bluuebunny/crossref_metadata_2025 huggingface dataset:
TWO-SIDE IMMUNOASSAYS OF ENZYME-INHIBITOR COMPLEXES FOR THE DIAGNOSIS OF THROMBOPHILIC STATES
Authors: Bleyl H
Published: August 2018

The diagnosis of prethrombotic states requires methods which detect products of intravasal activation of the coagulation cascade.

Two-side immunoassays for antithrombin complexes with clotting factors were developed (IXi-AT, Xi-AT, IIi- AT). These sandwich assays permit the diagnosis of hypercoagulability in the presence or absence of heparin. The biological half live time of the thrombin-antithrombin-complex was found to be about 15 min. Healthy young men 20-25 years old (n=30) have a thrombin-antithrombin-complex concentration of 0.4 ± 0.2 mU/ml thrombin equivalent (S 2238). Patients with acute myocardial infarction (n=40) showed at the time of admission to the hospital up to 10-fold (n=14), up to 100-fold (n=13) more than 100-fold (n=13) elevated thrombin-antithrombin-complex concentrations. Patients with gastrointestinal cancer showed sometime excessive elevated enzyme-inhibitor complexes.

No correlation was found between thromboplastine time (Quick) and complex concentration in patients under anticoagulant therapy with dicumarole. In patients under dialysis as well as in patients under open heart surgery with extracorporal circulation, the biocompatibility can be monitored by inhibitor complex measurement.
FIBRINOLYSIS AFTER HEART TRANSPLANTATION
Authors: Páramo J A , Arcas R , Fernández J , Herreros J , Llorens R , Rocha E
Published: August 2018

Some aspects of the function of the fibrinolytic system were investigated in 12 patients undergoing cardiac transplantation. Plasminogen, euglobulin fibrinolytic activity (EFA), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor activity (PAI),α2-antiplasmin (α2-Ap) and fibrinogen degradation products (FDP) were determined preo-peratively and on postoperative days 1 and 5. Results showed a significant decrease of plasminogen (p <0.005), EFA (p <0.0001) and t-PA (p ^.0.001) on postoperative day 1 as compared to the baseline value, followed by recovery on day 5. There was a significant increase of PAI (p < 0 . 005) , α2-AP (p <0.0001) and FDP (p < 0.02) on postoperative day 1 as compared to the preoperative value. PAI and FDP reached the baseline value on postoperative day 5, but α2AP also increased on postoperative day 5. Our data show that there is an impairment in blood fibrinolytic activity early after cardiac transplantation, mainly related to a decrease of plasminogen and t-PA and a increase of PAI and α2AP. The clinical relevance of these data needs further evaluation.
REDUCED FIBRINOLYTIC POTENTIAL IN PATIENTS WITH ARTERIAL OCCLUSIVE DISEASE (AOD) IN COMPARISON WITH NORMAL SUBJECTS
Authors: Hartl D , Tiso E , Anderle K , Philapitsch A , Anger G
Published: August 2018

When combining angioplasty and local lysis with urokinase (UK) in treatment of peripheral arterial occlusions we have observed marked differences in the individual patient's response irrespective of the age of the thrombus. The extensive arteriosclerotic changes revealed by angioghraphy in some of these patients suggest a reduced fibrinolytic potential depending on the underlying disease.

In the standard in vitro test system we measured the UK-dependent thrombolysis in blood samples from 10 normal controls at UK concentrations of 150, 200, and 300 IU/ml of whole blood. In comparison we determined the whole blood thrombolysis time (WBTT) of 10 patients with A0D using UK concentrations of 150, 200, and 300 IU/ml of whole blood. The mean WBTT values for normal controls obtained at UK concentrations of 150 IU/ml, 200 IU/ml, and 300 IU/ml, respectively, amounted to S.5, 5.5, and 3.5 minutes, respectively, while in patients mean values of 20.7, 8.1, and 5.5 minutes, respectively, were found.

Studies on plasma samples had shown that the lysis time could be shortened in a dose-dependent manner by addition of lys-plasminogen and to some extent also glu-plasminogen. Since lys-plasminogen gave clearly superior results we tried to improve the lytic potential in terms of a shortening of the WBTT by adding different doses of lys-plasminogen (0.14 - 0.56 CU/ml whole blood) to each patient sample. Although the individual response varied, the addition of lys-plasminogen to the patient samples resulted in a clear dose-dependent improvement of pathologically Pronged lysis times. We conclude from these in vitro findings that the failure of fibrinolytic therapy may in some cases be due to a reduced lytic potential, which may be enhanced Dy administration of lys-plasminogen, even when the plasminoqen level is normal.
DIURNAL FTRRINOLYTIC RHYTHM IN PATIENTS WITH FIXED HEART RATE
Authors: Peternel P , Možina H , Pušnar S , Keber D , Stegnar M , Vene N
Published: August 2018

It has been established that fibrinolytic activity fluctuates during the day. Diurnal rhythm is possibly related to hemodynamic changes caused by daily activities. This presumption was tested in 10 patients with pacemaker and therefore fixed heart rate and limited changes in heart minute volume. Ten age and sex matched volunteers with normal heart rate served as controls. Venous blood was sampled at 8 a.m., noon, 4 p.m., 8 p.m., midnight and 4 a.m. Fibrinolytic activity was measured by three methods: spectrophotometric assay for tissue plasminogen activator (t-PA) activity, with euglobulin clot lysis time (ECLT) and on fibrin plates (FP). In patients with fixed heart rate no significant diurnal fluctuation in fibrinolytic activity was observed. In subjects with normal heart rate, diurnal rhythm was confirmed by all three methods. The highest fluctuation in activity was observed by t-PA assay with peak activity at noon (80 fold increase compared to midnight value). Also FP showed the highest activity at noon.With ECLT highest activity was detected at 4 p.m.

