bluuebunny/crossref_metadata_2025 huggingface dataset:
ACQUIRED RISK FACTORS AND DEEP VEIN THROMBOSIS IN SYMPTOMATIC OUT PATIENTS
Authors: Prandoni P , Lensing A W A , Zambon G , Breda A , Cuppini S , ten Cate J W
Published: August 2018

Previous studies revealed a number of acquired risk factors predisposing to acute deep vein-thrombosis (DVT). Unfortunately many of these clinical or epidemiologic studies were not proper designed, since they didn't include consecutive patients, used no or different types of objective endpoints or collected the data retrospectively. In a prospective trial we evaluated 307 consecutive out-patients with clinically suspected DVT by using ascending venography, which confirmed suspicion in 136 (44%). A history of prior thrombotic episodes as well as factors predisposing to DVT including advancing age, obesity, smoking habits, cancer, chronic lung and/or heart disease, immobilization, pregnancy, childbirth, chronic liver disease, systemic lupus erythematosus (SLE), nephrotic syndrome, varicose veins, fractures or trauma or chronic arteriopathies of the legs, diabetes mellitus (DM), recent surgery and estrogen therapy were recorded in all patients. The results of our comparison of these risk factors with the outcome of venography indicate clearly a significant difference (chi-square test) between patients with and without DVT for the following: -previous documented thromboembolism, cancer (p < 0.01); -chronic lung and/or heart disease, age > 65 years, immobilization (p < 0.05). The frequency of pregnancy, childbirth, nephrotic syndrome and chronic liver disease among our patients was too low for providing sufficiently narrow confidence limits. Surprisingly the presence of varicose veins will decrease the possibility of DVT (p < 0.01). In all patients (n=3) affected by SLE clinical suspicion was confirmed. Obesity, smoking habits, recent trauma or fracture or chronic artheriopathies of the legs, DM, recent surgery and estrogen therapy were not associated with an increased risk of thrombosis, since their presence in both groups was approximately the same.
ALCOHOL- AND TOBACCO CONSUMPTION IN PATIENTS WITH SUSPECTED DEEP VENOUS THROMBOSIS (DVT)
Authors: Lagerstedt C , Fagher B , Olsson C G , Ögvist B
Published: August 2018

Alcohol has several effects on hemostasis that might predispose for thromboembolism. The association between alcohol consumption and venous thrombosis is sparesly documented and we have therefore conducted a prospective study.

Methods: 395 consecutive patients with suspected DVT entered the study. The patients were questioned about consumption of beer, wine and liquor and the average weekly consumption of ethanol was estimated. The total weekly consumption of tobacco was also estimated. 302 of the patients (130 men and 172 women) underwent phlebography, which was classified either as normal or as fresh DVT or/and postthrombotic changes.

Results: 31 of the 130 men reported a weekly consumption of 100 g ethanol or more. In men below 65 years of age (62 men, see table) there was a significant relationship between ethanol consumption and the presence of fresh DVT or/and postthrombotic changes at phlebography

In men above 65 years of age the incidence of DVT/postthrombotic changes was not higher among those who consumed more than 100 g ethanol per week (9 of 14) than among them who consumed less than 100 g per week (35 of 54). Only 5 of the women admitted a consumption of at least 100 g ethanol per week, one of them had a DVT.

Tobacco consumption was not related to the occurrence of DVT

Conclusion: Ethanol seems to be a risk factor for DVT in young and middle-aged men
PHYSICAL ACTIVITY RELEASES TISSUE PLASMINOGEN ACTIVATOR FROM EXERCISING PARTS OF BODY
Authors: Keber I , Potisk K , Keber D , Stegnar M , Vene N
Published: August 2018

To determine the origin of tissue plasminogen activator (t-PA) release during physical activity, we studied the separate and combined effects of venous occlusion and acute physical activity on t-PA release in arm and leg. In 15 healthy volunteers 20 min venous occlusions of arm and leg were performed simultaneously before physical activity ( maximal stress testing on treadmill)(occlusion I), immediately after physical activity and 45 min later (occlusion II). Blood samples were drawn from unoccluded arm before occlusion and after physical activity, and from occluded arm and leg after occlusion. Fibrinolytic activity was measured by euglobulin clot lysis time (ECLT) and t-PA activity assay. The amount of released t-PA during different stimuli (fibrinolytic potential) was calculated as the difference between post- and prestimulation fibrinolytic activity. Before physical activity there was a great increase in fibrinolytic activity due to t-PA in the occluded arm but no increase in the occluded leg. Physical activity itself caused a similar increase of systemic fibrinolytic activity as arm occlusion locally. After physical activity arm occlusion evoked equally good response than before it. Fibrinolytic activity during leg occlusion behaved differently: there was an increase in t-PA activity in the occluded leg which persisted one hour after physical activity, when systemic fibrinolytic activity already fell to initial level.These results demonstrated that walking and running triggered t-PA release from the leg vessels. Since leg occlusion was not a stimulus for t-PA release, it served only as a method to demonstrate the effect of physical activity.
DISCREPANCY IN FIBRINOLYTIC RESPONSE TO VARIOUS STIMULI IN HEALTHY MEN
Authors: Stegnar M , Keber I , Keber D , Vene N
Published: August 2018

Absent or low fibrinolytic response to stimulation is believed to reflect thrombotic tendency. However, it has been observed, that poor response to one stimulus is not always associated with poor^ response to other stimuli. Therefore, correlation between fibrinolytic responses to various stimuli has been studied in two groups of healthy men. Nine volunteers of the first group were subjected to 20 min upper arm venous occlusion (VO) followed by vasopressin analogue (DDAVP) infusion (0,4 ug/kg body weight in 10 min). In the second group (16 volunteers) VO and physical exercise (EXER, discontinuous test on treadmill up to maximal heart rate) were applied successively in this order. Venous blood was sampled before stimulation and in the 20 min of VO in both groups. The third blood sample was obtained 20 min after start of DDAVP infusion in the first group and immediately after EXER in the second group. Fibrinolytic activity was determined by euglobulin clot lysis time and by specific tissue plasminogen activator (t-PA) activity assay and increase in activity above prestimulation value calculated. No significant correlation between fibrinolytic response to VO and DDAVP was observed in the first group. Also in the second group responses to V0 and EXER showed no correlation.

