bluuebunny/crossref_metadata_2025 huggingface dataset:
ROLE OF DISULFIDE BONDS NEAR THE CALCIUM BINDING SITES IN FIBRINOGEN
Authors: Procyk R , Blomback B
Published: August 2018

Incubation of fibrinogen with 0.5 mM dithiothreitol in the presence of .20 mM calcium chloride cleaved disulfide bonds located at: the N-terminal end of the Aα-chain (either Aα28-Aα28 or Aα45-γ23), the C-terminal end of the Aα-chain (Aα442-Aα472) and the N-terminal end of the γ-chain (either of the symmetrical γ8, γ9 disulfides or the Aα45-γ23 disulfide bond). In the absence of calcium ions two additional disulfides, γ326-γ339, and one in the N-terminal end of the γ-chain were reduced.

Plasmin digestion of the reduced fibrinogens in buffers containing calcium chloride produced fragments D and E, except that smaller fragments of D were generated from the fibrinogen in which the γ326-γ339 disulfide bonds were reduced and alkylated. In these samples calcium did not protect the C-terminal end of the γ-chain from extensive digestion.

Addition of thrombin to partially reduced and alkylated fibrinogen prepared in the presence of calcium gave a clotting time similar to control unreduced fibrinogen. However, when the γ326-γ339 disulfide bonds and another γ-chain disulfide bond most likely in the N-terminal region were cleaved in reduced fibrinogen prepared in the absence of calcium, the thrombin clotting time was extremely prolonged. Apparently the disulfide bonded structure supported by γ326-γ339 was important both for binding of calcium and also for normal clotting.
A NEW AT III VARIANT WITH DEFECTIVE PROTEASE BINDING SITE
Authors: Aiach M , Roncato M , Sorin G , Dezellus P , Fiessinger J N
Published: August 2018

A decreased plasma anti thrombin activity in presence heparin cofactor (hep-cof) or in absence of heparin (AT) was discovered in a 47 year-old patient presenting with recurrent venous thromboembolism. The immunoreactive material (AT III-IR) was normal. The same biological abnormalities were found in two relatives of the patient, leading to the diagnosis of hereditary qualitative AT III deficiency.

The propositus'AT III was coeluted with normal AT III from an heparin sepharose column. An additional step of ion-exchange chromatography on a Mono Q column using a FPLC system (Pharmacia, Bois d'Arcy, France) allowed the purification of a protein which was homogenous in SDS - 10 % polyacrylamide electrophoresis gel (PAGE). AT III purified from propositus'plasma, normal plasma and the plasma of a patient known to have an AT III variant with defective protease binding (1) were compared. The specific activities measured as hep-cof AT or factor Xa inhibition in absence of heparin (anti Xa) were respectively 6.4 and 4.8 U/mg for the propositus 'AT III and 13.6 and 8.5 U/mg for the normal AT III (one unit is the activity of 1 ml of a plasma pool prepared from 30 normal subjects). The formation of protease inhibitor complexes was studied by incubating purified AT III with purified thrombin (in molar ratio 1:4, 1:2, 1:1) during 5 minutes at 37 °C and submitting the mixture to PAGE. The densitometric scan showed that in equimolar ratio the percentage of an AT III-thrombin complex (with 92 kA Mr) reaches 70 for normal AT III and respectively 30 and 23 for the propositus'AT III and the already described variant AT III Charlevillé (1). A 70 % proportion of free AT III (58 kA) remained for the propositus. As previously observed an unidentified 63 kA compound appeared for AT III Charleville. This results strongly suggest that in the patient described here, half th the AT III molecules are normal, the others having a defective protease binding site. We propose to call this new variant AT III Avranches, the town where the propositus was born.

(1) Thrombosis Research 1985, 39, 559-570.
THROMBOLYSIS AND ACUTE MYOCARDIAL INFARCTION (AMI)
Authors: Villemant D , Barriot P , Bodenan P
Published: August 2018

AMI is a major cause of morbidity and mortality in modern society Conventional treatment has no benefic effect on the size of infarct, alteration of left ventricular (LV) function and mortality. Intravenous (IV) thrombolysis reduces in hospital mortality by 23 % if infused within 3 hours of ischemia, 47 % if within 1 hour. It reduces the size of infarct by 51 % if reperfusion occurs within 1 hour of ischemia, 31 % if between 1 and 2 hours and 13 % if between 2 and 4 hours. The preservation of LV function is of 28 to 42%. These benefic effects, thanks to IV thrombolysis, can be obtained only if reperfusion occurs within 3 or 4 hours of ischemia. Unfortunately, a french prospective study “ENIM 84” estimates that the mean delay between onset of chest pain and arrival at hospital is 10,3 hours.Goals of the study were to show that “at home” thrombolysis: 1) is a feasible and a safe technique, 2) is responsible of a significant saving of time, 3) preserves LV function according to the precocity of treatment.Two groups of patients (pts) are compared : group A : 62 pts had “at home” thrombolysis by a trained medical staff aboard a mobile emergency care unit. Group B : 53 pts had thrombolysis at arrival at CCU. Protocol is simular in both groups : An IV infusion of 1 5 M iu of streptokinase over 45 to 60 min after an IV bolus of 100 mg Hydrocortisone. Criteriae and contra-indications are those usually used for thrombolysis. Radionuclide angiography was performed 4 days and 1 month after AMI to evaluate global and regional ejection fraction (EF). Only 1 hemorrhagic complication (a mild melaena) and 2 reversible ventricular fibrillations were reported. Reperfusion arrythmias were frequent (55 %) but do not need treatment. The number of candidates for thrombolysis is then increased. The saving of time is 73 min. Difference between the 4 days and 1 month EF is not significant in pts with conventional treatment or if reperfusion occurs after 4 hours of ischemia 48 ± 11 % vs 51 ± 13 %.But it is significant if before 4 hours 49 ± 11 % vs 56 ± 12 % and highly significant if before 2 hours 48 ± 12 % vs 59 ± 10 %.
PREHOSPITAL THROMBOLYTIC TREATMENT: A FEASIBILITY STUDY
Authors: Bossaert L , Demey H , Colemont L , HRM H
Published: August 2018