Absence of diurnal rhythm in patients with fixed heart rate suggested importance of heart minute volume in regulation of blood fibrinolytic activity.
THE EFFECTS OF PHYSIOLOGICAL CONCENTRATIONS OF VASOPRESSIN ON COMPONENTS OF THE FIBRINOLYTIC PATHWAY
Authors: Hariman H , Hughes J R , Grant P J , Davies J A
Published: August 2018

Evidence from studies in man suggests that vasopressin (aVP) at physiological concentrations activates the coagulation pathway, increases plasminogen activator activity and may have a role in the regulation of haemostasis under conditions of physical stress. Infusion of aVP in normal subjects increases plasma factor VIII concentrations and shortens the euglobulin clot lysis time (ECLT), but the mechanisms involved in these changes and their haemostatic significance are unclear. The aims of this study were to investigate the effects of aVP on the fibrinolytic pathway and to evaluate whether thrombin or plasmin are generated in vivo by aVP. After 30 min 0.9% saline infusion, vasopressin (20iu in 250ml 0.9% saline) was infused at 2.0 u/h for 1h in 9 normal subjects to achieve plasma aVP concentrations comparable to those attained during stress. Venous blood samples were taken before saline infusion (time 0) and every 30 min for 2h for assay of aVP, activated partial thromboplastin time (APTT), fibrinopeptide A (FPA), FPA generation time, FPBB15-42 ECLT, tissue-type plasminogen activator (t-PA) and t-PA inhibition. Plasma aVP rose frcm 0.5 pg/ml at time 0 to (median) 70.7 pg/ml at 90 min. The APTT shortened from 43.8 ± 1.9 to 34.4 ± 1.6 (SEM) seconds (p < 0.001) at 90 min. Plasma FPA and the FPA generation time remained unchanged (p > 0.05). Plasminogen activator activity rose from 36.4 ± 15.2 to 587.5 ± 206.6 units (p < 0.005), t-PA increased frcm 229.8 ± 20.4 to 1107.4 ± 224.1 ml.U/ml (p < 0.005) and t-PA inhibition fell frcm 7.9 ± 1.1 to 3.9 ± 0.9 I.U/ml (p < 0.05) in response to the aVP infusion. FPB815-42 increased frcm a baseline value of 1.7 ± 0.4 to 2.2 ± 0.7 pmol/ml after 90 min (p < 0.05). The results suggest the effects of aVP on fibrinolysis are due to an increase in t-PA and decrease in t-PA inhibition. The increase in FPBB 15-42 with no change in FPA supports the hypothesis that plasmin was generated by non-fibrin dependent pathways.
VASOPRESSIN AND THE REGULATION OF FIBRINOLYSIS: HAEMOSTATIC RESPONSES TO HYPOTENSION IN THE ABSENCE OF ADRENALINE RELEASE
Authors: Hampton K K , Grant P J , Boothby M , Davies J A , Prentice C R M
Published: August 2018

Vasopressin (aVP) infusions resulting in plasma concentrations that simulate those during stress cause increases in factor VIII (EVTII) and plasminogen activator activity (PAA). During apcmorphine induced nausea and abdominal surgery similar increases in aVP are associated with elevated FVIII and PAA suggesting aVP is a mediator of haemostatic function. However during stress there is release of adrenaline which has similar effects on haemostasis. The aim of this study was to investigate the effects of endogenous aVP release on haemostasis in patients with postural hypotension (a potent stimulus to aVP release) due to autonomic neuropathy who have absent circulating adrenaline. The study was performed in i) three patients with progressive autonomic failure (PAF) who release aVP but not adrenaline ii) three patients with PAF with multi-system atrophy (MSA, the Shy-Drager syndrome) who have deficient release of both aVP and adrenaline. After lying down overnight patients stood for 2 minutes on 3 occasions at 1 hour intervals. Samples were taken for aVP, adrenaline, ECLT and FVIII. In PAF mean blood; pressure fell from 108 lying to 47 irmHg standing. Pulse did not change. aVP rose from 1.2 lying to 56 pg/ml standing. Plasma adrenaline was sub-normal throughout. Median PAA (106 /ECLT2 ) lying was 92 and rose on standing to 376 units and correlated with plasma aVP (r = 0.87, p < 0.0001). In PAF with MSA mean blood pressure fell from 107 lying to 61 irmHg standing. aVP levels did not change and adrenaline remained below normal. PAA rose from a median 30 lying to 87 units standing. No change in FVIII was observed in either group. The results suggest that aVP regulates plasminogen activator activity in the absence of adrenaline. The lack of a rise in FVIII suggests that circulating adrenaline may be necessary for aVP to exert an effect on FVIII concentrations.
CAN THE RESPIRATORY PATTERN INFLUENCE FIBRINOLYSIS ?
Authors: Eriksson E , Risberg B
Published: August 2018

Pulmonary endothelium produce large amounts of tissue plasminogen activators (t-PA) and presumably activator inhibitors (PAI). Any systemic role of this pulmonary production is unknown. Previous studies in dogs have demonstrated that respiratory pattern influenced the fibrinolytic activity in blood. Plasminogen activators were thought to be released from lungs. Too study if similar conditions existed in man we evaluated the fibrinolytic response in surgical patients during various pattern of mechanical ventilation. Twelve patients undergoing infrarenal aortic reconstruction were evaluated. Blood samples were taken from the pulmonary artery and a radial artery before and at various times during surgery. Mechanical ventilation with varied levels of PEEP (0, 5, 10 and 15 cm H2O) was used. Samples were taken in ice-cooled EDTA vacutainers and the blood was centrifuged at 8000 g for 2 min. Plasma was frozen at −70° C for later analysis. Fibrinolytic activity was monitored by analysis of ECLT, t-PA activity, t-PA antigen (ELISA) and PAI activity. During surgery ECLT and t-PA activity were below detectable levels. t-PA antigen was at baseline 9-18 ng/ml and increased transiently early during surgery with 3.9 ± 1..0 ng/ml in the radial artery and 3.2 ± 1.9 ng/ml in the pulmonary artery (mean ± SEM). PAI was at baseline 2.0 - 35.2 IU. During surgery it increased significantly with 43.6 ± 8.6 IU/ml in the radial artery and 36.9 ± 9.6 IU/ml (mean ± SEM). There were nc gradients over the lung in either t-PA antigen or PAI. PEEP at levels from 0-15 cm H2O did not affect the measured parameters. Thus, during surgery there was a transient increase in t-PA antigen and a progressive increase in PAI, both pulmonary independent. The respiratory pattern did not influence the fibrinolytic system and could unlikely be an important factor in resolution of pulmonary emboli.
RELATIONSHIP BETWEEN AGE AND t-PA ANTIGEN, PA INHIBITOR ACTIVITY AND PA ACTIVITY
Authors: Hashimoto Y , Kobayashi A , Yamazaki N , Sugawara Y , Takada Y , Takada A
Published: August 2018