The observed discrepancy in fibrinolytic responses to various stimuli rise an important question: Which fibrinolytic response is more relevant to development of thrombosis?
IN VITRO EFFECTS OF RECOMBINANT TISSUE TYPE PLASMINOGEN ACTIVATOR ON FIBRINOLYTIC AND COAGULATION PARAMETERS AND ITS PREVENTION BY SPECIFIC ANTIBODY, D-PHE-PRO-ARG-CTUCl AND APROTININ
Authors: Seifried E , Tanswell P
Published: August 2018

Monitoring of systemic effects during rt-PA therapy has shown of depletion of fibrinogen-antiplasmin, plasminogen and other hemostatic factors. Because in vitro activation of plasminogen may occur between blood collection and freezing and thawing before assaying we analysed the influence of 0,0.2, 2.0 and 10.0,ug rt-PA/ml citrate blood (final conc.) on hemostatic and fibrinolytic parameters and its inhibition by 3 different inhibitors. Addition of rt-PA to citrated whole blood without an inhibitor induced a concentration-dependent depletion of Fbg, Plgα2-Apl,α2-M, C1 - I, α2-Atrp, a loss of activity of FV, VIII,IX, XIII and alterations of the global coagulation assays. No effect of rt-PA was observed on F II, VII, X, XI, XII, AT III and Protein C. To prevent in vitro fibrinogenolysis 0.1, 0.5 and 1 mg/ml of a polyclonal sheep anti-rt-PA-antibody, 0.3, 1.0 and 10 Aimol/1 PPACK (D-Phe-Pro-Arg-CH2Cl), 75 and 150 KlU/ml aprotinin (final conc.) and saline as a control were added to pooled citrate blood.All samples containing rt-PA and/or inhibitors and/or saline were incubated for 45 min on ice, centrifuged, aliquotted, snap frozen and stored at ™20° C until analysis. Pretreatment of blood samples with anti-rt-PA IgG prevented interferences with all fibrinolytic and most clotting assays in plasma at a dose of 2 ,ug rt-PA/ ml. PPACK was of limited utility in clotting assays, but enabled correct analysis of fibrinolytic assays. Aprotinin was suitable only for a restricted range of both assay types. It is concluded that collection of blood samples on an appropriate antibody may be the most suitable procedure to get correct measurements of in-vivo effects of rt-PA on the hemostatic system in patients undergoing fibrinolytic therapy.
COMPARATIVE STUDY OF RADIOIMMUNOASSAYS FOR FRAGMENT E-NEOANTIGEN IN PLASMA AND E-ANTIGEN IN SERUM
Authors: Chen J P , Goldman M H , stegner M
Published: August 2018

Fragment E-neoantigen (Eneo) is a specific marker of structual and conformational changes associated with the plasmic degradation of fibrinogen and fibrin. The Eneo radioimmunoassay (RIA) is capable of determining normal and pathological plasma levels of fibrin(ogen) degradation products (FDP). However, the detection of the minute quantity of E-related FDP present in the blood of a normal individual requires subjecting the Eneo RIA to a delayed (18 hr) addition of labeled ligand. This step increases the already lengthy period of incubation (48 hr) for the primary antigen-antibody complex to 66 hr. An additional incubation period (24 hr) is required to precipitate the primary complex with goat anti-rabbit IgG.

In an attempt to simplify and speed up the diagnostic blood test for deep vein thrombosis (DVT), the serum fragment E-antigen (E:Ag) RIA has been compared with the Eneo RIA in plasma. Four patients with DVT were followed sequentially with respect to the pre- and post-operative change in response to vascular surgery. Both the Eneo RIA and E:Ag RIAs showed a gradation in plasma (132 - 463 ng/ml) and serum (171 - 1400 ng/ml) E-related FDP levels respectively. Significantly, the sequential measurements of plasma Eneo and serum E:Ag immunoreactivities for these patients appear to fluctuate in parallel with each other. Normal females gave higher plasma Eneo (15.2 ± 4.3 ng/ml) as well as serum E:Ag (42.7 ± 17.8 ng/ml) values than normal males (Eneo = 10.2 ± 5.5 ng/ml, E:Ag = 38.8 ± 19.5 ng/ml). Normal serum E:Ag levels represent the increase in Fg-E concentration, 3-4 times that of normal plasma Eneo levels.

Considerably higher plasma Eneo (2.2 - 7.1 pg/ml of Fg-E equivalent) and serum E:Ag levels were observed in DVT patients under thrombolytic therapy with streptokinase. Western immuno-blotting of patients1 plasmas and sera revealed that the Fg-E antigenic sites of both pathological plasmas and sera showed a similar pattern with a major immunoreactive FDP at Mr-360,000.

The serum E:Ag assay as currently practiced takes 24 hr to complete, but the time of the assay can be shortened considerably by adapting it to a solid-phase RIA.
DEVELOPMENT OF A NEW ASSAY METHOD FOR THE DETECTION OF DD/E COMPLEX
Authors: KUROSO K , IKEMATSU S , HADA M , FUJIMAKI M , FUKUTAKE K
Published: August 2018

