bluuebunny/crossref_metadata_2025 huggingface dataset:
IMPAIREMENT OF PRIMARY HAEMOSTASIS BY LMW-HEPARINS
Authors: Borowska A , Lauri D , Maggi A , Dejana E , de Gaetano G , Pangrazzi J
Published: August 2018

Low molecular weight (LMW) heparlns have been developed with the aim of reducing anticoagulant activity thereby minimizing the bleeding complications of conventional heparin. Unexpectedly, bleeding events were reported during treatment with some LMW-heparins, in clinical and experimental studies. We studied the effect of four different LMW-heparlns on primary haemostasis In male rats (CD COBS, Charles River) after l.v. administration of 0.75 mg/kg b.w. of the drugs. LMW heparin A was devoid of any activity on an experimental model of “template” bleeding time in rats (110.6±5.9 sec versus 108.7±4.1 control values) whereas LMW-heparins B, C and D prolonged the bleeding time to a different extent (228.7±19.9, 161.5±6.4 and 161.7±8.6 respectively). Pretreatment of animals with aspirin (100 mg/kg b.w. per o.s). resulted In a significant potentiation of the “template” bleeding time. In vitro platelet aggregation Induced by collagen (20 μg/ml) or by collagen in combination with ADP (5-10 μM) was strongly inhibited by LMW-heparln B, while LMW-heparln A showed no effect. LMW-heparins C and D exerted an Intermediate level of Inhibition of platelet aggregation. The same pattern of aggregating response was found when LMW-heparins A and B were given i.v. to rats (0.75 mg/kg b.w.) and platelet aggregation was studied “ex vivo” 15 min after drug administration.

These data may help explain the impairment of primary haemostasis associated with some LMW-heparin preparations.
CATABOLIC BEHAVIOUR OF RECOMBINANT TISSUE PLASMINOGEN ACTIVATOR (R-t-PA) IN THE RAT
Authors: Harris R , Garcia Frade L , Poole S , Mahmoud M , Curtis A D , Gaffney P J
Published: August 2018

To compliment ongoing experimentation on the use of tissue plasminogen activator (t-PA) as a thrombolytic agent in a rat thrombosis model, clearance data for labelled R-t-PA in the rat were investigated. Both Iodogen and Chloramine T labelling procedures for incorporation yielded similar biological activity in the resultant labelled t-PA, while the half-lives in the rat circulation for Iodogen- and Chloramine T-label led materials were 5 1/2 and 7 1/2 minutes respectively. Clearance data using promoter based and ELISA assays support that obtained with 125j_ labelled t-PA. Following fractionation of various timed rat plasma samples by HPLC on a gel exclusion column (TSK G-3000 SW), 90% of the labelled t-PA was distributed between two inhibitor/t-PA complex peaks m the 1 minute sanple. One of these peaks (about 30% of labelled t-PA) was compatible with a t-PA/ PAI-1 complex, having a molecular weight of about 120,000, while the other (comprising about 60% of labelled t-PA) had a molecular weight of about 350,000 and was undetectable by ELISA, bioimmunoassay or fibrin plate assay, while showing low activity by a promoter-type t-PA assay. The two major activity peaks in these HPLC profiles of rat plasma were associated with low levels of radiolabelled t-PA and were compatible with free t-PA and a complex of t-PA having a molecular weight of about 200,000. It was observed that free t-PA was retarded during the HPLC column separations and eluted as a broad trailing peak despite the presence of 0.1% - 0.5% Tween-80 in the column. Thus molecular weights of the various complexes formed are subject to further examination. All three t-PA peaks had the same initial half-life of 2-3 minutes. Since about 60% of the t-PA is contained in the high molecular weight inhibitor complex, we propose that the formation of this complex may be a major mechanism by which t-PA is cleared from the rat circulation, despite inactive catabolic breakdown products reappearing in the circulation following clearance of these complexes.
PLASMINOGEN ACTIVATOR ACTIVITY (PAA) IN THE ISOLATED PERFUSED RAT KIDNEY (IPRK)
Authors: Müllbacher W , Maier M , Binder B R
Published: August 2018