A positive correlation between age and the occurrence of thrombosis has been suggested. We studied relationship between age and fibrinolytic activities; namely levels of tissue plasminogen activator (t-PA) antigen, activity of t-PA inhibitor (PA inhibitor activity) and activity of t-PA (PA activity). Plasma samples were obtained from 88 apparently healthy volunteers. t-PA antigen was measured by enzyme immunoassays(EIA). Monoclonal and polyclonal anti-t-PA antibodies were employed as the first and the second antibodies for EIA, respectively. Polyclonal t-PA antibody was raised in a rabbit after injection of purified t-PA and was labeled with β-D-galactosidase. Monoclonal anti-t-PA was obtained from IMCO Co., Ltd (Stockholm, Sweden). PA inhibitor activity was measured by inhibition of purified t-PA by diluted plasma. PA activity in the euglobulin fraction of plasma samples was measured by the hydrolysis of S-2251 after the addition of Glu-plasminogen. PA activity was calculated from the standard curve of t-PA. Plasma levels of t-PA antigen increased with increase in age. The leveles of t-PA antigen increased from 4.31 ± 1.55 ng/ml (mean ± SD) at the age range of 10 to 20, to 9.18 ± 2.34 ng/ml at the age range of 50 to 60. Increase in levels of t-PA antigen was quite linear with respect to increase in age. In 30 healthy volunteers, plasma PA inhibitor activity also increased with increase in age. PA activity in plasma of 20 healthy volunteers decreased with age, indicating that free t-PA antigen levels decreased with age. In conclusion, dramatic increase in both t-PA antigen and PA inhbitor activity was shown in persons with increasing age. PA activity decreased with age. Therefore it is suggested that tendency of decreased PA activity with increasing age may be related to high incidence of thrombosis in older persons.
ASPIRIN FAILURE TO INHIBIT THE RELEASE OF PLASMINOGEN ACTIVATORS-INHIBITORS BY HUMAN PLATELETS
Authors: Woods A I , Lazzari M A
Published: August 2018

Platelet-PA-Inhibitors can be released by thrombin, Col laaen( Col) and others.If they are physiologically important,inhibition of their release might facilitate thrombolysis.Intrinsic PA were tested in euclobulins (eug)of PPP and PPP+Washed platelets(WP) ,with and without aspirinf ASA) .treated with UK,SK and Col(20 atfd l2uo/ml) Results(mm2)were:euaPPP:232+78;+3×106WP/ul:217+71;+10%7ul:188+/5 +2×106MP/ul: 157+69:With UK:eugPPP:283+76;+3×l0517P/ul :234+69;+106 WP/ul :172+55;+2×l(PWP/ul :154+48; With SK:euoPPP:303+99;+3×l05WP/ul 252+65;+1067P/ul:203+68;+2×106UP/ul: 174+85;Wi th Col (20ug/ml) :eur PPP:234+97;+3×105WP/ul:160+63;+106WP/ul:141+73;+2×l06WP/ul:129+81; +2×106WP/ul+ASA: 105+31;Wi th Col uc/ml) :euaPPP:230+56;+3×l05NP/ul: 160+52;+106WP/ul:139+44;+2×106wp/ul:126+21;+2×106WP/ul+ASA:118+28. EugPPP+l/P showed lower lysis area.Col induced more decrease of lysis area in euc with WP.ASA did not modify this effect.UK and SK produced higher lysis area only in euoPPP.No difference was observed between high and low doses of Col-effect upon WP.Combined treatment of eucPPP+WP+Col, with UK and SK showed:(mm2):UK-treated eugPPP+2×106WP/ul+Col(29ug/ml):134+25;+ASA and Col(20ug/ml):115+65;+Col(2uc/ml):157+50;+ASA and Col(2uo/ml):133+49; SK-treated eupPPP+2×106WP/ul:+Col(20uo/ml):144+49;+ASA and Col(20ug/ml):128+ 60;+Co1(2ug/ml):173+66;+ASA and Col(2ug/ml):142+36.

Col (20ug/ml) produced slight lower lysis area of UK and SK-treated eugPPP+WP. (p:ns). ASA did not modify this effect.Col (2uc/ml) did not produce changes in lysis area of UK and SK-treated eupPPP +WP. ASA produced slight lower lysis area(p:ns).

Conclusion: high dosis of Col could release anti UK,anti SK and intrinsic PA-inhibitors from platelets;low dosis of Col could only release fntrinsic PA-inhibi tors. It suggests that anti UK and anti SK release might be triggered by stronger stimuli than intrinsic PA inhibitors.ASA could not inhibit any inhibitors release.

We must consider that PA-inhibi tors could be released by different metabolic pathways other than cyclooxygenase pathway.
NORMAL FIBRINOPEPTTDE A (FPA) AND ELEVATED FIBRINOGEN DEGRADATION PRODUCTS AFTER LONG DISTANCE RUNNING
Authors: Bartsch P , Haeberli A , Straub P W
Published: August 2018

Physical exercise leads to a shortening of activated partial thrcnboplastin time (PIT) and euglobulin lysis time (ELT). Whether this activation causes in-vivo thrombin and plasmin action remains controversial however. 19 well trained long distance runners were examined 25 min (range 5-53) after termination of a 100 km race (post-race values) run in 577 min (457-755) and 5 days later after at least one day without physical exercise (control values). FPA, platelet factors and fibrin (ogen) split products (fragment E and BB 15-42) were measured with radioimnunoassays. ELT was assessed before and after venous occlusion (VO). The table gives mean values ±SD:

Thrombin time, platelet count, platelet factor 4 and haematocrit did not change significantly. FPA was normal in all post-race samples and did not correlate significantly with the time lag between arrival and blood sampling, indicating that activation of blood coagulation in exhaustive physical exercise of long duration does not lead to in-vivo fibrin formation. Activation of fibrinolysis, however, results in circulating plasmin activity as demonstrated by the elevation of the fibrin (ogen) degradation products fragment E and BB 15-42. Since the polyclonal antibodies used in the latter assay crossreact with BB 1-42, these results alone do not allow to jjdge whether plasmin degrades fibrin or fibrinogen. However, lack of fibrin formation suggests fibrinogenolysis rather than fibrinolysis.
PLASMA CROSSLINKED FIBRIN DEGRADATION PRODUCTS FRACTION D (D-DIMER) IN PATIENTS UNDERGOING ELECTIVE MAJOR ABDOMINAL SURGERY AND IN HEALTHY VOLUNTEERS
Authors: Hauch O , Nerstrøm H , Kølle T R , Jørgensen L N , Wille-Jørgensen P , Neerstand H
Published: August 2018

An increased plasma level of D-dimer has been suggested as an indicator of postoperative thromboembolism (TE). We studied the D-dimer level in patients undergoing major elective abdominal surgery and in healthy volunteers to evaluate a possible value of D-dimer as a screening test for TE. 18 patients and 5 healthy volunteers were studied. The patients received low molecular weight heparin (Logiparin TM) subcutaneously once . daily for 7 days. The 125 I-fibrinogen uptake test (FUT) was done in all patients. Blood samples were taken preoperatively, postoperatively and on the postoperative day 1, 3, 4, 5 and 6. The volunteers had blood samples taken before and 4 hours after subcutaneous Logiparin injection. D-dimer was assayed by enzyme-immunoassay (Boehringer Mannheim, Elisa D-dimer, Cat.no. 998117). Results are given as median with 95% confidence limits in brackets.

One patient developed deep venous thrombosis verified by phlebography. Two patients had abnormal FUT but normal phlebography. The plasma level for D-dimer in the 15 patients with normal FUT was 500 ng/ml (300-800) preoperatively. The D-dimer level increased postoperatively to 1700 ng/ml (900-3300) (p < 0.001). The following days the D-dimer level increased steadily to 4800 ng/ml (2600-6800) 6 days postoperatively. The plasma level of D-dimer in the volunteers were less than 110 ng/ml both before and after injection of Logiparin. The plasma level of D-dimer in the patient with deep venous thrombosis and the 2 patients with abnormal FUT were within the range of the patients with normal FUT.

The postoperative D-dimer level was as high as that reported in patients with diagnosed thromboembolism.

In conclusion, the D-dimer test does not seem to be a potential screening test for postoperative thromboembolism.
A MONITORING PARAMETER IN THROMBOLYSIS?
Authors: Roth R , Duckert F , Marbet G
Published: August 2018

D-dimers are specific derivatives of cross-linked fibrin. Their concentration may be used to conduct thrombolysis and predict its outcome. In this investigation we studied the relationship between D-dimer plasma levels during thrombolysis of deep leg vein thrombosis (DVT) and the phlebographic success.

METHODS: 17 patients (9 males, 8 females, age range 17-56 years) were studied. All patients had DVT and were treated with either an acylated streptokinaseplasminogen complex (n=lO) or streptokinase (n=5) or both drugs successively (n=2) for 4.6 days on average. The thrombolytic effect was assessed by ascending phlebography and the patients were divided into 3 groups: A complete clearance, B partial clearance, C no change. The D-dimers were measured by a non-competitive EIA assay with a monoclonal antibody against a conformational epitope on the plasmin-resistant D-dimer, fragment of cross-linked fibrin.

RESULTS: The table gives the mean D-dimer levels in the 3 groups of patients at 12 hours intervals.

The statistical analysis (Newman-Keuls multiple range test) suggests that low D-dimer levels, especially over the first 36 hours are related to treatment failures.

CONCLUSIONS: Despite some overlapping between successful and non-successful cases and lack of absolute specificity of the assay the D-dimer concentration may predict treatment failure in the first two days.
CLINICAL AND LABORATORY STUDIES ON THE EFFECTIVENESS OF DEFIBROTIDE IN PERIPHERAL VASCULAR DISEASE OF ATHEROSCLEROTIC ORIGIN
Authors: Ulutin O N , Cizmeci G , Cornelli U , Fareed J
Published: August 2018

Defibrotide is a derivative of polydeoxyribonucleotide extracted from bovine lung which has been found to produce profound activation of endothelial cells as evidenced by the increase of tissue plasminogen activator (t-PA) and prostacyclin levels in animal models. Administration of a 600 mg dose daily for six weeks to patients with peripheral obliterative diseases resulted in significant clinical improvements in post-treatment exercise tests and radionuclide arteriography. Results were consistent with improvements in the circulation. Ex vivo blood analysis showed marked increases in the production of t-PA and prostacyclin levels. Other laboratory tests were also suggestive of activated endothelial states. Platelet C-AMP levels were increased, whereas MDA and TXB2 levels were reduced. No systemic anticoagulation was observed in terms of alterations of the global coagulant tests (PT, APTT, etc.). These studies suggest that Defibrotide mainly acts via the modulation of the endothelial function and acts in a novel fashion in contrast to other drugs used in this area.
ENDOTHELIAL FUNCTION MODULATION AND CONTROL OF VASCULAR AND THROMBOTIC DISORDERS: EXPERIMENTAL RESULTS WITH A POLYDEOXY RIBONUCLEOTIDE AGENT DEFIBROTIDE
Authors: Kumar A , Fareed J , Wehrmacher W H , Hoppensteadt D , Ulutin O , Walenga J M
Published: August 2018