A new assay method for the detection of DD/E complex derived from crosslinked fibrin is developed. This assay is performed on a microtitre plate using capture/tag antibody technique, in which the monoclonal antibody against D dimer fragment (DD-3B6, MAbCO) is coated and anti-E fragment polyclonal F(ab)’2 conjugated with horse radish peroxidase is for a tag-anti body. Antigen dilution curve is drawn in the range of 0.01-1.0 pg/ml of purified DD/E complex. DD/E complex can be measured specifically and other high molecular weight derivatives from crosslinked fibrin show a little crossreaction, though fibrinogen and fibrinogen degradation products show no crossreactivities on this assay. D dimer fragment dissociated from DD/E complex after further plasmin digestion is less reactive in this assay, while this type of D dimer can be detected by DIMERTEST-EIA (MAbCO). These data suggest that an early stage of plasmin digestion of crosslinked fibrin can be detected by this method. A trace amount of DD/E complex curculating in plasma from a small thrombus is possibly detected, because this assay gives an excellent high sensitivity with the detection limit of 0.01 jug/ ml. Normal value of plasma DD/E complex (n=50) indicates below 0.12 pg/ml as 90 percentile. Patients with DIC (n=24) show high levels of DD/E complex between 0.6 and 40 g/ml. These elevated
levels of DD/E complex may suggest consequently the existence of the plasmic digestion of crosslinked fibrin in the cases with DIC. In summary, it is concluded that the development of this assay will add one technique to discriminate between fibrinolysis and fibrinogenolysis and this assay is useful for the quantitative detection of DD/E complex produced in an early stage of fibrinolysis seen in various thrombotic disorders, and for the evaluation of the efficacy of thrombolytic therapy.
POSTOPERATIVE CHANGES IN HEMOSTASIS, ANALYZED BY SERIAL DETERMINATION OF FIBRINOPEPTIDES AND D DIMER
Authors: Kambayashi J , Kang J H , Tanaka T , Tsujinaka T , Sakon M , Mori T
Published: August 2018

It is generally believed that hypercoagulable state occurs after surgery, followed by hyperfibrinolytic state. Though a considerable number of studies on the postoperative changes in hemostasis have been reported, the results are inconclusive, as it is difficult to detect in vivo activation of coagulation and fibrinolysis by conventional laboratory tests. Thereby, attempts were made to elucidate the changes by serial determination of fibrino-peptide A (FPA), fibrinopeptide B Beta 15-42 (BB) & D dimer in our surgical cases ([A]-27 hepatic resections, [B]-27 gastric resections, [C]-4 probe laparotomies). FPA & BB were determined by radioimmunoassay and D dimer was assayed by aggulutination of Latex beads coated with monoclonal antibody. FPA was elevated lh after surgery in [A](25.5± 7.6ng/ml) and [B](16.4± 3.3) and it returned to normal range (4.2± 2.1) by postoperative day 1 (POD 1). No such change was noted in [C]. BB in [A] was elevated (46.2± 10.6ng/ml) lh after surgery and the value remained high until POD 14. In [B], BB was likewise elevated at lh (50.9± 7.7) and it gradually decreased to 26.0± 4.8 by POD 2. Then, it again increased to 36.8± 5.1 and remained high until POD 10. The value in [C] was normal (6.8± 2.4) throughout the period. FDP was moderately increased (10-20ug/ml) until POD 14 & 7 in [A] & [B]. Regardless of the increased level of BB, the amount of D dimer was within normal range (<200ng/ml) until POD 1 & 3 in [A] & [B]. Thereafter, it gradually increased and reached above l,000ng/ml between POD 7 and 10 in both groups. From these observations, the following conclusions were obtained; (1) Unexpectedly, postoperative hypercoagulable state was transient and of low magnitude. (2) On the other hand, significant fibrinolysis was observed immediately after surgery and it continued over 10 days. The early phase (up to POD 3) is likely to be primary fibrinolysis as it was not accompanied by formation of D dimer. The late phase is considered to be secondary fibrinolysis as D dimer was also elevated. (3) Regardless of the different surgical procedures, these changes were basically similar between [A] and [B], though the intensity of the changes was different. Therefore, the postoperative changes in hemostasis presented herein may be the general physiological responce against at least abdominal surgery.
A COMPARISON OF D-DIMER AND SERUM FIBRINOGEN/FIBRIN DEGRADATION PRODUCT LEVELS (F.D.P.’s) IN THE INVESTIGATION OF HYPERCOAGULABLE STATES
Authors: Wilde J T , Kitchen S , Freston F E , Greaves M
Published: August 2018

D-Dimer assays measure specific breakdown products of crosslinked fibrin whereas FDP assays are not specific for these products. We have, therefore, measured D-Dimer levels (MabCo Dimer Test) semi-quantitatively in patients with clinical and laboratory evidence of disseminated intravascular coagulation, acute and chronic liver disease, acute leukaemia at presentation and acute venous thrombosis at diagnosis. We have also measured D-Dimer in the 3rd trimester of normal pregnancy and in pregnancies with complications. We compared these levels with F.D.P. levels measured by the Thrombo-Wellcotest. Patients with liver disease comprised mainly cirrhosis and acute viral hepatitis; those with venous thrombosis had this diagnosis confirmed by venography. Pregnancy complications included mainly pre-eclampsia, ante-partum haemorrhage and intra-uterine foetal death. D-Dimer levels were elevated ( 200ng/ml) in all 31 patients with D.I.C., in 34 of 40 with liver disease, in 13 of 16 with acute leukaemia, in all 10 patients with D.V.T., in 9 of 16 normal pregnancies and in 29 of 39 complicated pregnancies. Using a rank correlation method, there was correlation between D-Dimer and F.D.P. levels (Normal 8ug/ml) in the following groups of patients as follows (r is the correlation coefficient, levels of significance are shown in brackets):

D.I.C. r=0.72(0.2%), liver disease r=0.56(0.2%), acute leukaemia r=0.72(0.2%), D.V.T. r=0.83(1%) and complicated pregnancy r=0.42(1)6). There was no correlation between D-Dimer and F.D.P. levels in normal pregnancy. Very rarely were D-Dimer levels elevated when F.D.P. levels were normal and vice-versa. We conclude that a close relationship does exist between D-Dimer and F.D.P. levels in the clinical conditions that we have studied. We note the high incidence of elevated D-Dimer levels and the close correlation with F.D.P. levels in patients with liver disease and the high incidence of elevated D-Dimer levels suggesting increased activity of the coagulation system in patients with acute leukaemia.
LEVELS OF D-DIMER FRAGMENT OF FDP IN CASES OF ACUTE PROMYELOCYTIC LEUKEMIA WITH NORMAL LEVELS OF ANTITHROMBIN III AND PROTEIN C IN PLASMA
Authors: Matsuda T , Ito K , Asakura H , Saito M , Uotani C , Jokaji H
Published: August 2018

