bluuebunny/crossref_metadata_2025 huggingface dataset:
DEMONSTRATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (scu-PA) ON HUMAN PLATELET MEMBRANES
Authors: Park S , Harker L A , Levin E G
Published: August 2018

A role for platelets in enhancing fibrinolysis has been suggested. To elucidate the nature of fibrinolysis-accelerating effect of human platelets, different fractions of the platelets were tested for fibrinolytic activity by the fibr:i.n plate method in the presence of plasminogen and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography. The platelets were washed in Tyrode’s buffer containing 0.5 ug/ml prostaglandin Ef, 1 mM ethylenediaminetetraacetic acid (EDTA), and 1 mM phenylmethylsulfonyl fluoride by albumin density gradient separation and gel filtration techniques. Platelet membranes were isolated by differential centrifugation after disruption of the platelets by ultrasonication. The sedimented platelet membranes demonstrated plasminogen activation with subsequent fibrinolysis which was completely abolished in the presence of affinity-purified antibody to high molecular weight urokinase-type plasminogen activator (u-PA). Minimal u-PA activity was observed associated with the supernatant from sonicated platelets indicating that most of the detectable u-PA was membrane-bound. Fibrin autography following SDS-PAGE of the platelet membrane fraction revealed a lysis zone at the molecular weight level of 54,000. Single chain nature of the u-PA was demonstrated by immunoblot analysis of the platelet membrane after SDS-PAGE under reducing conditions. Treatment of the platelets with 1.2 u/ml alpha-thrombin eliminated membrane-associated u-PA activity and probably resulted from the cleavage and inactivation of scu-PA. u-PA activity was quantitated by the 125I_fibrin plate method: values in the range of 0.15-0.25 mU/109 platelets were observed. Most of the activity was liberated from the platelet membrane by exposure to 3 M KC1 or 0.1 M glycine, pH 2.3, but not to 10 mM EDTA. We conclude that human platelets have scu-PA on their membranes that may play a role as a carrier of scu-PA in blood circulation. Binding studies are in progress.
ULTRASTRUCTURAL LOCALIZATION AND CHARACTERISATION OF VON WILLEBRAND FACTOR (VWF) AND TISSUE PLASMINOGEN ACTIVATOR (t-PA) IN ENDOTHELIAL CELLS (EC) AND MEGAKARYOCYTES (MK)
Authors: Jeanneaus Ch , Sultan Y
Published: August 2018

The reciproqual localization of VWF and t-PA in EC and MK was analysed in EM using immunocytochemical techniques. In parallel, PAGE electrophoresis, zymographic analysis and immuno-blotting were also performed in cell extracts using specific polyclonal and monoclonal antibodies against VWF and t-PA. By immunofluorescence, VWF showed a dense granular pattern in EC, MK and platelets, although no fluorescence was observed with specific antibodies to t-PA. In contrast, at optical level, with peroxidase or alcaline phosphatase conjugated antibodies, t-PA antigen in EC, MK and platelets appear lightly distributed in the cytoplasm. More precise localization of both antigens was studies in EM after incubation of ultrathin sections with specific colloidal gold coupled antibodies. VWF was detected by specific antibodies in storage granules : γ -granules in MK and Weibel-Palade bodies in EC only reacted with specific antibodies when the storage granules were opened by thin section ; VWF antigen was associated with the tubular structures of these granules. In EC gold or peroxidase coupled antibodies showed the presence of t-PA antigen in rough ergastoplasmic reticulum (RER) saccules and total absence of this antigen in granule structures. After thrombin stimulation, stained vesicles revealed t-PA in the cytoplasm, vesicles which rapidly disappeared after being released. In cultured MK, t-PA was detected by immunoperoxidase in perinuclear cisternae and in small vesicles near the Golgi complex, suggesting the synthesis of t-PA by MK. Concommitantly to appearance of the t-PA, fibrinolytic activity was detected. Immediatly after platelet production fibrinolytic activity disappeared probably by association with an inhibitor. MK and EC extracts after electrophoresis, immunoblotting and zymographic analysis revealed the presence of biologically active t-PA at the same position. Thus the cells have a basal level of active t-PA which can also be seen in intact cells when deposited on fibrin films. However the fibrinolytic activity of t-PA is inhibited in absence of VWF as it is the case in EC of patients with severe forms of VW disease although t-PA antigen is normally present.
ABNORMAL EXPRESSION OF VON WILLEBRAND FACTOR BY ENDOTHELIAL CELLS FROM A PATIENT WITH TYPE IIA VON WILLEBRAND’ S DISEASE
Authors: Levene Richard B , Booyse Francis M , Chediak Juan , Zimmerman Therodore S , Livingston David M , Lynch Dennis C
Published: August 2018

Studies were conducted to characterize the biosynthesis of von Willebrand factor (vWf) by cultured endothelial cells (EC) derived from the umbilical vein of a patient with type HA von Willebrand’ s disease. The patient’ s EC, compared with those from normal individuals, produced vWf which had decreased amounts of large multimers and an increase in rapidly migrating satellite species, features which are characteristic of plasma vWf from patients with type IIA von Willebrand’ s disease. The typQ IIA EC produced a full spectrum of vWf multimers in both cell lysates and post-culture medium, although the relative amounts of the larger species were decreased. The large multimers were degraded in conjunction with the appearance of rapidly migrating satellites which contained =170 kDa proteolytic fragments. Kinetic studies demonstrated that the =170 kDa species is not a primary translation product Normal metabolically labeled vWf, incubated with either the patient’ s EC or medium conditioned by these cells, was not similarly degraded. These results demonstrated that this patient’ s clinical phenotype is due to abnormal proteolysis and not to a primary failure of subunit oligomerization. Moreover, the increased degradation is attributable to increased proteolytic sensitivity of an abnormal vWf molecule rather than to pathologically elevated levels of endogenous proteases. Experiments using monoclonal antibodies which recognize either N- or C-associated epitopes have localized the defect to the N-terminal portion of the vWf molecule, which is believed to be involved in the inter-dimer polymerization reaction. The type DA EC also contained a single vWf mRNA species which comigrated with that from normal EC. However, the type HA EC contained 8-10 fold more vWf mRNA than their normal counterparts. These results suggest that the functional defect in this patient is caused by a subtle mutation in the vWf coding sequence leading to increased proteolytic sensitivity of its protein product
PLATELET DEPOSITION ON ARTIFICIAL SURFACES: EFFECT OF PROTEIN PRECOATING, ENDOTHELIAL CELL COVERAGE AND SHEAR RATE
Authors: Poot A , Dekker A , Beugeling T , Bantjes A , Van Aken W G
Published: August 2018