Numerous approaches with single and multiple drugs modulating protease cascade, platelet function and blood viscosity and to reduce blood lipids to manage thrombotic processes have been tried. Defibrotide, a polydeoxyribonucleotide, (Mr =17,000) offers a new approach to vascular and related thrombotic processes as it acts via modulation of endothelial cell function. We have used a primate model (Macaca mulatta) to study the endogenous action of this agent after the oral (10-25 mg/kg) and intravenous (5-10 mg/kg) administration. This agent produced no effect on clotting tests and ex vivo laboratory findings but rather it elevated the t-PA (antigen and functional), protein C (antigen and functional), prostacyclin and decreased thromboxane, 01.2-antiplasmin (functional) and t-PA inhibitor (functional) in both studies. These observations suggest that Defibrotide modulates endothelial function. Hepatic isolation in rabbits totally blocked the antithrombotic actions of Defibrotide suggesting that this agent is converted into an active product endogenously. Pretreatment of Defibrotide with nucleases also resulted in a complete loss of its actions. Defibrotide produced dose dependent antithrombotic actions in animal models (rabbit venous stasis and rat vena caval ligation) after either intravenous or oral administration. Blood pressure, heart rate, respiration and kidney function were not altered by it. No effect on bleeding time was noted in any studies. Upon oral administration this drug produced pharmacologic action after 2 hours whereas after intravenous administration, the action peaked at 30 minutes. Defibrotide exhibited cytoprotective effects towards endothelial lining of the vascular smooth muscles characterized by microscopic studies. In summary Defibrotide is an endothelial support agent whose multicomponent actions are primarily mediated via the physical and functional modulation of the endothelial cells in the vascular system.
CHRONIC ORAL DEFIBROTIDE STIMULATES VASCULAR PGI2 AND INHIBITS ATHEROSCLEROTIC PLAQUE FORMATION IN CHOLESTEROL-FED RABBITS
Authors: Lobel P , Palmer M , Schor K
Published: August 2018

Defibrotide (DEF) is a polydeoxyribonucleotide fraction from bovine lung, possessing profibrinolytic and PGI2 stimulating properties. Male rabbits were fed for 4 months a standard laboratory diet (150 g/day) without (A) or with (B) DEF or a cholesterol (1%) supplemented diet without (C) or with (D) DEF (60 mg/kg x day) was administered orally (drinking water) and withdrawn 24-36 h prior to the acute experiments.

DEF did not change the elevated serum cholesterol: 18 ± 2 (C) vs. 26 ± 5 (D) mM but significantly reduced the plaque formation in the aorta from 4.5 ± 0.3 (C) to 3.3 ± 0.2 (D) (subjective score). Collagen induced (0.6 pg/ml) thromboxane formation and ATP release was significantly reduced by DEF: 55+2 (C) vs. 42 ± 2 (D) ng/ml TXB2; 152 ± 11 (C) vs. 74±5 (D) AU ATP (platelet rTch plasma). DEF significantly increased the basal and bradykinin (Bk, 30 nM) stimulated PGI2 release from rabbit aorta preparations in Krebs buffer, while the PGI2 forming capacity (arachidonic acid, AA, 30 pg/ml) was unchanged Furthermore, the iloprost (30 nM) stimulated cAMP was significantly elevated by DEF in both control: 115 ± 10 (A) vs. 155 ± 18 (B) pmoles/1 and cholesterol-fed rabbits: 120 ± 14 (C) vs. 172 ± 9 (D). DEF, directly added to the platelets in vitro did not inhibit platelet activation up to 100 pg/ml.

The data demonstrate a 2-3-fold stimulation of basal and hormone (Bk) induced PGI2 formation of control and sclerotic rabbit aorta after 4 months DEF treatment while the atherosclerosis per se does not significantly change these parameters. DEF treatment also significantly reduces platelet hyperreactivity at unchanged serum ch() lesterol. Both properties might be useful to prevent complication’s of atherosclerosis, such as myocardial infarction and stroke.
DEFIBROTIDE DECREASES CHOLESTEROL CONTENT IN HYPERCHOLESTEROLEMIC RABBIT AORTA, WITH NO MODIFICATION OF PLASMA OR LIPOPROTEIN CHOLESTEROL
Authors: Pescador R , Porta R , Niada R , Mantovani M , Prino G
Published: August 2018

Defibrotide was shown to stimulate the production of endogenous PGI2 from rat and hamster aortic tissue, as well as the production of PGi2 frcm the coronary vascular bed in the platelet perfused heart model. This effect was only seen in presence of platelets. Durirg the infusion period with Defibrotide thromboxane release remained unaffected while platelet cAMP rised. Defibrotide, was also able to reduce the secretion of ATP from platelets as well as to deaggregate platelet cluTps. It has been postulated that long-term administration of stable PGI^ metabolites or analogues could be a more useful means of enhancing cholesterol and cholesteryl ester mobilization out of the arterial “ foam” cell during early stages of cardiovascular disease. These observations prompted us to administer Defibrotide i.v. to cholesterol fed rabbits to verify if it could cause a decrease in cholesterol content of aortas. Aorta cholesterol was evaluated by gas chromatography. Plasma or lipoprotein lipids were assayed by enzymatic kits from Boehringer Biochemia. Plasma lipoproteins were separated by density gradient ultracentrifugation. Platelet aggregation was carried out with ADP using an optical aggregometer to find out the effective aggregatory concentration fifty (EC). Histology of the rabbit hearts was performed by optical microscopy on heart sections coloured with hematoxylin-eosine. Defibrotide caused a decrease (-49%, P < 0.05, vs. cholesterol fed rabbits treated with placebo) in cholesterol content of aortas with no modification of total plasma cholesterol, triglyceride and phospholipid. The cholesterol, triglyceride, phospholipid and protein of plasma lipoproteins were not affected too. The EC of rabbits treated with Defibrotide was 50 normalized (-9%, N.S. vs. control animals fed the normal diet and treated with placebo; cholesterol fed animals treated with placebo: -32%, P< 0.05 vs. control animals fed the normal diet). Vascular lesions in the hearts of animals treated with Defibrotide had a lower rate (33%) in comparison to that (87%) of animals fed with cholesterol and treated with placebo. It is concluded that the ability of Defibrotide to stimulate vascular PGI2 formation and to reduce platelet sensitivity could be helpful in reducing the amount of cholesterol in the cardiovascular system in atherosclerotic prone situations.
PLATELET-LEUKOCYTE INTERACTION: ACTIVATION OF RABBIT PLATELETS BY FMLP-STIMULATED NEUTROPHILS
Authors: Coeffier E , Joseph D , Vargaftio B B
Published: August 2018