16 cases of acute promyelocytic leukemia (APL), with prolongation of prothrombin time, depletion of fibrinogen in plasma and marked increase in fibrin/fibrinogen degradation products (FDP) in serum, were compared with 8 cases of disseminated intravascular coagulation (DIC) caused by other diseases than APL. In the cases of APL, levels of plasminogen and α2-antiplasmin in plasma strikingly decreased and concentrations of α2-antiplasmin-plasmin complex in plasma markedly increased. However, levels of antithrombin III and protein C in plasma of these cases were within normal limits or only slightly decreased. In these cases, hepatic or renal dysfunction was not observed. Further investigation revealed that a marked depletion in factor XIII activity and a striking increase in levels of D-dimer fragment of FDP (including YD fragment etc.) were present in the cases of APL. Significant correlations between levels of D-dimer and FDP measured by the conventional method were also observed. From these results, it is concluded that DIC is present in the cases of APL, although relevance to fibrinolysis is strongly suggested. However, it remains obscure concerning to etiology of normal or only slightly reduced levels of antithrombin III and protein C in the cases of APL.
EFFECTIVENESS OF DEFIBROTIDE IN THE TREATMENT OF ACUTE MYOCARDIAL INFARCTION
Authors: Milazzotto F , Carelli M , Citone C , Di Macro Tullio G , Gambelli G C , Giampaolo P , Malinconico U , Polizzi C , Cornelli U
Published: August 2018

Defibrotide (D) is an extractive polydesoxyribonucleotide. In preclinical studies the product was shown to be active as a pro -fibrinolytic, antithrombotic and thrombolytic agent while comply tely devoid of anticoagulant activity. In animal models, D was found to afford striking protection from the effects of acute lethal and non lethal myocardial ischemia as well as from myo -cardial injury following reperfusion. In this open single - blim trial, D was administered to patients with acute myocardial infaj ction (AMI) for the prevention of complicating arrhythmias; throy bus formation, pericarditis, etc.

Sixty patients with AMI were divided randomly into two groups of 30 patients each. One group was treated with D by 6-hour drip infusion for 3 consecutive days (2.8 g on the first day, then 2.4 g daily). The other group was treated with equal volumes of physiological salt solution. All patients received conventional trea_t ment for AMI. The two trial groups were sufficiently homogeneous in terms of AMI type, age and sex distribution, PCT, Forrester index, Holter, and Peel index. D treatment proved effective in reducing the incidence of severe arrhythmia (p < 0.05), thrombus formation (p < 0.05), and pericarditis (p < 0.01). CPK, TT and PTT readings were not modified by the treatment; the incidence of post-AMI angina and the number of deaths (4 in each group) were similar in the two groups. The results of this pilot study are encouraging; further clinical trials are currently in progress to assess D activity in larger groups of patients treated with the product at higher dosages.
PROFIBRINOLYTIC ACTIVITY OF DEFIBROTIDE IS PTOSTACYCLIN-INDEFENDEMT
Authors: Porta R , Pescador R , Niada R , Mentavani M , Prino G
Published: August 2018

The profibrinolytic, antithrombotic and thrombolytic activities of Defibrotide, a polydeoxyribonucleotide obtained from mammalian lungs, have been previously described. Defibrotide is also able to promote the generation and release into the circulation of prostacyclin and of plasminogen activator factor (tPA) from vascular walls. These two last activities, i.e., PGI2 and tPA stimulation, could be of importance in the profibrinolytic activity of Defibrotide. In an attempt to clarify the profibrinolytic mechanism/s of Defibrotide, the role of PGI2 generation in this activity was investigated in rabbits in which the cycloxygenase pathway was blocked with Indcmethacin. Male rabbits were treated with Indcmethacin, 10 ng/Kg, i.v., 20 minutes or 2 hours before Defibrotide administration. Defibrotide was injected i.v. into rabbits at two dose levels (16 or 64 rrg/Kg). Rabbits treated with Defibrotide only were used as controls. The profibrinolytic activity was evaluated measuring the lysed area of normal human fibrin plates induced by euglobulin fraction obtained from plasma. Results are reported in the Table.

Indomethacin (Indo) administered 20 min (*) or 2h (**) before Defibrotide.

AN3VA: N.S. vs. Vehicle + D 12.5 or D 50 ng/Kg i.v.; P< 0.01 vs. basal.

The results reported here demonstrate that the profibrinolytic activity of Defibrotide is independent of PGI2 generation: Indcmethacin pro treatment was unable to suppress or to reduce the fibrinolytic response of the rabbit to Defibrotide.
DEFIBROTIDE IN EXPERIMENTAL MYOCARDIAL ISCHEMIA IN THE CAT: EFFECTS ON HEMODYNAMICS, ENERGY METABOLISM AND INFARCT SIZE
Authors: NIADA R , Porta R , Tettamanti R , Pescador R , Mantovani M , Prino G
Published: August 2018