Isolated rat kidneys perfused with albumin elaborate PAA into the urine as well as into the perfusate as assessed by the fibrin plate technique. Urinay PAA can be inhibited by addition of a monospecific antibody directed towards urokinase (uPA) while the activity in the perfusate is inhibited by an antibody against tissue type plasminogen activator (tPA). Infusion of plasminogen (45ug/g/min) into the albumin containing perfusion fluid is followed by the appearance of plasmin in the urine and perfusate and doubles PAA in the urine. Kidneys perfused with both plasminogen and the anti-urokinase antibody (0.5mg) excrete similar PAA and plasmin into the perfusate while these activities in the urine are completely abolished. Replacement of the anti-urokinase antibody by an anti-tPA antibody (0.5mg) abolishes PAA as well as plasmin activity in the perfusate and PAA in the urine is significantly decreased. From these data it is concluded that PAA in the urine of the isolated perfused rat kidney is due to urokinase and PAA in the perfusate is caused by tPA. The results suggest that plasmin activity in the perfusate might trigger increased secretion of urokinase into the urine
URINARY TRYPSIN INHIBITOR RELATED SUBSTANCE (UTIR) IN THE PLASMA AND TISSUES OF STOMACH CANCER
Authors: Nishino N , Tokura Y , Sakaguchi S , Takahashi S , Takada Y , Takada A
Published: August 2018

Urinary trypsin inhibitor(UTI) is a single chain glycoprotein with molecular weight of 67,000. In SDS-PAGE a hand appeared at molecular weight of 48,000. Results of gel filtration indicate its molecular weight of 67,000, suggesting extensive changes in its conformation in SDS. Injection of UTI into rabbit did not result in the formation of anti-UTI antibody. Conjugation of rabbit serum albumin with UTI gave rise to antibody formation upon injection into rabbits. Anti-UTI did not cross-react with anti-inter(Xtrypsin inhibitor. A highly sensitive enzyme immunoassay of UTI was developed using β-D-galactosidase coupled to Fab' of anti-UTI. The inhibition of the hydrolysis of TAMe by trypsin was used for the functional assay of UTI. Functional assay of UTI showed a good correlation of UTI antigen (UTIR) to the UTI activity in the urine. UTIR levels in the plasma of the normal male volunteers were 198.0 ± 15.7 μg/ml, and did not change with increasing age. Cancer tissue of patients with stomach cancer showed higher UTIR than surrounding healthy tissue, and urine of such patients had higher UTI.

In conclusion, UTIR were present in the plasma and tissue of normal persons, and UTI levels in the urine of cancer patients increased, and cancer tissues of patients with stomach cancer seem to have higher UTIR than that in sorrounding normal tissues.
GENERATION OF ADP BY HUMAN AND MURINE TUMOR CELLS IS SPECIFIC BUT IS UNRELATED TO METASTATIC POTENTIAL
Authors: Jamieson G A , Grignani G
Published: August 2018