Aim of the study: Thrombolysis is the treatment of choice in acute myocardial infarction (AMI). The delay between onset of symptoms and administration of thrombolytic drugs is critical for successful reperfusion and myocardial salvage. We studied the feasibility and safety of early prehospital thrombolytic treatment of AMI “at home”

Methods: Eminase(R)(APSAC;BRL 26921 ) was used as thrombolytic agent: its long half-life allows administration as a single IV bolus. The study was performed in collaboration with a well organised group of GP's, extensively retrained in ECG and CPR for the purpose of this study. Whenever a GP made the tentative diagnosis of AMI of less than 2 hours, inclusion and exclusion criteria were reviewed using a check-list, and the mobile intervention team of the hospital (MITUZA), consisting of CCU physicians and emergency nurses, was activated. After rechecking all criteria, including a 12 lead ECG, brief medical history and physical examination, a single IV bolus of 30 U Eminase(R) was given, followed by transferral to the CCU. Follow-up included estimation of infarct-size and LV function using biochemical, ECG, radionuclide and angiographic methods.

Results: Up till now, 15 male patients (age=57±9) have been treated using this protocol. Initial prehospital treatment consisted of Eminase(R) IV in 10 (reperfusion 9/10). Subsequent in-hospital treatment was streptokinase in 3 (reperfusion 2/3). In the 11 reperfused patients, PTCA was performed in 7 and CABG in 4. After onset of symptoms, the GP arrived after 55±24 min (15-90 min), Eminase(R) was administered after 95±28 min (75-130 min). The total calculated time gain (interval between treatment at home and admission in the CCU) was 42±15 min (20-75 min). There were no adverse events.

Conclusion: This pilot study indicates that prehospital thrombolytic treatment of AMI is feasible and safe, resulting in a considerable time gain. Early reperfusion was obtained in 90%. Collaboration with a well organised and trained group of GP's, clearly defined inclusion and exclusion criteria and administration of the thrombolytic drug exclusively by experienced critical care physicians are mandatory.
PROTECTIVE EFFECT OF CARNITINE (ST-261, SIGMA-TAU) IN ACUTE MYOCARDIAL INFARCTION IN DOGS
Authors: Kordenat K , Leasure J
Published: August 2018

Limitation of infarct size (IS), using ST-261, was evaluated in a group (I) of eight dogs, during acute MI. Another group (II) served as the control group. The protocol for both groups was the same except that each dog in the treated group was ST-261 as a single bolus (25 mg/kg, in 20ml normal saline), before inducing an occluding thrombus in the mid-LAD, using a closed-chest model, under x-ray visualization. Percentages of total (gms) myocardium at jeopardy (TMJW) and myocardial necrosis (TMNW), delineated by fluoroscein and TTC dyes, respectively, were calculated and compared to the total ventricular myocardial weight (TVMU), by computer technique for both groups at 3 Hrs post-occlusion of the LAD. Mean serum total CPK (CPK-t) and isozymes (mb-band) were measured before and up to 3 Hrs post-occlusion, as were various hemodynamic and mean precordial (21 lead) ST-segment and T-wave amplitudes. There was 14% less TMJU (p<0.05) and 41% less TMNW (p<0.01) in Group I compared to Group II. The mean % of CPK-mb/CPK-t decreased in I and increased in II over the 3 Hrs of observation. Mean HR decreased (p<0.01) in I compared to II at 3 Hrs postocclusion. The sum of the mean T-wave amplitudes from the precordial electrode sites was less in I at 3 Hrs. It is felt that ST-261 had a protective effect on the myocardium during acute myocardial infarction.
DECREASED FIBRINOLYTIC CAPACITY IN SURVIVORS OF ACUTE MYOCARDIAL INFARCTION
Authors: Schoenfeld P , Therville E , Bethume P , Leautaud P , Wachel D , Rodzynek J J
Published: August 2018

Disturbances of several markers of fibrinolytic capacity have been reported in patients with an history of thrombotic pathologies. In this work we assessed Euglobulin Lysis Time (ELT) before and after venous, stasis, t-PA release (t-PAr) expressed as the ratio of t-PA antigen after versus before venous stasis, and presence of t-PA inhibitor (t-PAi) in 2 populations: group 1, normal controls (n=50) and group 2, survivors of acute myocardial infarction (AMI)(n = 17).

The results were as follows :

1) ELT shortening after venous stasis = 46/50 in group 1 compared to 3/17 in group 2 (p < 0.01, X2 test);

2) t-PAr insufficient in 1/50 patients of group 1 compared to 15/17 patients of group 2 (p < 0.01, X2 test);

3) presence of t-PAi 3/50 in group 1 compared to 9/17 in group 2. The 3 normal controls with t-PAi were also characterized by absence of ELT shortening after venous stasis. In group 2, the relationship between absence of ELT shortening and other parameters of fibrinolysis appeared as follows :

In patients of group 2 without ELT shortening after venous stasis, insufficiency of t-PAr was consistently more pronounced in those without detectable t-PAi.

We conclude that :

1) our data confirm the marked abnormalities of fibrinolysis in post-AMI patients;

2) these abnormalities appear to be related to insufficency of t-PAr, either alone or in combination with the presence of t-PAi.
IgE-MEDIATED IMMUNOLOGICAL RESPONSE IN MYOCARDIAL INFARCTION
Authors: Szczeklik A , Sladek K , Dropinski J
Published: August 2018