In the present study the in vitro capillary perfusion system according to Cazenave was used. This system consists of a capillary tube connected to a syringe containg the perfusate which consisted of washed human platalet (111 In-labeled or native) and washed red cells in Ca2+ /Mg2+ -containing Tyrode-albumin buffer. Perfusate flow is controlled by a syringe pump. Polyethylene tubes (PE, 0.75 mm ID, 25 cm long) were precoated with purified human von Willebrand factor (vWF), fibrinogen (Fb), fibronectin (Fn), immunoglobulin G (IgG), albumin (HSA) or plasma. Compared with uncoated PE, platelet deposition increased after precoating with vWF, Fb or Fn, and decreased by pre adsorbed IgG, HSA or plasma. Platelet deposition was positively correlated with shear rate only on surfaces precoated with vWF, Fb or Fn. Scanning electron microscopy showed platelet aggregates on IgG—coated PE, whereas on all other surfaces single adherent platelets were observed. Complete platelet spreading was only observed after precoating with Fn. In contrast with PE coated with vWF, Fb or Fn, platelet adhesion on uncoated PE did not increase further after 5 minutes of perfusion probably due to passivation of the surface by albumin present in the perfusate. This could be overcome by addition of Fb to the perfusate. Perfusates prepared in this way were used for studying the effect of human endothelial cell (HEC) coverage on platelet deposition. HEC were seeded at different densities in PE tubes precoated with partially purified Fn. Platelet deposition decreased with 2increasing HEC coverage, and was negligible at 60,000 HEC/cm . Our results indicate the applicability of this perfusion model for the in vitro testing of artificial surface thrombogenicity.
FIBRINOLYTIC PEPTIDES IN ARTIFICIAL HEART RECIPIENTS
Authors: Corrigan J J , Jeter M , Smith R G , Levinson M , Copeland J G
Published: August 2018

Patients who are recipients of total artificial heart (TAH) implants develop significant thromboembolic disease (TED). These events cause significant morbidity and are the leading impediments to the use of the TAH. Recently, molecular markers of activation of the hemostatic mechanism have been employed in patients with other causes of thromboembolic disease for earlier detection and for assessing management. We serially measured three fibrinolytic markers (B-beta 1-42 and B-beta 1542 fragments and crossed-1inked D-dimer, XDP) in 3 recipients of the Jarvik-7 artificial heart. The markers were assayed in plasma samples using monoclonal antibodies using ELISA methodology. Normal ranges for our lab are: B-beta 1-42, 3-14 picomoles/ml; B-beta 15-42, 15-32 picomoles/ml; and XDP 6-26 ng/ml. The TAHs were in place for 8, 9, and 252 days. Two patients died and two had strokes. Two of the 3 artificial devises had thrombosis at the time of removal. XDPs rose significantly at the time of TED (>1000 ng/ml). A significant rise in B-beta 1-42 and 15-42 was noted prior to and during the TE events. The ratio's of 1-42 to 15-42 did not change suggesting that these levels were a result of thrombin activity with secondary fibrinolytic activation and not from primary fibrinolysis. These preliminary data suggest that the use of hemostatic molecular markers may be useful for the detection of TED and in assessing control of the hypercoagulable state in TAH recipients.
AMINO ACID SEQUENCE OF THE THROMBIN-BINDING SITE ON PLATELET GLYCOPROTEIN Ib
Authors: Yamamoto K , Kawasaki H , Suzuki K , Tanoue K , Kosaki G , Yamazaki H
Published: August 2018

Amino acid sequence of the thrombin-binding site on platelet glycoprotein (GP) Ib was determined and the effects of the synthesized analogous peptides on thrombin-induced aggregation were studied. Crude platelet membrane fraction solubilized with 5mM dithiothreitol and 0.2% Tween 20 was applied to a WGA-Sepharose. The bound fractions were eluted with 0.3M N-actylglucosamine, then applied to a TM60 ( a monoclonal antibody against GP Ib)-Affi-gel column. Only one GP was bound to the column and was eluted with 50mM glycine-HCl, pH3.0, containing 0.2% Tween 20. SDS-PAGE showed a single band of 130kDa, corresponding to GP Ib alpha chain (GP Ibα). When the purified GP Ibα was digested with trypsin, two fragments (94kDa . and 43kDa) were obtained. The 43kDa fragment was shown to bind to both affinity colomns of TM60- and thrombin-Affi-gel, while the 94kDa fragment did not bind to either Affi-gel. When the same experiment was performed using chymotrypsin, three fragments (94kDa, 45kDa and 39kDa) were observed. On TM60- and thrombin-Affi-Gel columns, the smaller fragments (45kDa and 39kDa) were bound to both columns. However, on. thrombin-Affi-Gel column, 39kDa fragment was found in both unbound and bound fractions. It showed that the 45kDa fragment interacts with thrombin with a higher affinity than the 39kDa fragment. These results indicate that the thrombin-binding site is located on the "tail" portion of GPIba, especially on a chymotryptic cleavage site.