The ability of tumor cells to activate platelets may facilitate the metastatic process. It has been generally assumed that the production of ADP by tumor cells is due to non-specific damage during harvesting in vitro or, in vivo, by frictional interactions with the capillary wall. The present work shows that tumor cell ADP arises not from cell damage but by a specific process under metabolic control. The human 253J urinary carcinoma cell line activated heparinized human platelets by an ADP-dependent mechanism based on inhibition by CP/CPK and the identification of aggregating concentrations (1 uM) of ADP in the cell-free supernatant by HPLC. Tumor cell damage during harvesting was shown not to be a factor since (i) the amount of ADP secreted was unrelated to the appearance of LDH, (ii) was similar when measured in confluent monolayers, in tumor cells after detachment and resuspension or following crossover studied in HBSS and MEM, and (iii) was constant at varying tumor cell concentrations. Metabolic control of ADP generation or transport was indicated by the fact that it was reduced 50 in tumor cells treated with p-chloromercuribenzene sulfonate and was completely abolished in those treated with iodoacetic acid. In order to determine whether this metabolically controlled generation of ADP was related to metastatic potential, we carried out identical experiments with the FI (low) and F10 (high) metastatic variants of the Bl6 murine melanoma line. The amounts of ADP produced by the B16 cells were about twice as great as with the human 253J cells but there was no significant difference between the amounts of ADP generated by FI and F10 variants. These studies demonstrate that ADP production by tumor cells is a discrete process under metabolic control but is not directly related to the metastatic potential of individual tumor cell lines.
FACTOR XIII AND ITS NATURAL PLASMATIC TARGETS:FIBRINO-GEN,FIBRONECTIN AND ALPHA2-ANTIPLASMIN IN MALIGNANT DISEASE
Authors: Kioczko J , Wojtukiewicz M , Bielawiec M , Pilecka E
Published: August 2018

The relevance of fibrin formation and its stabilization in tumour growth and metastasis formation seems to be important but it still remains unclear.The role of F.XIII in this process may emerge from a variety of mechanisms it is involved in,including crosslink of fibrin molecules,α2AP incorporation into fibrin and reciprocal crosslink of fibronectin,fibrin and collagen. We studied F.XIII and its natural targets such as fibrinogen , fibronectin and α2AP in 61 patients with various inoperable neoplasms (21 with Ca mammae,18 with Ca ventriculi and 22 with Melanoma malignum).F.XIII activity was measured by dansylcadaverine incorporation method acc.to Lorand et α2F.XIII subunit “a” and “b”, f ibronect in , α2AP , AT III, α22M , α2AT and C-I plasma concentrations were estimated by means of rocket immunoelectrophoresis acc.to Laurell using monospecific antisera (Behringwerke AG,Marburg).The following tests were performed in all cases:fibrinogen concentration, euglobulin lysis time and serum FDP.In comparison with healthy subjects the patients revealed statistically significant decrease in F.XIII activity and its subunit “a“ and “b“ concentrations concomitant with lowered fibronectin and α2AP levels.No statistically significant correlations were found between F.XIII and its natural plasmatic substrates.Furthermore,the malignant patients showed decreased AT III concentrations,whereas α2CiI and α2AT levels were elevated.Prolongation of ELT concomitant with an increase of FDP concentration were also found. Differences in F.XIII level and in other haemostasis parameters were stated between different tumour types. Our data indicate that the role of F.XIII in malignancy is not limited to fibrin stabilization but its interactions with fibronectin and α2AP should be taken into account.
PROTHROMBIN AND ANTITHROMBIN III IN PATIENTS WITH HEPATOCELLULAR CARCINOMA
Authors: Leone G , De Stefano V , Ferrelli R , Barone C , Garufi C , Bizzi B
Published: August 2018