Mast cells have been described in adventitia of coronary arteries; their number increases with progression of atherosclerosis. We reasoned that, if these cells were to contribute to cardiac pathology, the signals turning them on should be detectable in the blood. We, therefore, measured systematically serum IgE in 25 patients (18 men and 7 women, mean age 55 years) with unequivocal diagnosis of recent transmural myocardial infarction. All patients survived. When specifically interviewed, only two gave a history compatible with atopy. In 14 patients, whose initial values ranged from 44-750 IU/ml, and on average 172 IU/ml, serum IgE increased by more than 25%. It raised gradually after the infarction,reaching the peak by 7th day (mean increase by 92%), then began to decrease, but was still elevated on 14th and 21st day. Such behaviour was in striking contrast to mean serum IgG which at no time showed changes differing by more than 10% from the initial values. Thus, in at least half of the patients with acute myocardial infarction there occurs an immunological reaction characterized by distinct release of IgE into circulatory blood. It might be that IgE sensitizes mast cells and facilitates the release of chemical mediators. Histamine, prostaglandin D2 and sulfido-peptide leukotrienes are all powerful vasoconstrictors of human arteries. On the other hand, heparin exerts potent anticoagulant effects through activation of antithrombin III, and PGD2 inhibits platelet aggregation. This complex interplay of the mediators at the target site might affect blood clotting, cardiac function and the course of myocardial infarction.
THROMBELASTOGRAPHIC EVALUATION OF THE EFFECT OF ADDED PLASMINOGEN TO PLASMA ON FIBRINOLYSIS USING VARIOUS FIBRINOLYTIC ACTIVATORS
Authors: Summaria L , Yang G G , Holloway D S , Caprini J A
Published: August 2018

When lysis of peripheral thrombi requires infusion of plasminogen activators for longer than 24 hours duration, slow resolution of an obstructive thrombus can result in tissue injury and loss of limb. This study evaluated the effects of increasing the plasminogen concentration on the kinetics of clot lysis induced by plasminogen activators. By adding varying concentrations of any plasminogen activator to plasma, clot lysis can be continuously monitored using thrombelasto-graphy (TEG). Parameters measured during the lysis portion of the pattern are the final amplitude reached (Af), various time parameters (T, T’ and T50% ) and the absolute change in amplitude ( Δ A). By adding different amounts of Lys-Plasminogen (LYS-PLG) to plasma, we were able to measure the effect of added LYS-PLG on the lysis pattern with different activators (streptokinase, the plasminogen-streptokinase complex, or the B-chain-streptokinase complex). Statistical analysis of the plots of T50%(time for 50% lysis, where the TEG amplitude is reduced to naff that of the maximum amplitude) versus LYS-PLG concentration showed that a second order polynomial regression gave R values of better than 0.969. The addition of a small amount of LYS-PLG (20% of the normal plasma concentration) resulted in a two-fold decrease in T50% with all activators. Maximum clot-lysis enhancement was reached when 60% excess LYS-PLG was added. This amount caused a 2.9 to 3.9 fold decrease in T50%. Values above 60% did not significantly reduce clot- lysis time any further when very low levels of plasminogen activator were added to plasma. Plots of clot-lysis (T50%) versus activator or LYS-PLG concentration showed that tne amount of activator could be decreased by at least two-fold if between 40 to 60% extra LYS-PLG is added

Thus, the addition of LYS-PLG to a plasma clot produced a two- to three-fold increase in the rate of clot lysis (2 to 3 fold decrease in T50%) or permitted a two-fold lesser amount of activator to produce the same lysis pattern.
INCREASED THROMBOTIC TENDENCY IN A FAMILY WITH HEREDITARY ANGIONEUROTIC EDEMA ( HANE )
Authors: Kirschstein W , Hoffmann U , Simianer S , Dempfle E , Kortsik C , Heene D
Published: August 2018

Biochemical hallmark of HANE is a reduction of C1-inhibitor. We observed a family with type II disease ( non-functional protein ), in which 3 of 6 affected members had arterial thromboembolic events at young ages. For evaluation of alterations in the hemostatic system analysis included: fibrinogen, FVII, FVIII, FIX, FXI, FXII, prekallikrein PK, antithrombin III ATIII, protein C PC, α2 antiplasmin α2AP, cl α2 macroglobulin α2 MG, plasminogen activator inhibitor PAI, plasminogen PG, euglobulin clot lysis time ECLT and tissue plasminogen activator tPA at baseline and after venous occlusion. The results are shown in part in the table:

There is evidence of nearly no response to venous occlusion in 2 and a diminished response in 1 out of 4 patients.

We conclude, that the increased thrombotic tendency in this family is related to the increased potential of prephase coagulation factors and impaired fibrinolytic response to venous occlusion concomitantly with the reduction of the main inhibitor of the contact activation system.
LOCAL ACTIVATION OF COAGULATION AND FIBRINOLYSIS IN LUNG DISEASE IS REFLECTED IN BR0NCH0ALVE0LAR LAVAGE FLUID
Authors: Nakstad Britt , Lyberg Torstein
Published: August 2018

Fibrin deposition in the alveolar space and the lung interstitium is a prominent feature of many types of pulmonary inflammatory diseases.Ce11s of the monocyte/ macrophage lineage have a dualistic role in the balance between coagulation and fibrinolysis having the ability to synthesize both procoagulant factors and plasminogen activators. In the present studies bronchoa1veo1 ar lavage were performed on 128 patients with various lung diseases and 29 healthy controls.Both lung alveolar macrophages (LAM) and the supernatant bronchoalveolar lavage fluid (BALF) expressed two types of procoagulant activities a)thromboplastin and b) a direct factor X activator.The procoagulant in BALF was associated with membrane vesicles which sedimented at 100000g,lh.By electron microscopy the BALF ultrasediment was seen to consist for a large part of membrane material and this was confirmed by monitoring the content of different marker enzymes for specific subcellular structures. LAM from patients had significantly higher specific thromboplastin activity than LAM from controls (4,36 -0.98(SEM)vs.0.81± 0.14 U/mg cell protein).BALF collected from patients had significantly higher levels than BALF from controls of a ) thrombop1 astin(0.66-0.18vs.0.07 ±0.01 U/ml) b ) factor VII activity(1.33±0.31vs.0.48±0.06 U/ml) c)fibrin degradation products(presentin 28,7vs.0% of the cases) and d) fibronectin(491±103 vs.35±5 ng/ml ) In addition,the level of plasminogen activator was higher in controls than in patients(294±68 vs.!02±14 m U/m1).

