation, and then fixed in methanol:acetone. Individual cells were then staged, geometric meon cell diometer (size) determined with on opticol fylor, end FY end DNA levels measured. FV expression in a large area of the MEG (38 urn in diameter) was semi-quantitated by microspectrophotometry (MSPEC) using a sensitive Texas Red labeled streptavidin-biotin detection system and a monoclonal antibody probe (B10) directed against the non-binding FY activation peptide (150,000 kd). Cells were then reacted with Chromomycin A3 to allow for simultaneous DNA quantitation by MSPEC in the same cell. Correlation coefficients (r) and coefficient of determination [r2] were computer generated to discern potential relationships between FY expression, and MEG maturation stage, size or ploidy level in a total of 532 MEGS. Characteristics of the population, and r/r2 vs. MEG FV content ere shown below:

r/r2 values did not significantly change when total MEG FY content was measured in 100 additional MEGS. Therefore, MEG FY levels varied independently with cell size and ploidy, and did not appear to correlate with degree of mature MEG maturation as determined by standard critera. In two of four preliminary experiments, low dose (8nM ql2h × 4 doses) tetradecanoyl phorbol acetate augmented both the number of MEGS expressing FV, and the level of FY expressed per individual cell. We conclude from these data that once FV synthesis is initiated, FY synthesis and events relating to final cell size and ploidy development are regulated independently. Our data also suggest that MEG FV synthesis may be regulated in part by the phosphoinositide-protein kinase C system.
PROPERTIES OF A F VII CONCENTRATE.
Authors: Chabbat J , Pejaudier L , Kichenin-martin V , Hampikian-Le Nin S , Steinbuch M
Published: August 2018

A concentrate of factor VII obtained by adsorption onto an inorganic adsorbent using a by-product of routine fractionation as starting material This fraction contains only small amounts of the other constituents of the prothrombin complex ( PC ) but it is enriched in proteins C and S. It contains much less VIII CAg than the usual even activated PC concentrates. The amounts of isoagglutinins anti-A and B have also been checked. The concentrate of partially versus completely activated factor VII ( by factor Xlla ) has been submitted to animal experiences including those effected on hemophilic dogs. The concentrate and highly purified factor VII prepared from it were checked on a serie of chromogenic substrates and inhibitors of proteolytic enzymes of plasmatic and other origin. The results obtained are compared with those given by the two most commonly used activated PCCs. The correlation between active F VII and by-passing activity has been studied. This concentrate might thus be indicated for the treatment of hemophilia A-patients with inhibitors.
ANALYSIS OF LEUKEMIA PELT. TISSUE FACTOR BY WESTERN BLOTTING TECHNIQUE.
Authors: Tanaka H , Narahara N , Sadakata H , Andoh K , Kobayashi N , Maekawa T
Published: August 2018

It has been reported that the tissue factor(TF) of monocytes and leukemic leukocytes is one of the trigger substances of disseminated intravascular coagulation(DIC) in leukemia patients. To assess the properties of TF of leukemia cells, their TF was analyzed by the method of Western blotting. Placenta TF was purified using Concanavalin-A affinity chromatography. Briefly, human placenta TF was extracted from placenta acetone powder using Triton X-100 extraction and purified by Concanavalin-A affinity chromatography and SDS-preparative PAGE. Hie final product of the purified placenta TF-apoprotein exhibited a molecular weight (MW) approximately 46 kD in non-reduced condition and 47 kD in reduced condition and showed single band on analytical SDS-PAGE. Activity of purified placenta apo-TF after relipidation with placenta phospholipid was 4500 units/mg and was 6300 times purified from the starting material. Serum IgG fraction was separated from rabbits after 8 times weekly injections of purified placenta TF. This anti-TF-IgG fraction showed single precipitin line against purified placenta apo-TF and inhibited procoagulant activity of saline extract of the placenta as well as those of homogenates of the endotoxin(LPS)-stimulated Molt-4 cells and U-937 cells. Using this antibody, analysis of TF of cultured leukemia cells(Molt-4 and U-937) and acute promyelocytic leukemia(APL) cells was done by the Western blotting technique as follows: LPS-stimulated Molt-4, U-937 or APL cells were homogenized, delipidated and solubilized with 1% SDS-PAGE. Thereafter, Western blotting was done by the method of Towbin and TF of the blotted protein was immunologically identified. The nitrocellulose paper was soaked with anti-TF-IgG for 18 hours’, then peroxidase-conjugated anti-rabbit-goat IgG was used as an indicator of the antibody and o-dianisidine as substrate. In either case of APL cells, LPS-stimulated Molt-4 or U-937, single band which showed procoagulant activity was detected and the MW of the protein was approximately 48 kD in non-reduced condition. These results indicate that TF of all these leukemic leukocytes has common antigenic determinant and equal MW to placenta TF.
MONOCYTE/MACROPHAGE TISSUE FACTOR: ROLE OF ITS N-GLYCOSYLATED CARBOHYDRATE MOIETY.
Authors: NAKAMURA Shin
Published: August 2018