Defibrotide was able to prevent the hemodynamic and biochemical alterations caused by acute myocardial ischemia (AMI) induced by coronary occlusion in the cat when infused 3.5 h before and 5 h after left anterior descending coronary artery (LAD) occlusion. In the platelet perfused heart, Defibrotide was a selective stimulator of coronary vascular PGI^ but not of platelet thromboxane formation. The present study was designed both to investigate the effects of Defibrotide injected 30 min after the induction of acute myocardial ischemia (AMI) in the cat and to evaluate the ability of this drug to reduce infarct size. In the first set of experiments a permanent ligature (5 hours) was placed around LAD. ST segment from ECG, mean aortic pressure (MAP), heart rate (HR) and the pressure-heart rate index (PRI) were considered. Plasma and tissue creatine phosphckinase activity (CFK), tissue lactate and ATP were measured by enzymatic kits from Boehringer Biochemia. 30 min after coronary occlusion a loading dose of Defibrotide (32 ng Kg-1 ) was administered i.v. immediately followed by an infusion (32 ng Kg-1 h 4.5 h-1) MAP, HR and PRI were not modified either by AMI or by the infusion of Defibrotide. AMI-ST segment increases were reduced by Defibrotide from 0.5 h after the beginning of the treatment (—49% vs. AMI control) to the and of experiments (-83% vs. AMI control after 5 h occlusion period). Plasma CFK was reduced from 2.5 h after the beginning of the treatment (-29%) till the end of experiments (-52%). Ischemic tissue CFK, lactate and ATP were normalized by Defibrotide. In the second set of experiments the animals were infused with Defibrotide (50 or 200 mg Kg-1 h-1 , i.v.) starting 2 hours before coronary ligature. The infusion was maintained throughout the 5 h occlusion period. The risk and infarct areas were measured by Evans blu and nitroblue tetrazoliun staining. The 51 ± 3% of risk area was infarcted in AMI control cats. Defibrotide at the two tested doses significantly reduced these infarct areas to 42 ± 4% and 34 ± 2% of risk areas respectively. The beneficial effects of Defibrotide observed in AMI could be attributed both to its ability to enhance PGI2 release from vascular walls and to improved local tissue oxygenation and energy supplies. However it could be taken into account a direct cytoprotective action.
CHARACTERIZATION OF A NEUTROPHIL CPYMOTRYPSIN-LIKE ENZYME THAT ACTIVATES PLATELETS
Authors: Selak M A , Chignard M , Smith J B
Published: August 2018

Communication between neutrophils and platelets was previously investigated by measuring platelet aggregation, serotonin release and changes in cytosolic free calcium subsequent to specific stimulation of neutrophils by fMet-Leu-Phe (FMLP) in a suspension of both cell types. The addition of the chemotactic peptide was shown to elicit secondary platelet activation as a consequence of primary stimulation of neutrophils. Cell-free supernatants from FMLP-stimulated neutrophils were capable of inducing platelet activation thus demonstrating that a factor released bv neutrophils was responsible for the observed platelet responses. After eliminating classical platelet agonists as the acitive agent, it was shown that an enzyme termed neutroohilin induced platelet calcium mobilization, secretion and aggregation. The current studies were conducted to characterize the mediator released bv neutrophils. Neutrophilin bound bo cation exchange resins but failed to bind to anion exchangers. The biological activity associated with neutroohilin was unaffected by leupeptin, only very weakly diminished by N-bosyl-Lvs-chloromethvl ketone and was strongly inhibited by N-tosvl-Phe-chloromethvl ketone, aloha-l-antitrvpsin, soybean trypsin inhibitor and Z-Glv-Leu-Phe-chloromethvl ketone. Neutroohilin was released from stimulated neutrophils only after cytochalasin B treatment, as was beta-glucuronidase, suggesting that both enzymes are located in azurophilic granules. Neutroohilin-induced platelet activation was inhibited bv antiserum to human catheosin G in a dose-deoendent manner but was unaffected by antiserum to human elastase or alpha-fetoprotein. The inhibitor sensitivity, immunological cross-reactivity, ionic properties and probable subcellular localization indicate that neutrophilin is a cationic chymotrvosin-like enzyme related, if not identical to, catheosin G. Neutroohilin-induced platelet activation could explain different pathological events in which platelets and neutroohils are known to be involved.
PR0C0AGULANT ACTIVITIES IN HUMAN ALVEOLAR MACROPHAGES:
Authors: Nakstad Britt , Lyberg Torstein
Published: August 2018

The coexistence of fibrin and tissue macrophages is a common finding in the histopathology of chronic inflammatory diseases of the lung.Fibrin deposition may occur as a result of activation of the extrinsic coagulation system,initiated by procoagulants generated by alveolar macrophages.In this study human alveolar macrophages (LAM) obtained by lavage of healthy donors were shown to express procoagulant factors,thromboplastin (tissue factor) and a direct factor X activator,probably a thromboplastin/ factor VII complex.In contrast to blood monocytes, LAM were only slightly susceptible for in vitro induction of thromboplastin activity (11,3 ± 2,6 (SEM)-fold and 1,3 t 0,2-fold activity increase after endotoxin stimulation).

LAM were separated into four subpopulations by density gradient centrifugation.The specific thromboplastin activity of subpopulation cells varied inversely with their density (3,08 ± 0,42 U/mg cell protein for the least dense vs.0.49 ± 0.03 for the most dense subpopulation ).Low-density subpopulations of LAM released membrane material to the culture medium,which was sedimentable in the u1tracentrifuge and which expressed procoagulant activities with the same characteristics as the LAM procoagu1 ants.These findings suggest that alveolar macrophages and the membrane vesicles shed from their surface can contribute to local fibrin deposition in the lungs by expressing procoagulant factors.
MECHANISM OF ACTIVATION OF BLOOD COAGULATION BY LEUKOCYTE PROCOAGULANT ACTIVITY
Authors: Narahara N , Sadakata H , Uchiyama T , Andoh K , Tanaka H , Kobayashi N , Maekawa T
Published: August 2018