These studies show that activation products of the coagulation and fibrinolytic systems can be detected in BALF and that lung disease often is associated with abberations in the balance between these systems.

Fibrin serves as a substrate for fibronectin and the increases in lavage fibronectin may reflect the development of lung fibrosis.
FIBRINOPEPTIDE B6 15-42 IN LIVER CIRRHOSIS: A SENSITIVE INDICATOR OF MILD FIBRINOLYSIS ACTIVATION
Authors: Marongiu F , Acca M R , Mulas G , Conti M , Sorano G , Balestrieri A
Published: August 2018

In order to detect even minimal fibrinolysis activation in liver cirrhosis and to investigate whether an increased plasmin activity is related to a mild blood coagulation activation, we measured fibrinopeptide A (FPA) (Mailinckrodt) and fibrinopeptide BB 15-42 (BB 15-42) (IMCO and SORIN Biomedica) in 26 patients (16 men and 10 women, mean age 55.8 ± 13.1 years) with histologically proven liver cirrhosis..Mann-Whitney test, Student’s t test and correlation coefficient r were employed for statistical analysis when appropriate.FPA and BB 15-42 were not normal distributed and thus their levels were exprsessed as median and range.FPA values were significantly different in cirrhotic patients (3.9, 0.9-24.2 ng/ml) from those of the controls (2.5, 0.5-3.9 ng/ml) (p < 0.01).BB 15-42 levels were significantly higher in cirrhotic patients (19.4, 7.1-103.1 ng/ml) than in controls (10.4, 5.1-15.4 ng/ml) (p < 0.01). A posteriori the patients were divided in two subgroups according to whether their FPA levels were high (subgroup 1, n=10, FPA>4.0 ng/ml) or normal (subgroup 2, n=16, FPA < 4.0 ng/ml).In patients with high FPA levels we found higher levels of BB 15-42 (22.2, 9.9-103.1 ng/ml) than in patients with normal FPA (13.6, 7.1-30.7 ng/ml ).Thvis difference was significant (p < 0.02) .There was no relationship between FPA and BB 15-42.Our data indicate that in liver cirrhosis a mild fibrinolysis activation may occur.The role of a chronic intravascular coagulation appears to be significant in this regard.However the impaired clearance of plasminogen activators, the decreased synthesis of fibrinolysis inhibitors and the decreased levels of hystidine rich glycoprotein may be also involved in determining fibrinolysis activation as suggested by the lack of correlation between FPA and BB 15-42.
HOW DOES CALCIUM ALGINATE ACHIEVE HAEMOSTASIS IN SURGERY?
Authors: Jarvis P M , Galvin D A J , Blair S D , McCollum C N
Published: August 2018

Calcium alginate (Kaltostat, Cair Ltd) is a new absorbable material for topical haemostasis in surgery. The possible mode of action, release of calcium ions in exchange for sodium was investigated in human blood.

Calcium release was measured in 15 mg samples of calcium alginate placed in 20 ml of 0.9% saline, for intervals of 1, 3 or 10 minutes. To assess the effect on platelets, 3 mg of calcium alginate or surgical gauze were added to 5 ml of Heparinised (100 units) fresh blood for 2 minutes and platelet counts then made using plain blood as a control. Finally using a thrombelastograph, the activation of whole blood coagulation was assessed after a 2 minute contact with 3 mg of calcium alginate, surgical gauze or no additive as control.

When calcium alginate was placed in saline, 26% of calcium ions were released in 1 minute giving a calcium ion concentration of 4.62 t 0.02 mmol/L, with only slight further release after 10 minutes to 4.82 ± 0.004 mmol/L. There was a corresponding decrease in sodium ion concentration. Adding calcium alginate to whole blood reduced the platelet count from a control value of 248 i 16 × 109/L to 222 f 15 × 109/L (p< 0.05) compared to 241 ± 15 × 109/L for surgical gauze. Similarly calcium alginate shortened whole blood coagulation time from 17-7 i 1.0 minutes control, to 12.9 ± 1-32 mins (p< 0.001) compared to 15.0 ± 1.5 mins (p< 0.02) for surgical gauze.

Calcium alginate rapidly releases calcium ions in exchange for sodium on contact with blood stimulating both platelet activation and whole blood coagulation, significantly more than simple contact activation by surgical gauze.
IS ATHEROSCLEROSIS A DISEASE OF THE OUTSIDE OF ARTERIES?
Authors: Booth R F G , Martin J F , Moncada S , Honey A C
Published: August 2018

The early stages of atherosclerosis are characterised by: (i) Intimal infiltration by macrophages, (ii) Intimal smooth muscle cell (SMC) proliferation and (iii) Accumulation of cholesterol within the vessel wall. Many studies have suggested that these changes occur subsequent to endothelial cell (EC) damage and platelet deposition. We now describe a new animal model with characteristics of early human atherosclerosis but without observable EC damage or platelet deposition. 12 rabbits were separated into 2 groups; each was fed normal laboratory chow and one group was supplemented with lg/day cholesterol. At day 0, under anaesthesia, a silastic collar (internal volume 0.3ml) was placed around the left carotid artery. The collar was filled with whole blood and sealed without causing Constriction of the vessel. The right carotid arteries were sham operated. The vessels were replaced and the animals allowed to recover. After 14 days both carotids were perfuse-fixed in situ. The left carotid arteries were separated into:(A)A mid-region from within the collar,(B)A region proximal to the collar and (C)A distal region. These regions were subdivided for histology and scanning E.M. Medial/Intimal ratios of transverse sections of each region were analysed by computerised image analysis and shown below.