Prothrombin (F.II) and antithrombin III (AT III) levels were measured in 11 patients (mean age 61 years) with hepatocellular carcinoma; F.II antigen (Ag) mean levels (Laurell) were 1.39±0.53 U/ml and F.II activity (Ac) (clotting method) 0.9±0.21 U/ml; AT III Ag mean levels (radial immunodiffusion) were 1.18±0.32 U/ml and AT III heparin cofactor (HC) (amidolytic method) 1.15±0.31 U /ml. In 5 patients F.II Ag was higher than 1.2 U/ml; no patient had F.II Ag lower than 0.8 U/ml (normal range 0.7-1.2 U/ml). F.II Ac was in the normal range in all patients. In 4 patients both AT III Ag and HC were higher than 1.2 U/ml; no patient had AT III Ag and HC lower than 0.8 U/ml (normal range 0.75-1.2 U/ml). Seven patients had a long history of liver cirrhosis and 2 of them sho wed AT III Ag and HC of 1.8 U/ml; one of these two patients had F.II Ag and Ac around 1.00 U/ml, whereas the other had F.II Ag 2.4 U/ml and F.II Ac 1.2 U/ml. In these two patients a prelimina ry more extensive study was performed. In both subjects AT III plasma crossed immunoelectrophoresis was normal in the presence and absence of heparin and AT III crossed immunoelectrofocusing (CIEF) showed a normal pattern of 6 peaks (pH 5.2-4.6) and two additional small peaks at pH 4.5 and 5.4. In the patient with in creased F.II Ag the CIEF of plasma prothrombin showed a large peak with asymmetric branches at pH 5.2-4.9, as in the control, and a large additional peak at pH 5.9; after plasma absorption with Al(OH), the F.II CIEF pattern showed only the abnormal peek. We conclude that in patients with hepatocellular carcinoma F.II and AT III are normal, independently of previous history of cirrhosis; moreover, in agreement with previous studies (N.Engl.J. Med. 310,1427,1984), an abnormal prothrombin, which we demonstra ted characterizable by the CIEF, can be synthesized.
A STUDY OF THE ANTI-THROMBOTIC POTENTIAL OF LOW MOLECULAR WEIGHT HEPARIN LHN-1 (NOVO) IN NORMAL VOLUNTEERS
Authors: Johnston R V , Orr M , Rumley A , McLachalan J , Forbes C D
Published: August 2018

Studies of low molecular weight heparin have shown a molecular sized dependency of the anti-coagulant activity. We studied the effects of a low molecular weight heparin LHN-1 (Novo) with a mean molecular weight of 5-7000 daltons on the coagulation mechanism and platelet function of normal volunteers. The heparin was given for 5 days on a once daily dose of 2500, 5000 or 7500 anti-Xa units to 3 groups of volunteers and in a twice daily regime of 2500 and 5000 anti-Xa units in 2 further groups of volunteers. After subcutaneous injection LHN-1 produced a significant (p<0.01) increase in anti-Xa activity which peaked between 3-4 hours after subcutaneous injection on both once and twice daily regime. On once daily regime there was no significant measure able anti-Xa activity 24 hours after the last injection. There was a small but significant increase in both KCCT and thrombin time (p<0.01) following injection, which was also dose related. Bleeding time did not change and there was no effect on platelet function. There was a significant (p<0.01) increase in fibrinolysis as measured by the fibrin plate method. There were no bleeding problems. These findings would suggest that LHN-1 merits further clinical evaluation to confirm its anti-thrombotic and profibrinolytic potential.
DIFFERENT LOW MOLECULAR WEIGHT HEPARINS (LMWHs) ARE NOT IDENTICAL IN THE IN VITRO SCREENING REGARDLESS OF POTENCY ADJUSTMENT
Authors: Hoppensteadt D , Fareed J , Walenga J M , Emnuele R M , Racanelli A
Published: August 2018

There is considerable controversy in the standardization and potency evaluation of low molecular weight heparins (LMWHs).

Since these agents are produced by fractionation or depolymerization procedures, the resulting material shows marked compositional variations regardless of the similarity in molecular weight. In order to address the question “are different LMWHs the same?”, we utilized a uniform multiparametric in vitro test battery. Seven commercial LMWHs Choay Fraxiparine (CY 216), Choay CY 222, Novo LHN, Kabi Fragmin, Opocrin 2123 (OP), Hepar RD 11885 (RD), Pharmuka Enoxaparine (PK) and an unfractionated porcine mucosal heparin (PMH) were studied at equigravimetric amounts in established assays. PMH and LMWHs were ranked acdording to the dose-dependent effects and the results are summarized in the following:

As evident from the above data, each of the agents studied showed a distinct profile in the in vitro test system studied. This data suggests that LMWHs are individual drugs with marked differences in in vitro actions, which may not be the true determinants of the relative safety/efficacy in clinical settings.
SUBCUTANEOUS ADMINISTRATION OF A LOW MOLECULAR WEIGHT HEPARIN (CY 222) COMPARED WITH SUBCUTANEOUS ADMINISTRATION OF STANDARD HEPARIN IN PATIENTS WITH ACUTE DEEP VEIN THROMBOSIS
Authors: FAIVRE R , NEUHART E , KIEFFER Y , TOULEMONDE F , BASSAND J P , MAURAT J P
Published: August 2018