The interaction of neutrophils and platelets may be important in inflammation. We have studied the effect of the chemotactic peptide, N-formyl-methionyl-leucy1-phenylalanine (FMLP) on cells in whole rabbit blood or in mixture of purified rabbit platelets and neutrophils. In whole blood, FMLP triggered an aggregation (measured by electrical impedance) dependent upon the concentration of FMLP (9.9± 0.7 and 5.2+1.2 ohms at 1 and 0.01 uM FMLP resp.). This aggregation was accompanied by a strong decrease in platelets counts (54.6± 6.0 and 45.6± 3.8% for 1 and 0.01 uM FMLP resp.) and by a smaller decrease in neutrophils counts (25.0± 1.9 and 12.9± 1.7% at 1 and 0.01 uM FMLP resp.). When platelets were incubated in the presence of neutrophils, the addition of 0.1 uM FMLP induced a marked aggregation (50.0± 1.6 versus 19.5± 1.6% of light transmission,n=8,p< .001), ATP secretion (8.4± 1.0 versus 0.1± 0.1 nmol/ml,n=6,p< .001) and a marked decrease of platelets counts. FMLP induced aggregation of purified neutrophils and release of lysozyme but lacked direct platelet-stimulating effect. The release of lactate dehydrogenase and lysozyme were unchanged under the interaction conditions. Our results indicate that the stimulation of neutrophils by FMLP induces platelet activation both in whole blood and on isolated cells. The platelet activation was reduced by about 30% with lOOuM aspirin or indomethacin and by about 70% with lOOuM BW 755C. The two PAF-acether antagonists, BN 52021 (lOOuM) and WEB 2086 (luM) suppressed platelet activation by 70-80%. Neutrophil supernatant induced platelet activation only when neutrophils were stimulated by FMLP in the presence of bovine serum albumin (BSA,0.25%). Rabbit neutrophils stimulated in the presence of BSA by 1 uM FMLP formed 2 nM PAF-acether of which only 50% were released to the extra-cellular medium. WEB 2086 (luM) inhibited totally the formation and the release of PAF-acether. These data indicate that both arachidonic acid-metabolites and PAF-acether participate in platelet activation by FMLP-activated neutrophils.
THE EFFECT OF SIX PROSTAGLANDINS, PROSTACYCLIN AND ILOPROST ON GENERATION OF SUPEROXIDE ANIONS (0J) BY HUMAN NEUTROPHILS (PMNs) ACTIVATED BY ZYMOSAN OR FMLP
Authors: Szczeklik A , Gryglewski R J , Wandzilak M
Published: August 2018

Human PMNs in a suspension (2.5 - 3.5 × 106 cells/ml of PBS) were activated by opsonized zymosan (2.5 mg/ml) or by FMLP (22 jjg/ml) in presence or absence of prostaglandins (PG) E1 E2, D2, 6-keto-F2α, 6-keto-E1 prostacyclin and Iloprost (3nM -30 pM). The generation of superoxide anions was measured as a SOD-sensitive reduction of ferrocytochrome c. In FMLP-stimulated PMNs an average production of 0-
2 of 18 ± 3.2 nmoles/10-
2 PMNs/10 min was suppressed by 25% at following concentrations of PGD2, PGE2, PGE1s 6-keto-PGE! and PGF2 : 0.1, 0.2, 0.5, 0.8 and>30.0 pM, respectively. No significant inhibition occurred in the presence of prostacyclin, 6-keto-PGF2 and Iloprost at concentrations as high as 30 pM. In zymosan-stimulated PMNs prostaglandins of E series were less potent inhibitors than in FMLP-stimulated PMNs by following factors: PGE2 - 20, 6-keto-PGE2 - 13, PGE1 - 4, whereas PGD2 was equally potent inhibitor in FMLP- and zymosan-stimulated PMNs. It is concluded that PGE2 is an antagonist of FMLP-receptor-mediated events which are responsible for the generation of superoxide anions in PMNs, whereas its isomer PGD2 has another mechanism of the 0-
2-suppressive action. Perhaps, PGD2 is a direct inhibitor of membrane-bound NADPH-oxidase or an antagonist of oxygen activation by leukotrienes and lipoxins or a promotor of scavenging of 0-
2 by mitochondrial membranes. The latter mechanism has been proposed recently by Gryglewski as explanation for cytoprotective action of stable biologically active metabolites of prostacyclin. Mechanisms of anti-0-
2 action of PGE1 and 6-keto-PGE1 in PMNs are likely to be intermediate between these proposed for PGE2 and PGD2.
GRANULOCYTE ELASTASE RELEASE DURING BLOOD COAGULATION
Authors: Miura T , Inagaki M , Taki M , Saito N , Meguro T , Yamada K
Published: August 2018

Granulocyte elastase (ELP) has a high-potency fibrinolytic activity. Hence, there is a possibility that ELP acts as a thrombolytic enzyme like plasmin in thrombolysis. We investigated the release of ELP from granulocytes, especially during blood coagulation.

The biological activity of ELP was measured using a synthetic substrate, Suc-Ala-Tyr-Leu-Val-pNA. The immunological activity assayed as an alpha-l-antitrypsin-ELP complex was measured using an anti-ELP antibody (Merck), because more than 90% of ELP in blood forms alpha-l-antitrypsin-ELP complexes.

The ELP activity in granulocytes extracted by 2 mol/1 KSCN was 10 mU/106 cells. This fibrinolytic activity corresponds to 1-2 U of plasmin in the fibrin plate method.

The ELP release from separated granulocytes was observed by adding Ca2+, and the release was increased by Ca ionophore A 23187. The release was dose-dependent as far as 10 mM Ca2+ (final concentration) and the maximum release was obtained within 15 minutes. However, the ELP release was not produced by thrombin.

The level of alpha-l-antitrypsin-ELP complex in serum was twice higher and that in heparinized plasma was 1.5 times higher than that in sodium citrated plasma. ELP was not released from granulocytes incubated in both prekallikrein deficient plasma and Factor XII deficient plasma containing 10 mM Ca2+. But addition of normal plasma (about 10%) resulted in ELP release

These results suggest that the ELP release from granulocytes is dependent on Ca2+ and the release is relevant to the blood coagulation system, especially to contact factors.
THE EFFECT OF PHYSICAL EXERCISE ON MONOCYTE FUNCTION, COAGULATION FACTORS, FIBRINOLYSIS AND PLATELETS
Authors: Ûsterud B , Hansen J B , Olsen J O , Wilsgård L
Published: August 2018

Over a 2 years period, the Norwegian national team of cross country skiers, have been tested after strenous championships as well as before and after regular training.