Then 43kDa tryptic fragment was purified and its partial amino acid sequence was analyzed using gas phase amino acid sequence analyzer. Based on its amino acid sequence, several analogous oligopeptides were synthesized. Three peptides (#1-10, #11-23, #17-28) inhibited 0.05 U/ml thrombin-induced aggregations of washed human platelets in dose-dependent manners. IC50 were in the range of L50-550μM for each of these peptides.
IDENTIFICATION OF GLYCOPROTEIN Ib8 AS THE Mr = 24,000 PLATELET POLYPEPTIDE PHOSPHORYLATED BY AGENTS THAT ELEVATE CYCLIC AMP
Authors: Fox J E B , Reynolds C C , Boyles J K , Abel R A , Johnson M M
Published: August 2018

Platelet function is inhibited by agents that elevate intracellular cyclic AMP concentrations, presumably as a result of the cyclic AMP-stimulated phosphorylation of intracellular proteins. Polypeptides that become phosphorylated are of Mr = 250,000, Mr = 51.000 (P51), Mr = 36,000 (P36), Mr = 24,000 (P24), and Mr = 22.000 (P22). The Mr = 250,000 polypeptide is actin-binding protein, but the identity of the other polypeptides 1s unknown. In the present study, we identified the P24 polypeptide. Platelets were radiolabeled with [32P]P1 and then Incubated for 2-5 min in the presence or absence of 5 μM prostaglandin E1 (PGE1). The PGE1-induced phosphorylation of P24 was detected on autoradiograms of SDS-gels. Since P24 has been shown to be membrane-associated, its molecular weight was compared with those of known membrane proteins. P24 comigrated with the β-chain of purified GP Ib on reduced gels (Mr = 24,000) and also on nonreduced gels (when GP Ibβ is disulfide-linked to GP Ibα and migrates with Mr = 170,000). Like GP Ibβ, P24 was associated with actin filaments in Triton X-100 lysates. Both GP Ibβ and P24 were selectively associated with filaments of the membrane skeleton and were released from filaments when the Ca2+-dependent protease was active. Antibodies against GP Ib immunoprecipitated P24 from platelet lysates. Finally, exposure of Bernard-Soulier platelets (that lacked GP Ib) to PGE1 resulted in phosphorylation of actin-binding protein, P51, P36, and P22, but not P24. We conclude that P24 is GP Ibβ. To determine whether phosphorylation of GP Ibβ is responsible for the inhibitory effects of PGE1 on platelets, we compared the action of PGE1 on control platelets with that on Bernard-Soulier platelets. One of the ways in which PGE1 inhibits platelet activation is by inhibiting the polymerization of actin. While PGE1 inhibited actin polymerization in control platelets, it did not in Bernard-Soulier platelets. We conclude that GP Ibβ is phosphorylated by agents that elevate cyclic AMP and that phosphorylation of this glycoprotein results in inhibition of platelet function.
A DEFINITION OF HEPARIN ANTICOAGULANT POTENCY APPLICABLE TO ALL HEPARINS AND HEPARIN-LIKE SUBSTANCES AND ITS PRACTICAL APPLICATION IN ASSAYING HEPARIN
Authors: Jackson Craig M
Published: August 2018

Heparins increase the rate of inactivation of proteinases by antithrombin without being consumed in the inactivation reaction. The anticoagulant activity of any heparin or heparin preparation is thus determined by the increase in the inactivaton rate which it produces. This rate increase is dependent on the concentration of the heparin in the sample and on some now well known structural properties of the individual heparin molecules that produce high affinity for antithrombin . All proteinases are not inactivated by antithrombin equally rapidly in the absence of heparin, nor are heparins and heparin derivatives of different molecular weight equally effective in the inactivation of the same proteinase. Under appropriate conditions, the observed rate constant (kObs) for the heparin catalyzed proteinase inactivation reaction is simply related to the intrinsic potencies and concentrations of the individual high affinity heparin molecules in the sample. The intrinsic potency of a high affinity heparin molecule is the efficiency with which it catalyzes the inactivation of the particular proteinase, e.g. Factor Xa or thrombin, i.e., it is a second order rate constant, (designated k*) . After k* has been determined from kobs for a known heparin or heparin preparation and a particular proteinase, the concentration of heparin in an unknown sample can be calculated from the equation

[H] = [HAT] = kobs/k*

In general terms, the appropriate conditions, i.e.,the antithrombin and proteinase concentrations, the pH, and ionic strength, required for this equation to be used are those conditions for which all of the high affinity heparin is bound to the antithrombin and pseudo first order kinetic behavior occurs. At very low heparin concentrations, a correction for the inactivation of the proteinase by antithrombin alone is necessary, but is easily made.

Supported by Organon Teknika Corporation and an Established Investigator Award from the American National Red Cross
INDUCTION OF OSTEOPOROSIS IN RATS BY STANDARD HEPARIN AND LOW MOLECULAR WEIGHT HEPARIN
Authors: Mätzsch T , Berggvist D , Hedner U , Nilsson B , Østergaard P
Published: August 2018

Long-term treatment with heparin can induce osteoporosis. This complication is suspected to be related to the dosage of heparin rather than to duration of therapy, but the mechanism by which heparin induces osteoporosis is unknown. In a previous study we reported the same degree of reduction in mineral bone mass in rats after treatment with 2 IU heparm/g bw for 33 and 65 days (Thromb Haemostas 1986,56:293-4). Using the same animal model we compared the effect of a high-dose standard heparin (SH) and a low molecular weight heparin (LMWH) in a high and a low dose on the mineral bone mass in the femur of rats.