68 patients (pts) with acute deep vein thrombosis (DVT) were randomized to either CY 222 (Choay Institut, Cl, France) (n=33) in a dose of 750 u anti-Xa Cl/kg/day or standard heparin (SH) (n=35), 500 ui/kg/day, both given by 2 daily subcutaneous (sc) injections for 10 days. In SH group, dose was monitored by daily activated partial thromboplastin time to maintain a prolongation between 2 to 3 times the control value ; in CY 222 group dose remained unchanged during the 10-day treatment. Venography was repeated at day 10. Initial DVT localization and derived Marder’s score was similar in both groups (14.1 ± 11.7 in CY 222 group and 14.3 ± 10.6 in SH group, p = NS).

The results show that : 1/ no bleeding complication was observed in CY 222 pts, but large hematoma occured in 3 SH pts, 1 patient in each group developed a recurrent pulmonary embolism and 2 others pts (1 in each group) did not undergo control phlebography ; 2/ in CY 222 group, 8 pts obtained complete lysis (CL, thrombolysis more than 90%), 11 partial lysis (PL, thrombolysis between 30% and 90%), and 11 no change (NC, less than 30%) ; 3/ in SH group, 4 pts obtained CL, 15 PL, 8 NC and extension was seen in 2 pts. Thus, phlebographic improvement (thrombolysis more than 30%) was observed in 65% in both groups.

In conclusion, these preliminary results suggested that sc administration of CY 222 and SH, both given by 2 daily injections, was equally effective in pts with acute DVT, and CY 222 seemed to be safer than SH.
TREATEMENT OF PULMONARY EMBOLISM BY A VERY LOW MOLECULAR WEIGHT HEPARIN FRAGMENT (CY 222), A DOSE RANGE STUDY
Authors: Quilliet L , Charbbonnier B , Raynaud PH , Delahousse B , Toulemonade F , Brochier M L
Published: August 2018

Experimental studies showed that CY 222 (MW 2500 - 250 U AXAIC/ mg - 25 Ul/mg) kept the profibrinolytic properties of heparin with a considerable reduction of bleeding risk and that the optimal dose was about 1 000 U AXAIC/kg/day in rabbits. We have tested the dose related effectiveness and tolerance of this drug in 47 patients (pts) with acute pulmonary embolism (PE) less than 5 days. Pts were divided into 3 groups receiving : Group I : 500 (n = 16), Group II : 750 (n = 17), Group III : 1 000 (n = 14) U AXAIC/kg/day by continuous intravenous infusion for ten days. Effectiveness was appreciated by Miller’ s index with pulmonary angiography (PA) performed before, on the 5th day and on the 10 th day of treatment. The decrease of Miller’ s index mean value was comparable in the 3 groups. But the improvement was faster in the group III

We noticed 5 recurrent PE (2 in Group I, 1 in Group II and 2 in Group III) of which one was fatal (Group I), and 2 hematoma at the venous punction site in Group III.

In conclusion, CY 222 is a safe treatment of PE and the most effective dose seems to be 1 000 U AXA IC/kg/day in spite of minor bleeding.
STUDIES ON THE ANTICOAGULANT AND ANTITHROMBOTIC ACTIONS OF DERMATANS AND THEIR SULFATED DERIVATIVES
Authors: Hoppensteadt D , Kumar A , Fareed J , Mardigian J
Published: August 2018