Monocytes/macrophages and related cells are known to generate tissue factor (TF) , a membrane associated lipid-glycoprotein complex, following activation with LPS or other stimuli. Monkey (M. fuscata) mononuclear leukocytes (MNL, 3 × 106/ml) cultured with LPS (lµg/ml) in FCS-free RPMI medium were stimulated to produce the glycoprotein (TF-Apo). After a lag period of 2 h the TF-Apo production was initiated, and its accumulation reached the plateau after 12 h and then declined to approximately half of the maximum level after 24 h. A time course of the TF activity was strictly in accord with that of the TF-Apo accumulation. Tunicamycin, an antibiotic that blocks the first stage in formation of N-linked oligosaccharides of glycoprotein, affected to reduce the TF expression by 15 to 65 %, when monkey MNL (3 x 106/ml) were co-cultured with LPS (1 µg/ml) and the antibiotic (10 to 100 ng/ml) for 10 h. Similar reducing effect of tunicamycin to the TF expression was observed, when RET-1, a macrophage*related cell line that generates spontaneouly TF, was cultured with the antibiotic. Interestingly, leupeptin, an inhibitor to trypsin-type proteases including cathepsin B, protected completely the tunicamycin-induced reduction of the TF expression upon its addition to the culture medium at the concentration of 7 /iM. Chymostatin, an inhibitor to chymotrypsin-type proteases, also showed the protective effect. These results indicate that TF-Apo of monocytes and RET-1 has N-linked oligosaccharides and that defect of the oligosaccharide chain causes TF-Apo to be susceptible to proteolysis during intracellular processing. Thus, the N-glycosylated carbohydrate moiety of TF-Apo of these macrophage related cells has a roll to stabilize and/or protect it against proteolytic inactivation.
TISSUE FACTOR OF A HUMAN CELL LINE, RET-1: ITSPRODUCTION, PURIFICATION AND PROPERTIES.
Authors: NAKAMURA Shin , SUZUKI Yukio , HARADA Takayuki , MORIKAWA Shigeru , KAWABATA Shunichiro , IWANAGA Sadaaki
Published: August 2018

A dendritic cell-like cell line, HIN-Ret-1(RET-1), was found to produce the highest level of tissue factor (TF) among several established cell lines from human lymphoma or leukemia. The TF level expressed by this cell line exceeded 5.6 times that expressed by a monocyte-like cell line, U-937. Unlike other TF producing cell line, i.e. RET-2, HL-60, ML-3, and U-937, the TF expression by RET-1 was spontaneous and unaffected with TP A, PHA, LPS, or MAF. The TF activity of RET-1 was markedly inhibited by Con A as well as that produced by LPS-stimulated monkey monocytes, whereas the TF activity of monkey brain and lung was hardly inhibited by the lectin. Hence, the RET-1 cell lysate solubilized in Triton X-100 was subjected to affinity chromatography on a Con A-Sepharose column, and TF-apoprotein (TF-Apo) was completely bound to the column and eluted with TBS containing 0.15 M α(-methylglucoside and 0.1 % Triton X-100. Further purification of this material was performed with combination of FPLC on a DEAE-5PW column and affinity chromatography using a factor VII-Sepharose column. By these methods, TF-Apo preparation with purification-fold of 9,400 and over-all yield of 7 % was obtained. Its apparent molecular weight was estimated to be 120 kDa by gel filtration in TBS containing 0.1 % Triton X-100. SDS-PAGE gave the value of 47 kDa, which was almost compatible with that of TF-Apo from brain or placenta. TF-Apo frcm the monocytes also bound to the lectin column, suggesting that the apoprotein of these macrophage-related cells has an oligosaccharide chain interacting with the lectin. RET-1 TF-Apo was unstable under acidic condition (18 %) and acetonitrile (>8 %).
INTRAVENOUS BACTERIAL LIPOPOLYSACCHARIDE IMPAIRS MONOCYTE TISSUE FACTOR GENERATION IN RABBITS
Authors: Edwards R , Brande W
Published: August 2018

Bacterial 1ipopolysaccharide (LPS) ts a potent stimulus for monocyte tissue factor (MTF) generation jn. vitro. We have examined the effect of small amounts of LPS on MTF expression in vivo. LPS (0.1 - 50ug) was injected into the ear veins of 66 New Zealand White rabbits. Arterial blood was collected immediately prior to LPS (TO), and 5 (T5) and 30 (T30) minutes later. Mononuclear cell suspensions (MC) were assessed for monocyte (Mo) content and MTF generation was determined in both unstimulated and LPS-stimulated MC cultures (18 hours incubation). As seen in the table, T5 cells obtained following in vivo exposure to 5 or lOug LPS demonstrated spontaneous MTF generation in the absence of in vitro stimulation. By T30, the Mo content of MC decreased from 28% to 17% and MTF generation was decreased in both stimulated and unstimulated cultures. However, this decreased MTF could not be explained by reduction in Mo number. In reconstitution experiments, T30 lymphoctes (Ly) did not suppress TO MTF generation and TO Ly did not restore T30 MTF generation, suggesting that the decreased MTF generation is not mediated by increased Ly suppressor activity. Neither heparin, warfarin nor hydrocortisone had any effect on Mo count or on MTF generation. These experiments suggest that small amounts of LPS activate a subpopulation of Mo, which is quickly lost from the circulation, while the remaining Mo are resistant to further LPS stimulation. Direct activation of this subpopulation of competent Mo by LPS may contribute to activation of blood coagulation in sepsis.
THE IDENTIFICATION OF A NOVEL FACTOR X ACTIVATOR ACTIVITY IN Mg2+ - ANTICOAGULATED PLASMA
Authors: Saundry R H , Khumprayoon S , Savidge G F
Published: August 2018