Histology demonstrated an intense proliferation within 14 days which resembled early atherosclerosis. There was intimal monocyte infiltration and SMC proliferation and loss of SMC from the medial layer. Scanning E.M. revealed no observable EC damage or platelet adhesion. The lesion was highly focal and generally restricted to the collar region. Dietary cholesterol did not enhance proliferation but did cause foam cell formation. Sham operated vessels showed no significant atherosclerotic changes. Thus, early atherosclerotic lesions may be the result of damage to the outer layers of blood vessels.
INTERACTIONS OF CULTURED HUMAN ENDOTHELIAL CELLS WITH MODIFIED AND UNMODIFIED POLY(ETHYLENE TEREPHTHALATE); AN ULTRASTRUCTURAL STUDY
Authors: van Wachem P B , Schakenraad J M , Feijen J , Beugeling T , Blaauw E H , Nieuwenhuis P , Molenaar I , van Aken W G
Published: August 2018

A fundamental study of the influence of polymer surface properties on interactions with cultured human endothelial cells (HEC) may lead to the development of improved i.e. endothelium-lined vascular grafts.

We have previously demonstrated that HEC adhere preferably onto moderately wettable polymers.

Here we present the results of spreading measurements and scanning- and transmission- electron microscope investigations on the interactions of HEC with moderately wettable tissue culture polyethylene terephthalate (TCPETP) and more hydrophobic PETP. The numbers of adhering HEC on TCPETP were always found to be much higher than the numbers of HEC adhering onto PETP. However, similar adhesion behavior, occurring in both the presence and absence of serum in the incubation medium, was observed at both TCPETP and PETP surfaces during the first 30 minute period after cell seeding. In contrast with PETP, which showed a decrease in cell spreading during further incubation in the presence of serum, cell spreading increased on TCPETP, resulting in a confluent monolayer at two hours. The decrease of spreading on PETP was a result of detachment of cells. On PETP different structured cells, such as cells with detached central portions or cells, which internalized another cell were observed.

When HEC were first seeded onto PETP in the absence of serum, no decrease but rather an increase in cell spreading occurred after the addition of serum and during further incubation.

HEC spreading on PETP also occurred, when PETP was first precoated with cell adhesion promoting protein(s), such as whole blood, plasma or fibronectin. However, proliferation results indicated that HEC did not form a confluent monolayer after the pretreatment of (TC)PETP with plasma.
INTERACTION OF HUMAN ENDOTHELIAL CELLS WITH HEPARIN-LIKE POLYMERS : INSOLUBLE SULPHONATED POLYSTYRENE RESINS
Authors: Najab M , Jeanneau Ch , Serne H , Jozefowicz M , Sultan Y
Published: August 2018

It was previously demonstrated that insoluble sulphonated polystyrene resins possessed an anticoagulant heparin-like activity in the presence of plasma. The antithrombic activity is dependent on the surface density of the sulphonated groups and involves plasmatic anti T-III. This anticoagulant activity makes this material interesting for blood compatibility concerning its effect on the coagulation system. As these anticoagulant biomaterials may be used “in vivo”, their compatibility with endothelial cells (EC) is of great interest. In the present study, EC from human umbilical cord vein were cultured in 96 well plates in presence and absence of cryoprecipitate considered as the reference culture surfaces, and in wells covered with sulphonated polystyrene beads (SPB). Cell growth in this various conditions was observed and the following parameters were compared : rate of growth of EC, presence of Von Willebrand factor (VWF) by immunofluorescence, release and synthesis of EC specific antigens : VWF, tissue plasminogen activator (T-PA) and tissue plasminogen activator inhibitor (PAI). On SPB, cellular growth was found to be in a normal range but cell morphology was somewhat different. VWF antigen was identified in cells grown either on SPB or on reference wells. Non stimulated cells, incubated in serum free medium released a basic level of VWF in the supernatant. Thrombin enhanced the release of VWF from cells cultured on coated or uncoated dishes and from cells cultured on wells covered with SPB as well. In paralell, VWF in cell extracts decreased after thrombin stimulation and no difference was observed with cells cultured in presence or in absence of SPB. Without stimulation a small amount of t-PA was only observed in the supernatant 24 H samples. Thrombin stimulation induced a comparable release of t-PA from cells cultured either on SPB or reference surfaces. t-PA synthesis, measured in cell extracts did not show significant differences. In contrast, SPB inhibited the release of PAI from EC stimulated or not stimulated by thrombin. Studies are in progress to determine whether PAI is released and absorbed by SPB or absent from the cells.
MOLECULAR MARKERS OF ENDOTHELIAL CELL DYSFUNCTION: OBSERVATIONS ON EXTRINSIC FIBRINOLYSIS IN SURVIVORS OF MYOCARDIAL INFARCTION AND IN TYPE-1 DIABETES MELLITUS
Authors: Nilsson T , Johnson O , Lithner F
Published: August 2018

We have studied the extrinsic fibrinolytic system in survivors, below 70 years, from myocardial infarction (AMI) treated in Umeci during 1983; in 43 type-1 diabetics; and in controls. Elderly controls underwent chest x-ray, ECG, EEG, brain CT scan to verify their health. Tissue plasminogen activator (tPA) activity was measured with a fibrin-stimulated rate assay, before and after a 10 min venous occlusion test (VO), tPA antigen (Ag) with an ELISA, and plasminogen activator inhibitor (PAI) by incubating samples with purified tPA and measuring remaining tPA with a polylysine-stimulated rate assay.

In the diabetics, PAI and tPA:Ag were similar to the controls. tPA:Ag correlated with age (r=0.6). Diabetics had much higher specific activity of tPA (61,300 vs 21,900), and had also much higher tPA activity after VO (2.2 vs 1.2 U/ml). The tPA activities after VO correlated well with HbA1c (r=0.39). A significant effect of smoking was disclosed. Smoking diabetics had higher PAI and tPA antigen but also lower specific activity of tPA (60,600 vs 115,700 U/mg). Ex-smokers were very similar to smokers, not to the non-smokers. Retinopathy, nephropathy, or hypertension didn’t appear to affect fibrinolysis independently.

In the AMI survivors (sampled 3 months after discharge from hospital), PAI was 6-fold higher than in elderly controls (p less than 0.0001). tPA activity after VO was much higher (3.2 vs 1.2 U/ml), as was tPA:Ag. tPA specific activity was lower. Among AMI patients with PAI over 10 U/ml, PAI correlated with triglycerides (r=0.4) and negatively with age (r=™0.4): these relations were not seen in the patients with PAI less than 10. The effects of smoking seen in diabetics were not observed among the AMI patients, von Willebrand factor was not increased among AMI nor diabetic patients, except for those with retinopathy.