Non-antithrombin III mediated effects such as interaction with heparin cofactor II, modulation of endothelium and polymorphonuclear leukocytes contribute to the overall antithrombotic effects of glycosaminoglycans. In order to study the role of these dermatans, we investigated their in vitro anticoagulant effects using the clot based (PT, APTT, TT, and Heptest), antiprotease (anti IIa and anti Xa) and Thromboplastin C activated fibrinopeptide A generation test. The in vivo antithrombotic actions were investigated, against activated and non activated prothrombin complex concentrates, and in combination with Russells viper venom in jugular and femoral vein stasis thrombosis models (rabbit). The dermatans studied consisted of a standard dermatan of porcine intestinal origin and four sulfated dermatans with varying degrees of sulfation. All of the dermatans studied showed weak anticoagulant effects on the routinely performed clot based assays. Marked variability was seen on the protease inhibition (anti Xa and anti IIa) assays. In the in vivo studies all dermatans studied showed varying degrees of antithrombotic actions against various thrombogenic agents in a modified stasis thrombosis model. Sulfation appeared to produce stronger anticoagulant effects as determined by in vitro assays, whereas the intravenous antithrombotic actions of native dermatan were stronger than sulfated derivatives. This data suggests that dermatans produce their antithrombotic actions via non-antithrombin III mediated pathways. Furthermore, in vitro testing methods are of limited value in the evaluation of the biologic actions of dermatans and their derivatives.
DERMATAN SULPHATE (DS) INDUCES t-PA RELEASE IN THE PERFUSED RAT HINDQUARTERS
Authors: Mussoni L , Abbadini M , Zhu G J , Maggi A , Pangrazzi J , Donati M B
Published: August 2018

Previous studies have shown that heparin and related glycosaminoglycans (GAGs) were able to Induce the release of plasminogen activator (PA) activity In perfusion models. Among GAGs, DS has recently attracted Interest due to Its antithrombotic effect devoid of haemorrhaglc complications In experimental models of venous thrombosis. We report here that highly purified DS (MF-701, Mediolanum Farmaceutici, Milano, Italy) is able to induce t-PA activity release. Non heparlnlzed animals were perfused with oxygenated Tyrode’s solution at a constant flow rate of 10 ml/min. PA activity of the perfusate (collected at various time Intervals from vena cava) was measured by a fibrin plate method and expressed as mtl/ml by comparison with a reference curve of standard t-PA (WHO). Three minutes perfusion of different doses of DS (0.1, 0.2, 0.4 mg/ml) Induces a dose-dependent Increase in PA, activity (from 35 to 60, 99, 127 mU/ml respectively), being maximal during the first and second minute of drug perfusion. A further increase In DS concentration (0.6-0.8 mg/ml) resulted in an inhibition of PA activity released. The PA activity present In the perfusate of animals treated with 0.4 mg/ml DS has been characterized as associated with t-PA-like molecules on the basis of the relative mobility In SDS-PAGE followed by fibrin autography (67,000 m.w.) and inhibition by an antiserum against t-PA In this system. This effect was peculiar for DS, since none of the HMW heparlns tested induced PA activity release In this experimental model. These data may suggest that during “In vivo” infusion of DS In a thromboembolic condition some t-PA could be released from the endothelial cells and bind to fibrin In the thrombus to favour its dissolution.
KINETICS OF EXPERIMENTAL ANTITHROMBOTIC ACTIVITY OF MF 701 DERMATAN SULFATE
Authors: Morani A , Gianese F , Bianchini P
Published: August 2018

In order to evaluate the intensity and duration of the prophylactic activity of dermatan sulfate against thrombosis induced by ligature of the inferior vena cava (Reyers'model), we treated 6 groups of rats at increasing intervals of time before ligature (1 min, 30 min, 45 min, 2 h, 16 h). Within each group the rats were injected subcutaneously with saline solution (controls) or increasing doses (2.5, 5, 10 and 20 mg/kg) of MF 701, dermatan sulfate (Mediolanum Farmaceutici, Milan, Italy). Two hours after ligature the presence of thrombi and their weight were recorded.

A significant, dose-dependent antithrombotic effect was observed for all the treatment times. The maximum effect occured when the compound was administered 45 min before ligature (total abolition of thrombosis at the dose of 5 or more mg/kg). Total inhibition was also observed at 2 h (with 10 mg/kg) and at 4 h (with the same dose). A significant antithrombotic effect, over 50% com pared with the controls, was still found at 16 h at the dose of 20 mg/kg.