Method: 60 female Wistar rats (207±1.8 g) were randomly allocated to treatment with either 2 Xal U/g bw of standard heparin (SH), 2 Xal U/g bw of LMWH ("high-dose"), 0.4 Xal U/g bw LMWH ("low-do-se") or placebo (saline). A standard sodium salt heparin of porcine origin was used, and the LMWH was an enzymatically depolymerized pork mucosal heparin (LHN-1, mean MW 4900 D). Treatment with s.c. injections was continued for 34 days. 24 hours after the last injection the rats were sacrificed and the carefully cleaned femora weighed in air and in water under standardized conditions. Volumes and densities were calculated from the weights. The bones were then incinerated for 48 hours at 600°C and weighed again to determine the ash content (expressed as ash weight per ml of unashed femur volume).

Results: There was a significant decrease in ash content (p<0.01) and density (p<0.01) of the femora in all heparin treated groups as compared with controls. High-dose LMWH caused the same reduction in bone mineral mass as standard heparin. Treatment with low-dose LMWH resulted in a significantly less pronounced reduction in ash content (p<0.001) and density (p<0.05) of the femora when compared with high-dose standard heparin and high-dose LMWH.

CONCLUSION: Daily injections of 2 Xal U/g body weight of standard heparin or low molecular weight heparin for 34 days causes the same degree of reduction of mineral bone mass in rats. The reduction of mineral bone mass in rats by treatment with low molecular weight heparin is dose dependent.
FAMILIAL TYPE I PROTEIN S DEFICIENCY ASSOCIATED WITH SEVERE VENOUS THROMBOSIS. A STUDY OF FIVE CASES
Authors: Boyer-Neumann C , Wolf M , Amiral J , Guyager A M , Meyer D , Larrieu M J
Published: August 2018

Protein S deficiency has been demonstrated in 5 members from the same family with a history of severe recurrent venous thrombosis over three generations. The propositus, a 16 year old female, had a first spontaneous thrombotic episode at age 15. Phlebography revealed a total obstruction of her left ilio-femoral vein with an extension to the vena cava. She was treated with heparin followed by oral anticoagulant therapy. The four other affected members (mother, aunts and uncle of the propositus) had also presented recurrent thrombosis with onset at a young age. The grandfather, not tested, had died from massive pulmonary embolism at age 54. The immunological assay of protein S was performed in plasma by Laurell, using a monospecific antiserum to human protein S, or by an ELISA, using a kit from Diagnostica Stago (Asserachrom Protein S). In order to separate free protein S, the functionally active form, from protein S complexed with C4-binding protein, plasma was adsorbed with 3.75 % polyethyleneglycol (PEG 6000). Following PEG precipitation, the levels of free protein S antigen remaining in the supernatant were quantitated by the usual immunological methods. In addition, two-dimensional immunoelectrophoresis (DDIE) also provided information on the distribution of both forms. In plasma protein S levels were decreased (40 to 55 % of the normal range) in two untreated patients and lower levels (17 to 20 96) were observed in the three others, including the propositus, who were under dicoumarol therapy. In PEG treated-plasma, protein S was undetectable (less than 5 %) in all patients, indicating a lack of free protein S. This was confirmed by DDIE : whereas protein S migrated as two distinct peaks, corresponding to free and complexed protein S in normal plasma, only a single peak of complexed protein S was observed in all affected patients. These results clearly demonstrate a total lack of free protein S which appears to be responsible for the thromboembolic disorder in this family as there was no deficiency of the other plasma inhibitors (antithrombin III, heparin cofactor II and protein C). According to the classification recently proposed by Comp et al., this family belongs to type I protein S deficiency, with an autosomal dominant mode of inheritance.
A CELLULAR RECEPTOR FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR
Authors: Belin D , Baccino D , Wohlwend A , Estreicher A , Hurate J , Vassalli J-D
Published: August 2018

Recent cell biological and biochemical studies on the urokinase-type plasminogen activator (u-PA) have revealed an unsuspected property of this protein: it binds with high affinity and specificity to the plasma membrane of a number of cell types. Hence, while the interaction of tissue-type plasminogen activator (t-PA) with fibrin suggests a preferred role for this enzyme in the maintenance of fluidity of the extracellular milieu, the cellular binding of u-PA results in the focalisation of plasmin generation to the close environment of the cell surface; this appears as an optimal configuration if u-PA is to participate in the enzymatic events required for cell migration.

The available information on the cellular binding of u-PA can be summarized as follows:

1. Human monocytes-macrophages, monocyte-like cell lines, fibroblasts, and a variety of other cell lines all express u-PA binding sites. The number of u-PA binding sites on a given cell type may vary as a function of the functional state of the cells. In some cases all sites are occupied by “endogenous” u-PA.

2. Binding does not require u-PA activity, and prou-PA binds with the same affinity as does the active enzyme.

3. The Kd for u-PA binding is between 1 and 10×10-10 M. The binding site appears to be specific for u-PA.

4. Binding requires the presence of the A chain of u-PA; the growth factor module of the A chain is involved in this interaction.

5. Bound enzyme does not dissociate readily, nor is it rapidly endocytosed; most importantly, it retains catalytic activity.

Studies in progress are aimed at further defining the u-PA determinants responsible for binding. In this context it is noteworthy that there is a tight species specificity of binding: human and murine u-PA, for instance, bind only to cells of the homologous species. Characterization of the u-PA binding site suggests that it is an integral membrane protein that includes at least one Mf 50.000 polypeptide chain.