The results suggest that the tPA/PAI system is a more sensitive indicator than vWF of endothelial cell dysfunction. It relates to effects of age, atherosclerotic vascular disease, and among diabetics also to degree of metabolic control and to tobacco smoking habits.
DISTINCTIVE FEATURES OF PROCOAGULANT RESPONSE OF MONOCYTES FROM DIABETIC PATIENTS
Authors: JUDE B , WATEL A , FONTAINE D , FONTAINE P , COSSON A
Published: August 2018

Hypercoagulability is one of the possible factors reported in genesis or aggravation of vascular complications in diabetes mellitus. We therefore examined procoagulant activity (PCA) of disrupted monocytes frcm 26 patients with Type I diabetes and 6 with Type II, versus 32 control subjects (male/ female ratio = 1 in each group).

Diabetes monocytes exhibited a slight but detectable PCA before any incubation or in vitro stimulation, whereas control monocytes did not. Data obtained with coagulation factor deficient plasmas or phospholipase C indicated that PCA was tissue factor (TF) alone in 22 cases and TF associated with a significant amount of factor VII/VIIa activity in 10 cases.

Incubation in serum free medium led to significant raise of PCA in diabetes cells when stimulated with endotoxin or not, and in control cells only after stimulation. Furthermore, PCA appeared earlier in diabetes monocytes than in control ones, (4 hours, versus 20 hours). PCA frcm control cells was FT-like. PCA frcm diabetes cells was FT-like when no VII/VIIa activity was present on non-stimulated cells, and prothrombinase-like when VII/VIIa activity was early associated with the cells. In the latter case, trace amounts of factor X activity were also detectable. Whether factor VII and factor X activities were of plasmatic origin and associated to the cells, or synthesized in vitro by the cells remains unclear. The characteristics of PCA were net correlated with clinical features (age, diabetic complications) nor with the type of diabetes.

Our data suggest that in diabetes patients, monocytes exhibit an increased PCA, possibly corresponding to a baseline stimulation, or at least a higher responsiveness to undergoing stimuli in vitro.
FAMILY WITH HYPOPLASMINOGENEMIA
Authors: Tengborn L , Johannessen M , Hedner U
Published: August 2018

The propositus, a 2 1/2 year old boy, was found to have splenomegalia at a routine control, but was otherwise in good health. He had in his neonatal period had osteomyelitis in his left leg, compl. with sepsis and shock. His mother had had DVT in her left leg extending to the iliac vein in the puerp. Investigation of the boy and his mother showed a decrease of plasma plasminogen to about 50% of the normal both when assayed immunochem. and amidolyt. using the chrom. subst. S-2251 after activ. with SK. The same was found when using UK as the plasminogen activ. Investigation of 23 other members of the family showed another 11 members with the same defect but none of them had had DVT. The defect was inherited in an autosomally dominant way. Plasminogen was purified from plasma withdrawn from the mother of the propositus on a lysine Sepharose column. A normal aff. to lysine was noticed but the yield was low (43 mg/l plasma). The purified plasminogen showed the same spec, activ. as normal plasminogen purified in the same way (8.4 CTA u/mg and 8.7 CTA u/mg resp.). SDS-PAGE and crossed immunoelectrophoresis in tandem with the patient’s plasminogen and normal purified plasminogen revealed no difference between the two plasminogens. Furthermore, the same kinetic constants for the activ. by UK were found for both plasminogens. It is concluded that the plasminogen found in this family with a low incidence of DVT seemed to be normal. The reduced plasma concentration may be the result of a decreased synthesis of a normal protein.
A NEW QUANTITATIVE ENZYME-IMMUNOASSAY FOR FIBRIN DEGRADATION PRODUCTS (FbDP) IN PLASMA, BASED ON MONOCLONAL ANTIBODIES
Authors: Koppert P W , Hoegee-de Nobel E , Nieuwenhuizen W
Published: August 2018

We have developed a sandwich-type enzyme immunoassay (EIA) for the quantitation of fibrin degradation products (FbDP) in plasma with a time-to-result of only 45 minutes. The assay is based on the combination of the specificities of two monoclonal antibodies (FDP-14 and DD-13), developed in our institute. FDP-14, the catching antibody, binds both fibrinogen degradation products (FbgDP) and FbDP. It has its epitope in the E-domain of the fibrinogen molecule on the BB-chain between amino acids 54-118 (Blood 68, 437, 1986). Antibody DD-13 was raised using D-dimer as antigen and was used as a tagging antibody, conjugated with horse-radish peroxidase.

A strong positive reaction is obtained with a whole blood clot lysate (lysis induced by tissue-type plasminogen activator) which is used as a standard.

The EIA does not detect FbgDP i.e. purified fragments X, Y, D:E complexes or FbgDP in plasma treated in vitro with streptokinase. This indicates that the assay is specific for fibrin degradation products.

We have successfully applied this assay to the plasma of patients with a variety of diseases. In combination with the assays previously developed by us for FbgDP (Thromb. Haemostas. 1987, in press) and for the total amount (TDP) of FbgDP + FbDP in plasma (J. Lab. Clin. Med. 1987, in press), we are now able to study the composition of TDP in terms of FbgDP and FbDP in patients.
CHARACTERIZATION OF SYNOVIAL AND PLASMA FIBRIN DEGRADATION PRODUCTS IN RHEUMATOID ARTHRITIS
Authors: Whitaker A N , Masci P , Hazelton R A , Morrison J J
Published: August 2018