These results confirm the experimental antithrombotic efficacy of dermatan sulfate already reported in a different animal model by F. Fernandez et al. (Br J. Haematol. 64, 309, 1986) and demonstrate the long duration of the prophylactic effect of the compound.
EVALUATION OF AN AUTOMATED COAGULATION ANALYZER
Authors: Johnston M , Andrew M
Published: August 2018

The ACL (IL-Automated Coagulation Laboratory from the Fisher Scientific Group) is the first microcentrifugal analyzer incorporating 2 reading channels, a coagulometric channel consisting of a laser light source and a chromogenic channel consisting of a halogen light source. We have evaluated the instrument for precision and accuracy using different reagents for both clotting and the chromogenic assays. Replicate samples were run in both the PT and APTT modes using 4 different reagents. The reagent with the least particles had the greatest precision. The mean values for APTTs and PTs using a particle free reagent were 68.7 ± .83“ for the APTT (C.V. 1.2%), and 30.2 ± .33” (C.V. 1.1%) for the PT. A fibrinogen assay measured on the ACL (delta light scatter of the prothrombin time) was compared to the Clause fibrinogen assay in three population groups: the adult, the newborn and patients receiving thrombolytic therapy. The correlation for the adult and newborn was good with r values of .911 (n = 51) and .96 (n = 36) respectively. The thrombolytic therapy group had poor correlation r = .32. Antithrombin III (ATIII) and a2 antiplasmin (AP) assays were measured on the ACL using IIs chromogenic method. These were compared to an ATIII chromogenic method (Kahle) using a Gilford SBA 300 analyzer and α2 AP assay using a Protopath (Dade). The correlations were ATIII, r=.95 and α2 AP, r=.82. The plasminogen method of Friberger was adapted to the ACL giving us comparable results to those read off the Gilford SBA 300 (r=.93). With the introduction of the ACL we have been able to: 1) reduce the technical time required for assays by one half; 2) reduce reagent costs by one half to three-quarters; 3) reduce the amount of plasma required for screening tests by half the volume, which has greatly facilitated neonatal coagulation testing
ANTITHROMBIN III, PLASMINOGEN, α2 ANTIPLASMIN, PREKALLIKREIN AND HEPARIN ASSAYS ON AN AUTOMATED COAGULATION LABORATORY (ACL 810) ANALYZER
Authors: Mammen E F , Farag A M , Sachs R J , Brown J M
Published: August 2018

A recently introduced automated, microcomputer controlled centrifugal analyzer ACL 810 (Instrumentation Laboratories, IL) performs both clot based and chromogenic substrate based assays. We compared the performance characteristics of this new instrument with the Multistat III Centrifugal Analyzer (MCA, Instrumentation Laboratories) and the Protopath system (American Dade). Antithrombin III (AT) and plasminogen (PLG) were assayed on the ACL 810 using II kits, on the MCA using Kabi kits, S-2238 and S-2251, and on the protopath Dade reagents. 02 Antiplasmin (AP) was determined on the ACL with kits from General Diagnostics and on the MCA with the Kabi kits S-2251. On the protopath we used Dade reagents. Prekallikrein (PK) was analyzed on the ACL and MCA using Kabi kits S-2302. We adapted the plasminogen channel of the ACL for this assay. The correlation data for these four assays were as follows:

AT and PLG assays on the ACL using Kabi kits correlated equally high.