In addition to allowing for the peri-cellular focalisation of u-PA catalysed proteolysis, expression of the u-PA binding site provides a mecanism whereby one cell type can acquire membrane-bound u-PA activity following secretion of the (pro)enzyme by another cell population. A striking example of this is the binding of u-PA, synthesized by the epithelial layer of the male genital tract, to the head region of murine spermatozoa.
CLINICAL RESEARCH ON SINGLE-CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR (SCU-PA) IN GERMANY. RESULTS IN PATIENTS WITH ACUTE MYOCARDIAL INFARCTION (AMI)
Authors: Welzel D , Wolf H
Published: August 2018

The German multicenter trial on scu-PA so far comprises 63 patients with AMI that received scu-PA intravenously in increasing dosages from 2mio units (15 mg) up to 9mio units (69 mg), combined in a bolus injection and a subsequent infusion over one hour. Another 18 patients received 200.000 IU urokinase combined with scu-PA 6.5mio units/h. All patients were anticoagulated prior to the lytic therapy with a bolus injection of 5000 IU heparin, and subsequently received heparin in a dosage maintaining the aPTT-prolonga-tion at 60 to 80 sec. The application of scu-PA up to 4mio units lead to a reopening of the coronary artery in only 5 out of 22 patients. Under the dosage regimen of 6.5mio and 9mio units the reopening rates were 50% and 70% respectively. The reopening time varied between 40 and 60 min. At the end of the infusion the fibrinogen levels were between 60% and 90% of baseline. In 10 out of 13 patients given the UK/scu-PA combination recanalisation was obtained within 22 ± 13,6 min. In another 5 patients on UK/scu-PA patency was controlled 90 min. after thestart of lytic therapy; 3 of these showed patent coronary arteries. Fibrinogen dropped by 25% on an average. No bleeding complications were observed. The subjective tolerance proved excellent. Reocclusion at 24 hours was evaluated in 18 patients but only found in one. On the basis of the evidence given so far the combination UK/scu-PA appears to be promising.
DIAGNOSIS OF DEEP VENOUS THROMBOSIS: NON-INVASIVE VS INVASIVE DIAGNOSTIC PROCEDURES
Authors: ten Cate J W , Huisman M V , Buller H R
Published: August 2018

The clinical diagnosis of deep vein thrombosis (DVT) in symptomatic patients is unreliable. The need for objective diagnostic tests is widely acknowledged. Contrast venography in experienced hands is considered to be the reference method. This method is invasive, uncomfortable to the patient, not easily repeatable and expensive.

For this reason several non-invasive tests have been developed and evaluated recently. Of the non-invasive tests impedance plethysmography (IPG) has been thoroughly evaluated in properly designed prospective clinical trials. Serial IPG in symptomatic outpatients is safe and effective. It was shown in longterm follow-up that anticoagulant treatment could be safely withheld in over 500 patients with repeatedly normal IPG (0.3-0.6% recurrence DVT). The specificity for proximal DVT was 92%. The feasability of IPG in symptomatic outpatients was over 95%. The safety of withholding anticoagulant treatment in symptomatic inpatients with a serial normal IPG is an unresolved issue. Preliminary results show that a similar sensitivity might be obtained in inpatients, however, the feasability was lower (87%).

Doppler ultrasound has been studied extensively, however, there is a great variation in reported sensitivity for proximal DVT due to the lack of objective diagnostic criteria and the safety of withholding anticoagulant treatment in patients with serial normal Doppler tests results is not es tablished.

strain gauge plethysmography has not been evaluated properly and therefore awaits further studies. 125I-fibrinogen legscanning has been shown to be sensitive for calf vein thrombosis (over 90%). In combination with IPG this method is a safe and effective alternative to venography.

Radionuclide phlebography has never been evaluated in prospective clinical trials in a broad spectrum of symptomatic patients, and can therefore not be recommended for routine use.

It is concluded that presently the management of patients with clinically suspected DVT should be performed with the use of serial IPG, IPG in combination with 125I—fibrinogen legscanning or contrast venog raphy.
A RANDOMISED DOSE RANGING STUDY OF TWO-CHAIN TISSUE-TYPE PLASMINOGEN ACTIVATOR (BW-t-PA) IN MYOCARDIAL INFARCTION: IN VITRO MONITORING
Authors: Kluft C , McNeill A , Adgey A A J , Rijken D C , Nieuwenhuizen W , Cohen A F
Published: August 2018

In a randomised study, thirty patients with myocardial infarction received either 20, 50 or 100 mg recombinant BW-t-PA in 90 minutes. Blood samples were taken just before the infusion, and after 90 min. In vitro effects of t-PA were prevented by addition of 250 μg/ml polyclonal t-PA antibodies, which were shown to block in vitro actions of up to 5 μg t-PA/ml during plasma handling.

There were dose related (MIU/kg/90 min) decreases of α2 -antiplas-min (AP, functional assay), plasminogen (PLG, streptokinase assay) and fibrinogen (FBG, Clauss) at 90 min (lineair regression: r = 0.67; 0.55 and 0.40, n = 30, all p < 0.05, resp.). Decreases for the 100 mg dose were for the respective components: 70 ± 10 (SD)%, 28 ± 7% and 33 ± 17% of pretreatment levels. The extent of the AP changes appeared to be the most sensitive parameters and these changes correlated with those in PLG and FBG. Pretreatment values of FBG were elevated (137 ± 37%) and at 90 minutes were 100 ± 23% of pooled plasma. Decreases in FBG were significantly (p < 0.001) correlated with absolute pretreatment levels, indicating preferential reductions of elevated levels of fibrinogen.