The presence of fibrinogen/fibrin derivatives in vasculitic and synovial lesions of rheumatoid disease has long been recognized, although a pathogenetic role for them has not been clearly defined. In this study species of fibrin (ogen) derivatives have been characterized from joint aspirates from 32 patients (40 samples) with rheumatoid disease, and from 12 cases of seronegative arthridities, using immunoadsorption and SDS PAGE (reduced and unreduced gels); and quantitated by immunoassays utilizing the D dimer specific monoclonal antibody DD-3B6/22. The dominant fibrinogen derivatives in synovial aspirates were identifiable as derivatives of the degradation of crosslinked (XL) fibrin and included large quantities of XL high molecular weight degradation products. By enzymeimmunoassays (EIA) levels of XL fibrin derivatives ranged from 12 - 194µg/ml. Elevated levels of XLFDP (0.5 - 11.4 µg/ml) were also found in the majority of plasmas studied (29/38). A significant correlation was demonstrated between synovial and plasma concentrations of XLFDP (r = 0.455, p <0.005). A significant correlation also existed between erythrocyte sedimentation rate and plasma XLFDP (r = 0.554, p< 0.001) but not between ESR and synovial XLFDP. Autologous mixed lymphocyte reaction, measured in 26 patients, did not correlate with XLFDP levels. The synovial fluid data are consistent with the sequential alteration of fibrinogen by thrombin, XIIIa and plasmin (and/or elastase). Although the activation of fibrinolysis in the joint may be protective, the capacity for XLFDP to mediate bone and joint damage or to initiate immune responses is as yet unknown, whether in inflammatory arthritis or in haeraophilic arthropathy. The plasma XLFDP level may provide an index of disease activity in rheumatoid disease, although the extent to which this measures transfer from synovial accumulations to plasma remains to be determined.
RISTOMYCIN (AGGRISTIN) PRECIPITATION TEST: A NEW, RAPID AND RELIABLE METHOD FOR THE DETECTION OF INTRAVASCULAR CLOTTING
Authors: Pfliegler G , Arnout J , Vermylen J
Published: August 2018

The rapid and specific detection of fibrin monomers (fm) and fibrin degradation products (fdp) is of major importance in the laboratory diagnosis of disseminated intravascular coagulation, deep vein thrombosis or pulmonary embolism. Most methods in use are either time-consuming, needing special techniques, or insensitive and poorly specific. Some time ago, Watanabe and Tullis described a simple and rapid, semiquantitative test to detect fm and fdp in plasma, based on the finding that ristocetin in low concentrations (1.0-1.5 mg/ml) can specifically precipitate fm and fdp. To 0.4 ml ACD plasma, 0.1 ml ristocetin (2.5 mg/ml) is added and vortexed. The mixture is then incubated for 30 min at 20°C and centrifuged at 50xg for 5 min. The test is considered to be positive when fibrin-like strands or small or large pellets are observed on the bottom of the tube. More recently, Pfliegler et al. reported that ristomycin (AGGRISTIN), a structural analogue of ristocetin, can replace ristocetin in this test.

Here we report on further results with the ristomycin (AGGRISTIN) precipitation test in 138 patients with various intravascular thrombotic events. The results of this test, performed on ACD plasma, were compared to the serum fdp values detected by immunoelectrophoresis (IEF) and by the haemagglutina-tion inhibition test (HIT). In all 30 cases with serum fdp above 30 ug/ml (HIT) or 28 pg/ml (IEF), the precipitation test was positive; at lower fdp concentrations, as detected by HIT or IEF, the test still was positive in 70 per cent of these thrombosis patients, suggesting a superior sensitivity. In 16 patients with elevated fibrinogen levels (but no evidence of thrombosis), the test was positive in only 3. No false positive results were detected in 16 healthy controls. Preliminary results show that the minor disadvantage of the test (blood collection on acid citrate dextrose) may readily be overcome by the in vitro adjustment of the pH of citrate plasma, commonly used for other haemostatic tests, to between 7.0 and 7.4.

On the basis of our results we suggest that the AGGRISTIN (ristomycin) precipitation test is a simple, rapid and reliable method for the laboratory diagnosis of intravascular clotting.
DIAGNOSTIC VALUE OF D-DIMER, FIBRINOGEN DEGRADATION PRODUCTS, ETHANOL GEL TEST AND TRANSFER TEST IN DEEP VENOUS THROMBOSIS AND PULMONARY EMBOLISM
Authors: Wachel D , Segers M , Leautaud P , Rodzynek J J
Published: August 2018

In order to determine the sensitivity and the specificity of laboratory tests in the diagnosis of thrombotic disease, D-Dimer (D-D) latex, Fibrin(ogen) Degradation Products (FDP), Ethanol Gel Test (EGT) and Transfer Test (TT) were performed in two groups of patients : group 1 = 199 consecutive patients without thrombotic disease, group 2 = 56 patients with thrombosis (Deep Venous Thrombosis confirmed by radiological phlebography, n=19, and Pulmonary Embolism confirmed by ventilation-perfusion lung scintigraphy, n=37). The sensitivity, the specificity, the positive and negative predictive values (PV+ and PV-) for the different tests appeared as follows :

Conclusion :

1) Among the different parameters which have been studied, D-Dimer latex appears to be the most useful in the clinical practice (sensitivity 98% and specificity 90%).

2) Easy to perform, cheap, the results can be obtained within 20 minutes (for instance during the night in emergency situations).
FIBRINOLYTIC RESPONSE TO VENOUS OCCLUSION, AND FIBRIN FRAGMENT D-DIMER AND FIBRONECTIN LEVELS IN NORMAL AND COMPLICATED PREGNANCY
Authors: Mombaerta P , Ballegeer V , Declerck P , Van Assche F A , Collen D
Published: August 2018

The fibrinolytic response to venous occlusion was assessed in pregnant women with measurements of total and free t-PA, using specific ELISAs based on monoclonal antibodies.

Total t-PA levels increased after venous occlusion with 11±8 ng/ml (mean ± SD) in healthy fertile non-pregnant women (n=6), with 0.8 ± 1.3 ng/ml in 2nd trim. (n=5) and with 3.8 ± 3.9 ng/ml in 3rd trim. (n=4) healthy pregnant women. The increase in free t-PA was 12 ± 11; 1.2 ± 0.9 and 0 ng/ml respectively. The difference in post- and pre-occlusion levels in 3rd trim, pregnant women with insulin dependent diabetes mellitus IDDM (n=4) was 3.2 ± 4.2 ng/ml, with intrauterine growth retardation (IUGR) (n=4) 2.6 ± 3.0 ng/ml and with preeclampsia (n=5) 3.2 ± 3.5 ng/ml for total t-PA and 0, 0 and 0 ng/ml for free t-PA.