Finishing a 50 km race generated a rise of white cells from 5.4 ± 1.0 to 19.3 ± 3.7 × 109 /I (n=14). The mobilization of new and more sensitive white cells may explain the resultant rise in monocyte response to stimuli in vitro after the race. Thus, monocytes from blood incubated with 2 ng/ml blood, drawn from the athletes just after finishing the 50 km race, possessed 6-7 fold higher specific activity of thromboplastin than monocytes from blood drawn and stimulated in a rest-period. There was a positive correlation between the inverse level of F. VII in plasma of the skiers after the race and the monocyte response to stimuli in vitro as expressed by the level of thromboplastin. Activated monocytes with exposed thromboplastin are probably pulling out F. VII from the circulation just as seen in patients with gram negative septicaemia.

A group of long distance runners were also tested after strenous jogging. High monocyte response to stimuli in vitro was associated with extremely active platelets that aggregated spontaneously after drawing the blood into heparin and tested it in a whole blood aggregometer. Those individuals with very active monocytes and platelets had also an extremely activation of their fibrinolytic system as judged by whole blood clot lysis. In contrast, everyone with low cell activation had hardly any change in their fibrinolytic activity after strenous running.

The clear trend in this study was that almost everyone of our top athletes had a very depressed blood-cell activation system as compared to non athletes. Low blood cell activation in vivo was also associated with a low induction of the fibrinolytic system.
CYCLIC THROMBUS FORMATION IN RABBIT AORTA: A NEW MODEL OF ACUTE ARTERIAL THROMBOSIS
Authors: Kuhn M , Müller T H , Eisert W G
Published: August 2018

Existing models of arterial thrombosis either require extensive surgical intervention or are limited by the short period of observation. Therefore we developed a new model in rabbits to monitor cyclic flow reductions (CFR) due to thrombus formation in a partially denuded and stenosed aorta.

An electromagnetic flowprobe was positioned proximally to a stenosis of the abdominal aorta in anesthetized New Zealand rabbits after limited denudation of the vessel using a hemostat. The aortic blood flow was spontaneously reduced due to formation of thrombi. It returned to the basal level after gently shaking the occluder. The 'free flow' (defined as mean area under the flow curve) as well as the frequency of CFR were monitored. CFR persisted in all control animals for 3 to 4 hours. Prostacyclin at an infusion rate of 100 to 250 ng/kg/min completely abolished CFR in 16 of 20 animals. I.v. application of nitroglycerine (0.7 mg/kg/h) or papaverine (2 mg/kg/h) did not affect CFR. This as well as histology indicates strong involvement of platelet aggregation rather than vasospasm. CFR were also reduced by the following drugs: phosphodiesterase inhibitors (AHP 719, 1 mg/kg/h, i.v.; UDCG 212, 5 ug/kg/min., i.v.), a cyclooxigenase inhibitor (sulphinpyrazone, 30 mg/kg, i.v.) or a thromboxane synthetase inhibitor (dazoxiben, 1 mg/kg, i.v.) as well as an alpha2 antagonist (yohimbine, 1 mg/kg, i.v.). Heparin (800 U/kg, i.v.) reduced CFR suggesting significant contribution of the coagulation system.

This new approach opens an avenue to long term observations of antithrombotic therapy in a fairly simple and highly reproducible model.
A NEW PAN SPECIES MODEL FOR THE MEASUREMENT OF IN VIVO THROMBOLYSIS
Authors: Hotchkiss A , Refino C , DeGuzman L , Rigter B , Eisert W
Published: August 2018

A new model suitable for the quantitation of Li vivo thrombolytic efficacy was developed which has significant advantages over currently available models. This model allows animals to be used repeatedly and produces data that are less variable.This is a species-independent model utilizing an arterio-venous shunt (AVS). This model was used in rabbits (3 kg) and beagle dogs (13 kg). Each animal was used repeatedly during a 7 day experimental period without evidence of antibody formation. Clot lysis data are less variable than the rabbit jugular vein clot lysis model (RJV). The average coefficient of variation at all doses ([SD/Mean]×l00) with the AVS model was 13±9 and 21±6 for dogs and rabbits, respectively, compared to 25±11 for the RJV model. The rate of data accunulation with the AVS is 3-fold greater than the RJV.

In the dog, catheters (25 cm long, 1.6 mm ID) were placed in the jugular vein and the carotid artery, tunneled under the skin to the back of the neck, exteriorized and plugged with a heparin lock. Thrombi were made from a donor dog by placing 0.7 ml of whole blood in a 1 cc plastic syringe barrel containing a cotton thread. The blood was clotted for 2 hr at 37.5 deg C, in vitro. The thrombus was rinsed and the syringe barrel was connected into the circulation by silicone tubing for a 90 min test period. Clot lysis was determined by weight. The flow rate across the thrombus was regulated to 60 ml/min by placing a teflon capillary tube (30 cm, o.7 mm OD) on the venous side. The procedure for the rabbit study differed as follows: Rabbit blood made larger clots therefore the blood was diluted with saline (4 to 1). Clots were incubated 90 min in vitro. The flow rate was 40 ml/min. Clot lysis was measured by the loss of 125-1 fibrinogen.