Mg2+ anticoagulated PPP (20mM MgCl2) was applied at RT to a Zn2+ immobilised biscarboxymethylamino Sepharose 4B column and eluted with 20mM Tris, 20mM MgCl2, 2.5mM CaCl2 0.15M NaCl buffer pH 7.4. Following collection of the wash-through fractions, bound proteins were developed through application of linear (0 to 35mM) imidazole gradients.

All fractions were screened for F.II, V, IX, X, vWF:Ag, Protein C, Fg, Fn and 2-macroglobulin by ELISA, VIII:Ag by IRMA, and VIII:C by both 1-stage and 2-stage bioassay methods. VIIIrAg (60-90% yield), vWF:Ag (100%), VIII:C 1-stage activity (35%), Fn (> 60%), Fg (> 80%), V (50%) and a2-macroglobulin (> 70%) were located only in the gradient fractions. Two distinct peaks demonstrated shortening of the 2-stage VIII:C assay:- one co-eluting with the 1-stage VIII:C activity and another major peak in the wash-through fractions where antigenic determinants of F.II, IX, X and part of the protein C were located and partially resolved from each other. Only F.II.-Ag co-eluted with the “ 2-stage VIII:C” activity. Similar observations were found in Mg2+ -anticoagulated severe Haemophilia A plasma and in citrated PPP developed with 20mM MgCl2, 20mM Tris buffer. A1(0H)3 treatment abolished the activity from citrate -, but not from Mg2+ -anticoagulated plasma.

The relevant fractions showed no activities in F.V, VII, VIII:C, IX or X 1-stage bioassays. They did not clot Fg and did not contain detectable Xa or Ila as assessed by S-2222, S-2238 or S-2288. Following incubation with specific antisera against IgG, II, V, VII:Ag, X, protein C, a- and g- lipoproteins, and plasminogen only anti-II inhibited this "2-stage VIII:C" activity. 2-stage VIII:C assays depend upon Ca2+ -dependent generation of Xa. Since the activity in the wash-through fractions could not be ascribed to VIII, Xa, or Ila the results would indicate the presence of a hitherto undescribed Factor X activator activity.
INHIBITION OF HUMAN FACTOR Xa BY ITS ACTIVATOR FROM RUSSELL’ S VIPER VENOM.
Authors: Latallo Zbigniew S , Jackson Craig M
Published: August 2018

In contrast to bovine Factor Xa, human Factor Xa is inhibited by its activator from Russell’ s viper venom (X-CP). This inhibition occurs only in the presence of Ca ions. When Xa activity is measured using chromogenic substrates at pH 7.8, inhibition is more apparent at 37° C than 25° C, but can be reduced to nearly zero at high NaCl concentration. The extent of inhibition depends on the source of X-CP. X-CP isolated from Vipera russelli siamensis, which activates human Factor X with a K = 0.0187 ptt and Vmax of 5.26 x 10’ 11 M’ s-1 ([X-CP] 0.15 nM, 0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES, 5mM CaCl2, 0.1% PEG, pH 7.8, 25° C) inhibits the Factor Xa about twice as effectively as X-CP from Vipera russelli russelli for which the human Factor X activation rate is about half that observed with V. russelli siamensis. The inhibition is of a mixed type and results in doubling the apparent Km value and decreasing V by 20%. By fitting of the hyperbolic, mixed inhibition model

where E is human Xa, I is V. russellii siamensis X-CP, S is Cyclohexylglycyl-glycyl-arginine p-NA, the following values were calculated Km = 120 /xM, k, = 250 s’ 1, K. = 1.0 x 10"8, a = 2.0 and p = 0.82, (0.22 nM Xa, 0.1 M NaCl, 0.01 M TRIS, 0.01 M HEPES, 5 mM CaCl2 0.1% PEG pH 7.8, 25° C). Activation of human prothrombin by human Factor Xa as well as inhibition of the latter by antithrombin III are similarly affected by the binding of X-CP in the presence of Ca . X-CP binding to human Xa may explain some of the enigmatic differences in functional properties of bovine and human Factor Xa preparations.

Supported by a Matching Grant from the American National Red Cross and by the S.E. Michigan Red Cross Blood Service