Using our new enzymimmunoassays based on monoclonals for degradation products of fibrinogen (FDP), fibrin (FbDP) and the total (TDP), these products were measured in plasma (not serum). Before infusion 14 patients showed TDP levels (range: 0.5 - 2.3 μg/ml); at 90 min 29 patients (0.6 - 13 μg/ml. The increase in TDP accounted for only 0.75% (range up to 5.3%) of the apparent FBG reduction (Clauss) at 90 min. Detectable FbDP were generated in 24 patients at 90 min. For the two highest dosages the level (median 2.0 μg/ml) was significantly higher than at the dosage of 20 mg (median 0.85 μg/ml), indicating weaker fibrinolytic effect of t-PA at the lowest dosage. To evaluate the fibrinolytic/fibrinogenolytic effects of the t-PA the ratio FbDP/FDP was calculated. For the 50 mg dosage all ratios were ≥ 1; for 100 mg only 6 patients showed a ratio ≥ 1 at 90 min. This may indicate relative specific increase of fibrinogenolysis at higher dosages.
A RANDOMISED DOSE RANGING STUDY OF DOUBLE CHAIN TISSUE PLASMINOGEN ACTIVATOR (BW t-PA) IN ACUTE MYOCARDIAL INFARCTION: CLINICAL RESULTS
Authors: McNeill A D , Cunningham S R , Koster R , Bucknall C , Sponzilli C , Kluft K
Published: August 2018

Despite extensive experimental use of t-PA in patients with acute myocardial infarction (AMI) a randomised dose ranging study has not yet been published. Fifty-five patients were randomised to BW t-PA 20 mg, 50 mg or 100 mg administered over 90 minutes. They received the drug 2.3 hours (range 0.3-4.3 hours) after the onset of AMI and underwent coronary arteriography to determine perfusion grade of the infarct related artery at 90 minutes. Responders were defined by TIMI perfusion grades 2 or 3. Response rates were:

A logistic model fitted to the data showed the probability of reperfusion to increase in a dose related manner to 70%. Fibrinogen concentrations were measured in 30 patients and decreased to 83.6% of the preinfusion value at 90 minutes after 20 mg, to 76.2% after 50 mg and to 67.1% after 100 mg. There was a significant correlation between the dose of BW t-PA in megaunits/kg and consumption of fibrinogen at 90 minutes (p<0.05). This study indicates that thrombolysis with double chain t-PA proceeds in a dose related manner and that systemic fibrinogenolysis appears to be mild.
THE COMBINED EFFECT OF u-PA AND t-PA ON A CANINE CORONARY THROMBUS MODEL
Authors: Nobuhara M , Ashida Y , Kato K , Horisawa Y , Kubota H , Yajima T , Shliraki S
Published: August 2018

The combination experiments of both PAs have been performed in in vitro and in vivo to know whether they act synergistically or not.

In in vitro experiments the additive combined effects of u-PA and t-PA were observed on human plasma clot lysis. In in vivo experiments, on a canine coronary thrombus model with a thrombin thrombus in LAD, the combined administration of u-PA and t-PA ( 1 × 104 IU/kg/30') gave significant thrombolysis without enhancing the reduction of Fbg, Pig and α2-PI.

In order to study the mechanism of in vivo synergistic thrombolysis, changes of plasma and blood dynamic viscosities by administration of u-PA and t-PA were investigated. Whole blood dynamic viscosity decreased significantly in both u-PA and the combination groups, while plasma viscosity was not changed. This lowered blood viscosity was rather notable at the lower shear rate condition than at the higher shear rate.

From these results it can be expected that u-PA could suppress the elevation of blood dynamic viscosity under the condition of retained blood flow, while t-PA might provide a thrombus specific lysis without any bleeding complications. And the synergism of u-PA and t-PA demonstrated here have a greate advantage on not only thrombolysis but also on lowering of blood viscosity in the coronary artery.
DISSOCIATION BETWEEN DDE LEVEL AND RECANALIZATION IN PATIENTS UNDERGOING THROMBOLYTIC THERAPY FOR MYOCARDIAL INFARCTION. A POSSIBLE EXPLANATION
Authors: Mirshahi Mc , Soria J , Soria C , Xi Ma , Mirshahi M , Samama M , Caen J P
Published: August 2018

The aim of this work was to determine whether plasma fibrin degradation products (FbDP) level may be used as a marker of recanalization in patients undergoing thrombolytic therapy for myocardial infarction. Samples were collected in patients treated by a 90 minutes infusion of 60 mg recombinant tissue type plasminogen activator (rtpA). Plasma FbDP (DDE complexes) were specifically and reliably determined by Elisa using a personal monoclonal antibody. Additional identification of FbDP and fibrinogen derived fragment D was performed by SDS PAGE followed by their detection using an anti D neo monoclonal antibody.

Fragment D was only evidenced up to the end of rtpA infusion. Concerning FbDP levels : prior to treatment, each patient presented FbDP levels within the normal range. After rtpA infusion, FbDP were increased in almost all cases, but our results clearly demonstrated that this increase was not correlated with coronary recanalization : on one hand, FbDP (D dimer and YD complex) were increased at the end of rtpA infusion in patients for whom the treatment was efficient as proved by angiography but also in five of seven patients who did not reperfuse. On the other hand, in most cases even in patients presenting a good recanalization at the end of rtpA infusion, a further increase in FbDP was noted even 6 hours after the end of the infusion.

It is therefore suggested that FbDP came either from a degradation of fibrin from different sources that coronary thrombus or coagulation in coranary artery was going on. A careful analysis of a possible activation of coagulation during thrombolytic therapy is under investigation.