To investigate the process of activation mechanism of blood coagulation by leukocytes, binding of radiolabelled Factor X and the activation of Factor X on the cell surface of leukocytes were studied by using cultured leukemia cell line, Molt-4 cells. Cells were cultured in RPMI 1640 medium with 10% inactivated fetal, calf serum at a concentration of 1x106cells/ml. After 6 hours' stimulation with 1 ug/ml of endotoxin(LPS: Escherichia coli 026:B6), cells were separated by centrifugation, washed three times with Tris containing NaCl buffer(pH 7.5, TBS), and then suspended in TBS containing 0.5% bovine serum albumin(TBS-BSA) to the concentration of 5x106 cells/ml. Factors X, VII and IX were purified from fresh-frozen human plasma by the method of Bajaj in a modified version. Factor VIII was purified from cryoprecipitate as starting material. Factor X labelled with 1-125 by the method of McFarlane showed a single band on autoradiography. Specific radioactivity was 0.3 mCi/mg. For the study on binding of Factor X, TBS-BSA solution containing 4 mM of CaClp, various amounts of radiolabelled Factor X with/without purified Factors VII, VIII and IX were added to the LPS-stimulated washed cell suspension and mixed well. N-butyl-phtalate was layered over the reaction mixture after incubation at room temperature for various minutes. Total amount of bound Factor X was calculated from the radioactivity of the cell pellet separated by centrifugation of the reaction mixture. The Xa activity generated in the supernatants was assayed using S2222.

Results: Factor X bound specifically to the LPS-stimulated Molt-4 cells. Amount of bound Factor X and the dissociation constant was 1.0 ng/5x10bcells (5.2x103sites/cell) and 5x106M, respectively. More amounts of Factor X bound when Factors VIII and IX were present in the reaction mixture than their absence. Five times more Factor Xa was generated when Factors VII, VIII and IX were present in the reaction mixture as compared with presence of Factor VII, alone. These results suggest that blood coagulation cascade proceeds on the LPS-stimulated leukocyte surface.
CHEMOTAXIS OF POLYMORPHONUCLEAR LEUKOCYTES BY VASCULAR PERMEABILITY FACTOR FROM BOVINE PLATELETS
Authors: Hoshikawa N , Niino H , Imura J , Ashihara Y , Shirasawa K
Published: August 2018

Bovine platelet a-granule acid extract (BPAE) contains vascular permeability factor (VPF) as human platelets. VPF plays as a mediator depending on polymorphonuclear (PMN) leukocytes and possesses a potent cell growth activity in cultured fibroblasts. This study was carried out to examine the chemotactic activity of VPF for PMN leukocytes of rabbits. For the study of chemotaxis, the PMN leukocytes were collected from the ascites after production by an injection of 0.1% oyster glycogen in 0.85% NaCl solution into the peritoneal cavity of rabbits. They were suspended in the Gey's balanced salt solution with supplementation of 2% human albumin. Partial purification of BPAE was performed by gel filteration using Sephadex G-50 so as to exclude the influence of PF-4 and 8-TG. BPAE, as the attractant solution, was prepared at the several concentration in the Gey's solution. Human serum activated with zymosan was used as the positive control. Effects of protamine sulfate, a competitive inhibitor of platelet-derived growth factor (PDGF), on the chemotactic activity of VPF were also studied. Chemotaxis of the PMN leukocytes was measured in the modified Boyden chamber with a 3jJm-pore filter. Five high power (x400) grids were counted per filter and cell migration was corrected by subtraction of blanks in which the lower compartment contained medium only. The most striking chemotactic response was obtained when BPAE was used at the concentration of 0.1μg/ml. However, the response was reduced at the concentration of more than 1μg/ml. The activity of BPAE (0.1μg/ml) was about 60% of the positive control level. The migration was reduced to 18% of the positive control by protamine sulfate (3μg/ml) at that concentration of BPAE

It is concluded that VPF induces chemotaxis to PMN leukocytes at the concentration lesser than that enhancing the vascular permeability response. Furthermore, protamine sulfate inhibits the VPF-induced migration of PMN leukocytes. Those findings may indicate that VPF acts as PDGF in function. Therefore, it is likely that the factor attracts PMN leukocytes and promotes cell growth in the inflamed region
MEASUREMENT OF ELASTASE-INDUCED FIBRINOGEN-DERIVED PEPTIDES IN VITRO
Authors: Eckhardt Th , Haas S , Lange B , Pfeiffer H
Published: August 2018

Leukocyte elastase (EL) cleaves peptides from the N-terminal fibrinogen (fbg) Aα and B6 chains. The Aα peptide (Aα 1-21) and the as yet unidentified BIB peptide contain thrombin (TB)cleavage sites. As we were interested in fbg proteolysis in sepsis and leukemia, we have tried to measure these peptides (generated by incubating a crude granulocyte extract with fbg) using available fibrinopeptide A (FPA) - und Bß 15-42-antigen-RIA techniques. As the indirect measurement of Aα 1-21 in terms of FPA releasable by TB treatment in vitro (TB-inducible FPA, TIFPA) requires the use of a highly specific anti-FPA-antiserum to avoid cross reaction of Aα 1-21 we tested the specificity of several antisera. Only R2, provided by Dr. J. Owen, proved suitable whereas two commercial antisera measured 6-15% of Aα 1-21 as apparent FPA. Simultaneous recovery of FPA and Aα 1-21 from plasma using R2 was between 80-100% after precipitation of cross-reacting fbg by ethanol, whereas bentonite adsorbed Aα 1-21 and the EL induced BIB peptide. TIFPA was fully recovered after incubation with plasma (37°C, 2 hr) whereas cross-reactivity of Aα 1-21 in the FPA-assay did not increase. EL-induced proteolysis in plasma in terms of TIFPA generation did not occur unless the normal granulocyte-plasma ratio was increased about 300-400 fold. The Bß 15-42 antigen RIA allowed quantitative measurement of the as yet undefined EL-induced Bß peptide, since the "Bß 15-42" concentration measured was equal to the amount of FPB (measured as Bß 1-13) releasable from the EL-induced BIB-peptide by TB. The immunoreactivity of this peptide is stable in plasma and completely recovered after ethanol precipitation. This finding suggests that the EL-induced BIB peptide is longer than Bß 1-4 2 and not susceptible to C-terminal degradation of the Bß41/42 region which is crucial for recognition of the peptide by the antiserum (supported by Deutsche Forschungs-gemeinschaft).
BEHAVIOUR OF ADREN0CHR0ME PATHWAY IN PATIENTS WITH CEREBROVASCULAR DISEASES
Authors: Alessandri C , Violi F , Rasura M , Caliendo C , Pelaia P
Published: August 2018