The dose of reccminant t-PA (ActivaseR, Actilyse™) that achieved 40% lysis (ED40) was determined to be 180 ugAg in the dog and 660 ugAg in the rabbit AVS model. Using the RJV model the ED40 was 340 ug/kg.
MONITORING OF HAEMOSTASIS DURING AND AFTER EXPERIMENTAL AUXILIARY LIVERTRANSPLANTATION
Authors: Porte R J , Knot E A R , Terpstra O T , Rodriquez Erena N F
Published: August 2018

Haemorrhagic diathesis is a major risk after human orthotopic livertransplantation. Theoretically, auxiliary livertransplan-tation (APLT) during which a partial livergraf t is inplanted next to the sick host-liver, should be acccnpanied with a minor haemo-rrhagic diathesis. In this study, haemostasis was monitored intensive during experimental APLT. Transplantation was performed in healthy pigs (n=6). There was no subtitutian of plasma products or platelet concentrates, nor use of heparin during the operation, thus making it possible to study the effect of the transplantation procedure and the (ischaemic) liver-graft only. Bloodsanples were taken just after induction of anaesthesia, after anastomosis of the Portal Vein, after anastomosis of the Hepatic Artery, 2 and 3 hours post-operative and several days after transplantation (max. follow-up 30 days). A sairple was also taken from the first blood outflow from the graft after recirculation. The following parameters were studied: APTT, PT, thrombintime, Normotest (R):, fibrinogen, AT-III, plasminogen, α2-AP, FDP's, platelet-oount and haematocrite. Sanples taken direct from the first blood outflow from the graft showed a decrease in platelet;-count and a prolongation of the APTT, PT and thrombintime, possibly indicating a oonsurrption of platelets and clotting factors in the graft. During operation there was a decrease of α2AP, AT-III and platelet-oount, while a remarkeble increase was seen during the first week after surgery to concentrations higher than the normal upper limit. At the end of the first week, in four animals an increase in APTT was seen (x=252%), together with a prolongation of the thrombintime ( 60 sec.). Because an anti-Xa activity was measurable, an endogenous heparin-like substance might be reponsible for this. None of the animals showed a bleeding tendency during or after transplantation, which indicates that the surgical procedure followed during APLT and the ischaemic graft do not give a serious disturbance of the haemostasis.
QUANTITATIVE DETERMINATION OF ENDOGENOUS PGI2 RELEASE IN ANAESTHETIZED BEAGLE DOGS
Authors: Hermán F , Hadházy P , Magyar K
Published: August 2018

A 30μm diameter pore size screen was inserted into an arteriovenous bypass system in anaesthetized, heparin-treated beagle dogs. The arterial blood was directed through the screen by a roller pump at a constant rate. As a result, the pressure proximal to the filter continuously increased (F.Hermdn et al.Thromb. Res.44 /1986/,575). The concentration of proximally infused PGI2 that stabilized the filtration pressure curve (pressure stabilizing concentration= PSC) was determined. If it was low enough (between 0.4 and 1.5 nmol/1) we administered the PGI2-releaser bradykinin (1/ug/kg), angiotensin II (0.5/ug/kg) or ADP (20/ug/kg) in bolus dose intravenously. Together with the cnanges in blood pressure, we observed a transient decrease in filtration pressure. From the pressure changes, based on the previously determined PGI2 concentration-res-ponse relationship, we estimated the amounts of released PGI2 as well as the time course of this release. Indomethacin (2 mg/kg i.v.) significantly decreased the PSC for exogenous PGI2 thereby increasing the sensitivity of the method; the release of PGI2 was abolished. The sensitivity of the method could also be increased by infusing BM.13.177 - an endoperoxide, thromboxane receptor antagonist - proximal to the filter (final concentration: 1-10/ug/ml). This substance did not affect the release of PGI2.

We conclude that by using this technique the endogenous release of prostacyclin can be continuously determined provided that PGI2 level exceeds 50 pg/ml.
FIBRINOLYTIC ACTIVITY IN MALIGNANCY
Authors: Aranda A , Páramo J A , Cuesta B , Fernández J , Paloma M J , Rocha E
Published: August 2018

Euglobulin lysis time (ELT), euglobulin fibrinolytic activity on fibrin plate (EFA), plasminogen, tissue-type plasminogen activator (t-PA) activity and antigen, ;2-antiplasmin (α2-AP), plasminogen activator inhibitor (PAI) and fibrinogen degradation products (FDP) were determined in a group of 149 patients (mean age 57 ± 13 years, 93 male), with different malignancies (digestive 61, lung 18, urological 24, ginaecological 16, others 30). Findings were compared with those of 44 age-sex matched healthy subjects. There was a significant prolongation of ELT (p <0.003), a decrease of plasminogen (p < 0.004) and an increase of PAI (p < 0.0001) in patients as compared to controls, being similar EFA, t-PA, α2AP and FDP. No differences in any ot these parameters were found in patients with metastatic disease (n= 65) as compared with those with local disease (n= 84). As regards to tumour localization, plasminogen and t-PA decrease were more pronounced in lung malignancies and PAI increase in lung and digestive malignancies. We conclude that there is an impairment in blood fibrinolytic activity in malignancy. Reduced fibrinolysis seems to be related to localization but not to degree of tumoral activity. That might have clinical complications.
PLASMINOGEN ACTIVATORS (t-PA and u-PA) IN HUMAN NEOPLASTIC CELL LINES AND THEIR MODULATION BY BASEMENT MEMBRANE COMPONENTS
Authors: Arnoux D , Boutière B , Pourreau-Schneider N , Martin P , Sampol J
Published: August 2018

Plasminogen activators (PA)may play an important role in the regulation of enzyme activation relative to basement membrane degradation associated with the invasive growth of tumors. In order to acquire a better understanding of the complex cascade reactions leading to the formation of plasmin, we have undertaken a comparative study of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in cellular extracts of 20 human cancer cell lines (13 malignant melanomas, 6 breast adenocarcinomas and 1 vulvar carcinoma). Four malignant cell lines,showing various t-PA or u-PA activity levels, were selected to study the modulation of proteolytic activity by laminin and fibronectin, major components of basal membrane. This study was performed in cellular extracts and conditioned medium. Our results showed that melanoma cells have high t-PA activity preferentially released into the culture medium. On the vulvar cell line, A 431, u-PA activity predominates and is also secreted into the medium. In contrast, breast cancer cells MCF-7 and MDA show u-PA activity, mostly recovered in the cellular extracts. An enhancement of respective PA activities occurs when cells are cultured on fibronectin or laminin, varying with the nature of the cell line.

Additional studies are needed to precise interrelation between tumor cells, basement membrane components and PA activities and the potential significance of proteolytic activities as markers of malignancy and invasive capacity.