In this way in patients under thrombolytic therapy, we propose to investigate the balance between coagulation and fibrinolysis by determining at the same time the level of plasma fibrinopeptide A and DDE complexes.
STREPTOKINASE-INDUCED, ANTIBODY-MEDIATED PLATELET AGGREGATION: A POTENTIAL CAUSE OF CLOT PROPAGATION IN VIVO
Authors: Vaughan D E , Loscalzo J
Published: August 2018

We identified a patient who exhibited paradoxical propagation of thrombus coincident with the administration of intracoronary streptokinase (SK) that was mediated by anti-SK antibodies. The patient had not been treated with SK in the past and had a plasma SK-neutralizing capacity of 160 U/ml. Using platelet-rich plasma (PRP) obtained from the patient, we found that SK initiated spontaneous platelet aggregation and secretion in vitro. Aggregation was specific for SK and not induced by urokinase or tissue plasminogen activator. In 14 of 15 controls, no platelet aggregation was observed in PRP with addition of SK. The addition of plasma or purified IgG from our index case to the PRP of all 14 controls supported SK-induced aggregation. This aggre-gatory response was not inhibited by aprotinin. Using purified proteins in a washed platelet system, we found that platelet aggregation was dependent on the presence of SK, specific anti-SK IgG, plasminogen, and platelets. These data demonstrate that anti-SK antibodies can promote platelet aggregation, presumably by binding to platelet-bound plasminogen-SK complexes. These data also imply that some individuals may possess anti-SK antibodies that are capable of inducing platelet aggregation in vivo and, thereby, promoting clot propagation or thromboembolic complications. In the absence of adequate, specific screening, this observation argues for the use of nonimmunogenic thrombolytic agents in the emergent setting.
SYSTEMIC THRCMBOLYSIS WITHIN THE FIRST 3 HOURS OF ACUTE CORONARY THROMBOSIS : APSAC VS STREPTOKINASE : A RAMDCMIZED STUDY
Authors: Momier P , Sigwart U , Bachmnn F , Ferret C , Kapperiberger L
Published: August 2018

Intravenous administration of thrombolytic agents in acute myocardial infarction may be preferable to intracoronary thrombolysis for practiced, reasons and to reduce time to reperfusion. As compared to the streptokinase infusion, the bolus injection of APSAC may be even more rational. We have therefore compared the effects of one intravenous bolus injection of APSAC (30 units in 5 min) to an intravenous injection of streptokinase (1.500.000 units in 60 min) within the first 3 hours (mean 141 min for APSAC and 135 min for streptokinase) after the onset of symptoms associated with acute myocardial infarction. All patients received i.v. heparin at therapeutic dosage for 24 hours starting 3-4 hours after the onset of therapy.

14 patients were randomized to the APSAC group and 11 to the streptokinase group. Coronary angiography carried dout 2.5 hours after beginning of treatment revealed a patency rate (complete opacification of the vessel at selective injection) of 92% (13 of 14 patients) for APSAC and 63% (7 of 11 patients) for streptokinase. Both thrombolytic agents produced similar fall in plasma fibrinogen levels. One death occurred in the streptokinase on day 4 due to rupture of the anterior ventricular wall. No other side effects were life-threatening, there consisted of local bleeding at the punction side. We conclude that APSAC is at least as efficient as streptokinase and easier to administer in patients with acute myocardial infarction.
INTRAARTERIAL THROMBOLYSIS BY STREPTOKINASE-GLU-PLASMINOGEN
Authors: Pilger E J , Lammer J , Bertuch H , Steiner H
Published: August 2018

In order to predict usefulness of streptokinase (SK), urokinase (UK) and streptokinase-glutamin-plasminogen (SK-Glu-Plg) for intraarterial fibrinolysis, an in vitro test was designed. Fibrin plates with and without plasminogen were incubated with SK, UK and SK-Glu-Plg (in molar ratios of 1:1, 2:1 and 1:2). On the fibrin plates containing plasminogen the highest fibrinolytic activity was observed with UK; on the fibrin plates without plasminogen, SK-Glu-Plg in a molar ratio of 1:2 was superior. We concluded, that plasmin would be synthesized by SK and Glu-Plg. In order to examine the in vivo efficacy of these different fibrinolytic agents, 120 patients suffering from peripheral artery occlusion were randomized into three treatment groups for local thrombolysis. The technique of Hess and coworkers was used for local thrombolytic therapy. In group I local fibrinolysis was performed with SK (2500 IU/5 min), in group II UK (4000 IU/5 min) was used and in group III the lytic agent consisted of SK-Glu-Plg (2500 IU/5 min). The primary recanalization rate was equal in all groups: 86%, 89% and 83% in group I, II and III respectively. However the duration required for the procedure and thus the total amount of fibrinolytic agent used was significantly different (p < 0,001) between the three groups: 2,3 ± 1,4; 2,1 ± 1,2 and 1,1 ±0,8 hours for groups I,II and III, respectively (mean ± SEM) . We conclude that SK-Glu-Plg or plasmin itself has the highest efficacy as a fibrinolytic agent for intraarterial thrombolysis. Since the intra-thrombotic concentration of Pig is unknown in an individual patient an empirically chosen dose of SK of UK may either be to high or to low for optimal thrombolysis.
FOUR CASES WITH STABILIZATION OF UNSTABLE ANGINA PECTORIS BY THROMBOLYTIC THERAPY
Authors: Gulba D , Lichtlen P
Published: August 2018