Histopathological studies in segments of cerebral ischaemia show local inflammation with leucocytes infiltration.This event has been confirmed in vivo by means of radiolabelled leucocytes. This inflammatory response could be of detriment to cerebral tissue since leucocytes release toxic substances such as oxygen free radicals.A free radical mechanism,in fact,has been supposed as an event worsening the evolution of ischemia.Evidence of neutrophil activation in stroke patients was shown by us in previous reports, where we have described that the plasma of these patients contained an excess of a neutrophil oxidase able to convert,in vitro, adrenaline to adrenochrome.Aim of present study was to evaluate if neutrophil activation can be observed in patients with brain hemor ragie (BH) also.Six patients (females 1,males 5;age 68-79 years) suffering from BH and 15 patients (females 5, Males 10;age 58-86 years) affected by brain infarction (BI) were studied within 20-48 hours from acute episode.Diagnosis of stroke was made by computerized tomography.Neutrophil activation was studied in plasma evaluating the oxidation of adrenaline to adrenochrome according to Matthews and Campbell method.20 matched for age and sex healthy subjects were studied as control.A significant rise of plasma adrenaline oxidase activity was observed in patients with BI.This preliminary investigation suggests that neutrophil activation could be restricted to patients with BI.In fact,patients with BH had plasma oxidase activity similar to controls.Clinical data should be necessary to evaluate if a relation between leucocyte activation and the natural course of cerebral ischemia does exist.
HEPARIN ANTICOAGULATION AND ITS EFFECT ON ARTERIAL THROMBUS FORMATION IN THE RABBIT
Authors: Shand R A , Butler K D , Davies J A
Published: August 2018

Injury to the rabbit carotid was induced by clamping the vessel for 5 minutes. Deposition of radiolabelled fibrinogen (F) or platelets (PL) on the injured carotid was monitored continuously, for 45 minutes, using an iodide crystal scintillation detector positioned over the vessel linked to an automated isotope monitoring system (AIMS 8000). The results were expressed as the maximal increase or as the area under the curve (AUC) for the recording period.

Following injury there was a simultaneous increase in both F associated and PL associated radioactivity of 29% (median value, range 10.9 - 4.37) and 11% (range 7.45 - 24.7) respectively above control values. The response as measured by the AUC was 47,000 ± 8,550 and 15,000 ± 5,600 respectively.

Heparin (250 IU/kg, i.v. bolus) did not influence F deposition following injury and only produced a transient prolongation of the prothrombin time. If the same bolus dose of heparin was followed by an infusion of 670 IU/kg/hr there was a significant reduction of both the maximal increase and the AUC of F deposition (45% and 55% respectively). There was also a sustained (2-3 fold) prolongation of the prothrombin time. Heparin (500 IU/kg i.v. plus infusion of 670 IU/kg/hr) significantly impaired both the maximal increase and AUC of F deposition (by 33% and 69% respectively) and PL deposition (79% and 100% respectively). No significant changes in either albumin or erythrocyte associated radioactivity in the injured carotid artery of control or treated rabbits were found.

This data demonstrates that anticoagulation, using heparin, inhibits fibrin(ogen) and platelet deposition, but not permeability changes, in the injured rabbit carotid artery. This highlights the interdependence in arterial thrombus formation of both the coagulation system and platelets.
EFFECT OF MCI-9038, A SELECTIVE THROMBIN INHIBITOR, ON CEREBRAL MICROCIRCULATION AFTER CEREBRAL ISCHEMIA IN RATS
Authors: Yamamoto T , Hirata T , Inagaki M , Kikumoto R , Tamao Y , Okamoto S
Published: August 2018

MCI-9038', a synthetic thrombin inhibitor No. 805, has been shown to be effective for various thrombotic diseases including cerebral thrombosis in acute stage. In this report, we studied the effect of MCI-9038 on disorders of cerebral microcirculation in rats generated by the method of Pulsinelli et al. One day after both vertebral arteries were electrocauterized with electrocautery needle through the alar foramina, bilateral carotid arteries were occluded with Vari-angle aneurythm clips to induce hemispheric ischemia, which was confirmed by electroencepharograms becoming isoelectric. Thirty min. after bilateral carotid artery occlusion, clips were removed to restore carotid blood flow and 5 min. later India ink was infused to detect no-perfusion region. The brain was removed and noperfusion area (NPA) was measured for 10 colonal sections of the brain. While NPA in the control group was 14.6 ± 0.7 % of the total area of 10 colonal sections, MCI-9038 significantly reduced NPA to 5.8 ± 2.1 % at 5 mg/kg i.p. and 6.3 ± 1.3 % at 10 mg/kg i.p. Heparin at 50 and 100 u/kg i.v. and tissue culture urokinase (TCUK) at 48,000 and 96,000 u/kg i.v. did not reduce NPA. Electronmicroscopical observation revealed the platelet aggregates occluding the micro vessels in the region of noperfusion, suggesting that disorders of cerebral microcirculation in this model resulted from the obstruction of blood flow by micro platelet aggregates. MCI-9038 is considered to improve the cerebral microcirculation by the inhibition of the formation of platelet aggregates. Since MCI-9038 does not inhibit platelet aggregation induced by collagen, ADP or arachidonic acid but by thrombin potently, thrombin is considered to take an important role to form the micro platelet aggregates in this model. The difference in the effectiveness between MCI-9038 and heparin is discussed.
PROTEIN C ACTS AS AN ACUTE PHASE REACTANT IN EQUINE LAMINITIS
Authors: Prasse K W , Moore J N , Duncan A
Published: August 2018