Fibrin fragment D-dimer levels in plasma measured with a specific ELISA were 130 ± 36 ng/ml in healthy fertile non-pregnant women (n=8). A significant increase was found in 4 out of 5 1st trim., 25 out of 25 2nd trim, and 21 out of 22 3rd trim, normal pregnant women. In these groups, plasma levels were 340 ± 160, 400 ± 170 and 440 ± 220 ng/ml respectively.

Fibronectin levels, measured with a Laurell electroimmunoassay and expressed as percentage of pooled human plasma (=330 yg/ml) were 83 ± 26% in 2nd trim, patients (n=24) and 102 ± 35% in 3rd trim, patients (n=17). Normal fibronectin levels were found in 4 patients with IDDM and in 6 with IUGR, whereas in 6 out of 8 preeclamptic patients significantly increased levels were observed.

These results confirm, with the use of a newly developed free t-PA assay, that the fibrinolytic response to venous occlusion is completely inhibited in the 3rd trimester of pregnancy. A reduced release of t-PA from the vessel wall during venous occlusion and/or an increased inhibition of released t-PA were observed. No difference was found in the fibrinolytic response between normal and complicated pregnancy. D-dimer levels are significantly elevated during pregnancy. Finally, the usefulness of fibronectin for the diagnosis of preeclampsia is confirmed.
D-DIMER AND SERUM FDP IN GYNAECOLOGICAL SURGERY AND DIC PATIENTS
Authors: Legnani C , Palareti G , Maccaferri M , Bianchini B , Boggian O , Poggi M , Coccheri S
Published: August 2018

We carried out in parallel plasma D-Dimer (Dimertest Latex assay, kindly supplied by Ortho Diagnostic Systems, Milan) and serum FDP measurements (ThrorriboWellcotest, Wellcome) in: 1) 35 gynaecological patients (Gyn. pts) pre and post- operatively (8th day); pts were treated with low dose heparin for thromboembolic prophylaxis and controlled with 1125- fibrinogen leg scanning (L.S.); 2) 20 pts from the intensive care unit (ICU) with DIC diagnosed by means of standardized laboratory criteria (DIC pts); tests were performed at admission to the ICU and 1 week later in the 15 survivors.

In all Gyn. pts both D-Dimer and serum FDP tests were negative pre- operatively. At the 8th post-op. day 16 pts (45.7%) had a positive Dimertest, 8 of which had also positive, even if transient, leg scanning (out of 10 L.S. positive pts).Serum FDP were present in 5 pts (14.3%), 2 with negative Dimertest and 2 with positive l.s.. All the 20 DIC pts showed at first observation positive Dimertest and 19 had also serum FDP. After 1 week Dimertest was still positive in 12 and serum FDP in 10 pts. Considering five range levels of results (from normal to highly positive for both tests) we found a significant (p<0.05) correlation (Kendall’s T .non parametric test) between the results of the two tests in all observations (35 samples). In conclusion, Dimertest, but not serum FDP, seemed to be highly sensitive to post-operative hypercoagulability, with a specificity of 5C% for subclinical fibrin deposition as detected with L.S.. No relevant differences were appreciated in the usefulness of the two tests in DIC pts.
THE EFFECT OF DIETARY COD LIVER OIL SUPPLEMENTATION ON LEUKOCYTE PHYSIOLOGY; A POSSIBLE MEDIATOR OF THE ANTIATHEROGENIC EFFECT OF MARINE OIL
Authors: Hoogasiam J , Fisher M , Levine P H , Weiner B W , Vaudreuil C H , Natale A , Johnson M
Published: August 2018

Leukocytes appear to be important in the pathophysiology of atherogenesis. Fish oil derived, omega-3 fatty acids suppress atherogenesis in experimental atherosclerosis models and select human populations. We assessed the effect of dietary cod liver oil (CLO) supplementation for 6 weeks on human monocyte and polymorphonuclear leukocyte (PMN) inflammatory potential in healthy controls and patients with a purported autoimmune disorder, multiple sclerosis (MS) in whom monocytes appear to be chronically activated. Baseline and 6 week venous blood samples were obtained from 6 stable MS patients and 6 healthy controls. . Monocyte hydrogen peroxide (HO) production (pMoles/minute/Ix10 monocytes) was measured in a spectrophotofluorometer after stimulation with latex particles. Baseline H2O2 production was 1.551.60 (mean 1 S.D.) in the MS patients and 1.19± .49 in the controls. Post-CLO the values were 1.021.24 and 1.091.27 respectively, representing a significant decline with CLO supplementation in the MS group (P< .01). PMN chemiluminescence (counts x10 /5min/PMN) levels was assessed by a liquid scintillation counter after stimulation with latex particles. Baseline levels were 17.416.1 in the MS group and 17.814.8 in the controls Post-CLO the levels were 12.812.3 and 12.313.3; both signifi-antly lower than baseline (P < .05). PMN superoxide (0 —) levels (nMoles/20min/lxl0 PMN) were measured by the reduction of cyto-chrome-c after stimulation with zymosan. Baseline O2- levels were 21.0±5.8 in the MS group and 22.115.9 in the controls. Post-CLO the O2- levels declined to 8.210.7 and 7.811.5, both significantly lower than baseline (P< .O2-). These data demonstrate that CLO supplementation reduces the intensity of PMN and monocyte reactions to a standard stimulus as measured by toxic oxygen metabolite production, although the monocyte effects were only observed in a population (MS) with increased baseline activity levels.

It has previously been assumed that omega-3 fatty acids might exert antiatherogenic effects via their inhibitory effects on platelet reactions. If leukocytes are important mediators of endothelial damage and/or cholesterol deposition in arterial walls, then our data suggest another mechanism by which fish oil may confer benefit in reducing the risk of arterial disease.