Acute myocardial infarctions in the vast majority of cases are caused by coronary artery thrombosis at the site of complicated arteriosclerotic plaques. By several trials evidence has been given, that myocard can be preserved, when thrombolytic therapy is started within a short period after thrombotic coronary occlusion. Recently, angioscopic evidence has been given, that the unstable angina pectoris syndrome frequently is assciated with coronary artery thrombosis, too. Thus, thrombolytic therapy should be of comparable benefit for patients suffering from unstable angina pectoris syndrome. Up to now, we have treated four patients suffering from unstable angina pectoris syndrome (two with documented spontaneous reversible ST-segment elevations, two with newly complained recurrent nocturnal episodes of severe angina) with thrombolytic therapy (Pat. 1: 1.5 Mio IE Streptokinase; Pat. 2: 100 mg rt-PA; Pat. 3: 150 mg rt-PA; Pat. 4: 60 mg scu-PA plus 200 000 IE UK). After thrombolytic therapy, all four patients were free of symptoms for at least 60 h. Pat. 3 had recurrance of chest pain with spontaneous reversible ST-segment elevations on the third day after therapy. Pat. 1, 2, and 4 were without clinical symptoms until angiography and secondary intervention (angioplasty (PTCA) /bypass operation (CABG)). Cardiac catheterization was performed within one week after thrombolytic therapy. In all four patients, ischemia related coronary artery was patent at angiography. We conclude, that in unstable angina pectoris syndromes with newly developed nocturnal symptoms and/or spontaneous reversible ST-segment elevations in the ECG can be stabilized by thrombolytic therapy. After thrombolysis, however, recurrance of chest pain may be soon, and PTCA or CABG should be performed as soon as possible.
PLATELET FUNCTION ASSOCIATED WITH EXERCISE INDUCED MYOCARDIAL ISCHAEMIA: MODIFICATION BY COMBINED BETA-BL0CKER AND CALCIUM ENTRY BLOCKER THERAPY
Authors: McGill D , McGuiness J , Ardlie N
Published: August 2018

The relationship between platelet activation and exercise induced myocardial ischaemia is controversial, and the presence of measurable effects of anti-anginal drugs on platelet function requires further clarification. This study addresses these questions in patients with coronary artery disease (CAD), treated with metoprolol and nifedipine. Twenty seven clinically stable males aged 35 to 69 years (mean 53) with proven CAD, ceased all medications for 5 days, were maximally exercised on a treadmill, and then commenced treatment for 4 weeks. They were exercised to the same workload on treatment and again without drugs one week later. Blood samples were collected immediately before and after exercise in each of the three tests, to measure serum thromboxane B2 (TXB2). Myocardial ischaemia was assessed by ST segment depression. Statistical analysis was performed on paired and grouped data using the appropriate T-test. Baseline TXB2 levels were significantly lower in patients with exercise induced ischaemia, and this group had a significant increase in TXB2 production after exercise (Table). This increase was inhibited by therapy. .Those with a negative test had higher baseline TXB2 levels which not increase with exercise.

It is concluded that myocardial ischaemia is associated with a lower potential for TXB2 production. It is also associated with an exercise induced increase in TXB2 which is prevented by anti-ischaemic drugs. It is suggested that continuous platelet activation may occur in CAD patients with ischaemia, depleting the potential for TXB2 production.
EARLY SPONTANEOUS FIBRINOLYSIS IS RELATED TO THE EXTENSION OF ACUTE MYOCARDIAL INFARCTION
Authors: Hanss M , Rousson D , Touboul P , Dechavanne M
Published: August 2018

The relative importance of spontaneous fibrinolysis was studied during the early stage of acute myocardial infarction (AMI). Ten consecutive male patients (52.1 ± 5.7 years old) with AMI and without haemodynamic or rythmic complications were selected. Blood samples were obtained less than 5 hours (3.4 ± 0.8 hours) after the onset of symptoms. Enzyme linked immunosorbent assay procedures were performed to quantify the D-dimer antigen (Dd-Ag) and tissue plasminogen activator antigen (tPA-Ag) plasma levels. The creatine phosphokinase (CPK) peak level was measured in serum as an index of the AMI extension. Mean ± S.D. (range) levels were respectively 368 ± 342 (118-1100) μg/1 for Dd-Ag, 12.9 ± 9.4 (4.5 - 29.1) μg/1 for tPA-Ag and 1117 ± 856 (256-2800) U/l for CPK. Coefficient correlation (r) between these parameters are given in the table.

A significant linear correlation was observed between tPA-Ag and Dd-Ag. Moreover, plasma levels of these two parameters were inversely correlated to the logarithm of the CPK peak level. Thus abnormaly high tPA-Ag levels are detected in plasma from peripheric venous blood when likely marked fibrin lysis occurs and if AMI size is limited.

These data suggest that impaired fibrinolysis is probably involved in the .progression of coronary occlusion during the early stage of AMI.
PLATELET FUNCTION AND PLASMA FIBRINOGEN IN YOUNG SURVIVORS OF MYOCARDIAL INFARCTION
Authors: Berglund U , von Schenk H , Wallentin L
Published: August 2018

An increased liability for thrombosis might be of pathogenetic importance in young survivors of myocardial infarction (MI). In 73 (58 men and 15 women) patients with MI below 45 years of age and 73 matched healthy controls plasma fibrinogen and platelet function tests were studied 3-6 months after the MI. At the time of the MI 77% of the patients were smokers but at the time of the investigation 27% of the patients smoked compared to 37% of the controls. Platelet aggregabi1ity was measured in vitro in platelet-rich plasma (PRP) as maximal aggregation to ADP and collagen. The platelet sensitivity to the inhibitory effect of prostacyclin (PG12) was tested by preincubation of PRP with PG12 before inducing aggregation with ADP 5 μM. Plasma levels of beta-thrombog1obuIin (BTG) and platelet factor 4 (PF4) were measured by RIA methods and plasma fibrinogen by heat precipitation. The table presents the results (means ± SE). * is p<0.04, ** is D<0.02 and ns is non significant.

Severe emotional stress preceeding the MI occured in 7 patients - these cases had an increased platelet reactivity to ADP. The fibrinogen level was also elevated by smoking and obesity (multivariate analysis). Conclusion: young MI patients have elevated levels ol fibrinogen and reduced platelet sensitivity to PGI2. This might cause an increased thrombotic tendency.