Equine Colic Syndrome is a disease of horses whose complications include laminatis.This term describes a situation where microvascular damage to the hoof causes degeneration of the interphalangeal laminae,leading to lameness. Vascular studies have suggested that microthrombosis involving the delicate vessels in the hoof,coupled with changes in the platelet count, coagulation factors & elevated FDP's implicate DIC as a potential etiology. Limited test capability in the horse has limited further evaluation of this hypothesis. We have developed an assay for equine protein C activity,our normal range being 70-60% (Mean+/- 2SD). We studied 12 horses with the disease for 5 consecutive days,drawing 1 blood sample per day. Our expectation was that protein C levels would decrease.if DIC was significant,as would be expected in humans. No significant decrease was noted in any horse. However there was a significant increase in the protein C levels beyond the upper limit of the normal range in 10 of the 12 horses by the third day. Five of the 10 horses maintained this elevation beyond the 5th day. Thus protein C changes were more consistent with an acute phase reactant response,rather than reflecting the decrease we anicipated,if the equine DIC parallels human DIC. We are measuring other acute phase reactants to see if equine protein C parallels those. Since our assay is still being evaluated,more data needs to be obtained in this and other equine disease states before any definative role for protein C in equine pathology can be determined. In our laminitis horses,we are devolping assays for antithrombin III and plasminogen which should allow us to evaluate the disease state more completley for any involement of elements of intravascular thrombosis and fibrinolysis in the equine colic syndrome.
A DOG MODEL OF PERIPHERAL ARTERIAL THROMBOSIS: POSSIBLE PHARMACOLOGICAL CONTROL OF VASCULAR OCCLUSION IN A STENOTIZED FEMORAL ARTERY
Authors: Prosdocimi M , Finesso M , Zatta A
Published: August 2018

It has been described by several authors that a critical stenosis of a dog coronary artery causes cyclical blood flow variations (CBFV), which are caused by platelet plugging of the vessel. Little information is available on the mechanism of thrombus formation in a stenotized peripheral artery and on the pharmacological control of this phenomenon. Male beagle dogs were anesthetized with sodium pentobarbital, artificially ventilated and prepared for the recording of arterial pressure, heart rate and femoral blood flow. A cylinder of Lexan with internal diameter of 1.6-1.8 mm and a length of 2.0 mm was placed on the femoral artery near the flow probe. This procedure induced CBFV in about 15% of the experiments. If the arterial vessel was previously squeezed with a forcep at the site of stenosis, CBFV were present in 100% of the experiments. Femoral flow at its peak averaged about 30% of control flow and was characterized by a gradual decrease followed by spontaneous abrupt restorations. CBFV were rather constant for at least two hours in dogs without pharmacological treatment thus allowing, by measuring the changes in frequency and severity of CBFV, the evaluation of a drug action on thrombus formation after its intravenous administration. Heparin (50 I.U.Ag) was ineffective while ASA (20 mgAg) reduced or prevented CBFV in about 50% of the experiments. CBEV were always completely prevented by an inhibitor of thromboxane synthetase (UK 38485, 0.5 mgAg) r by chloropromazine (0.5 mgAg) and by a serotonin antagonist (ke-tanserin, 0.5 mgAg). On the other hand, dipyridamole (1.0 mgAg) and an alpha 1 antagonist (prazosin, 0.1 mgAg) were not effective. These results are quite similar to those previously reported in the coronary model of CBFV. They suggest some similarity between the process of thrombotic occlusion in different arterial districts and emphasize the importance of thronboxane and serotonin in this particular model of vascular occlusion. Moreover, the striking increase in the percentage of animal showing CBFV after vessel damage is in keeping with the view that vessel wall properties are essential modulators in the process of arterial thrombosis.
PLATELET ASSOCIATED IMMUNOGLOBULINS IN PATIENTS WITH NON - HODGKIN'S LYMPHOMAS
Authors: Lawandowski K , Zawilska K , Komarnicki M , Zozulińsks M
Published: August 2018

Levels of platelet associated IgG and IgM were studied in 42 patients with non-Hodgkin'a lymphomas / NHL / using rocket inaunoelectrophore8i8 in agarose gel . Twenty percent of patients with NHL had elevated PAIgG and fifty percent had elevated PAIgM levels.

Both PAIgG and PAIgM ware strongly correlated with the extend of disease . When patients with NHL and elevated PAIgM levels were treated by chemotherapy , PAIgM levels normalized aa tumor load diminished . No similar correlation axsited between response to the therapy and levels of PAIgG in patients with NHL. These data suggest that PAIgM may be useful as a marker for disease activity and may be an early indicator of relapse when measured periodicaly in patients who have completed therapy.
CHARACTERISATION OF PLATELET AGGREGATING MATERIAL EXTRACTED FROM HUMAN LUNG ADENOCARCINOMA CELL LINE WHICH METASTASIZED IN NUDE MICE S,C, IMPLANTATION
Authors: Inufusa H , Sagara N , Nakano K , Yasutomi M
Published: August 2018

It has been reported in animal experimental system that Platelet Aggregating Material (PAM) of cancer cell play important role in cancer metastasis. Many human cancer cell lines has been also studied the platelet aggregation activity of PAM. But correlation between the platelet aggregation activity and metastatic potential of human cancer cells were usually unkncwn. We established a human lung adenocarcinoma cell line KUM-LK-2 which produce spontaneous lung metastasis when cells implanted into subcutaneous of nude mice. PAM was extracted from KUM-LK-2 cells following the method of D.Mbhanty and P.Hilgard. Platelet aggregation study of PAM was performed by human Heparinized platelet rich plasma useing NIKO Hematracer PACT2D. Character of PAM were examined by physical and chemical treatment. KUM-LK-2 PAM shew 80% of platelet aggregation in maximum after 150 sec lag time, and aggregation was not found by Citrated PRP.

It is concluded that PAM is high moleculer protein and contain Phospholipid and aggregation activity is concerning with ATP composition of platelet. PAM dose not contain Fibrinogen and Sialic acid, and not concern with Throrriboxan composition. This study is first case of platelet aggregation activity of PAM extracted human canoer cells which contain metastatic potential.