We also compared heparin assays, both anti IIa and anti Xa, on the ACL (anti IIa, II kits, anti Xa, Stago kits) with the MCA (anti Xa) and a spectrophotometer (anti IIa). The data are summarized below:

These data suggest excellent correlations between the new ACL 810 and other instruments for these assays.
EFFECT OF ORAL ANTICOAGULANTS ON STABLE ROSETTE FORMATION
Authors: Aydar S , Alataş S , Numanoğlu L , Sönmezdağ A
Published: August 2018

Human peripheral blood T lymphacytes when cultered in the presence of mitogen Phytohemogglutinin (PHA) acquire the capacity to form E rosettes with sheep erythrocytes that are resistant to incubation at 37° C. Whereas human thymus lymphocytes form 37° C stable E rosettes. On the other hand, it is shown that the use of anticoagulants can prevent cancer metastases which brings forth the importance of explaining the relationship between the lymphocyte functions and anticoagulant action mecha-nismus. In order to investigate this relationship, we did a group af experiments with lymphocytes of normal children and of children with severe burn wounds. Peripheral blood lymphocytes were seperated by “Lymphoprep” centrifugation technique. The lymphocytes of normal children and patients with burn were divided in two groups: A-Activated lymphocytes: 1×106 /ml lymphocytes were cultured and activated by PHAfor 48 hours at 37° C in RPMI 1640. B-Non activated lymphocytes were in culture witout PHA. 1×10™6 M/ml warfarin sulfate was added to some of the cultures of each group prior to the culture conditions. At the end of the 48 hour incubation, heat stable rosette formation was determined by the method of Wauve and co-workers. Significantly elevated levels of heat stable rosette forming cells were found in the PHA activated culture treated with warfarin sulfate in normals and patients with burn. Although the blastic transformation of T lymphocytes was found to be depressed, heat stable rosette formation of warfarin sulfate treated lymphocytes abtained from burn patients was observed to be significantly elevated. It is concluded that warfarin sulfate increases the activity of T lymphocytes by interfering with the resynthesis of heai stable E receptors.
COMPUTER PREDICTION OF ANTICOAGULATION STATUS AND WARFARIN DOSE FOLLOWING CARDIAC SURGERY
Authors: Crow M J , Latif A B , Critchley A I , Stainton C , Nealon P , Rajah S M
Published: August 2018

Fluctuations are freguently seen in the anticoagulant status of patients in the immediate post operative period following prosthetic heart valve replacement. These patients are at high risk of haemorrhage or thromboembolism. We have used a pharmokinetic model of warfarin metabolism to develop a computer programme to predict the maintenance dose of warfarin from early prothrombin activity determinations. This will enable controlled anticoagulation to be achieved. The expression for warfarin kinetics employs 4 constants determined by the residual sum of the sguares, which are used immediately to redefine dosage predictions. In a pilot study data obtained from 16 patients post operation 3, 5 and 7 days after commencing treatment, has been used to predict the reguired maintenance dose at 21 days. These predicted doses were then compared with the maintenance dose achieved by clinical practice. The programme was told to optimise its dose to achieve a PT ratio of 3 whereas clinically the ratio was allowed to vary in the therapeutic range of 2 to 4. Predicted doses at 21 days are shown.in the table:

Correlation between predicted and clinical maintenance doses after 3 and 5 days treatment was poor but had improved significantly by 7 days, despite similar levels of prothrombin activity. Predicted prothrombin activity never exceeded the upper limit of the therapeutic range, and the predicted dose can be uprated on addition of further data within 2 minutes.

After 7 days computer predicted warfarin dose has produced a good correlation with the clinical maintenance dose (the doses of only 3 patients varying by more than 1 mg/day). The significant fluctuations seen in the prothrombin ratio during clinical dosage were not observed with computer dosing and we now feel it is safe to use this programme to anticoagulate patients post operatively.
THROMBIN KINETIC PARAMETERS WITH TRIPEPTIDE p-NITROANALIDES UNDER PHYSIOLOGICALLY RELEVANT CONDITIONS
Authors: Fenton J W , Witting J I , Miller T M
Published: August 2018

The Michaelis-Menten (Km ), catalytic (kcat ), and specificity (kcat/Km ) constants were determined for humSft α- and β-thrombins with the chromogenic substrate S-2238 (H-D-Phe-Pip-Arg-pNA), Chromozym-TH (Tos-Gly-Pro-Arg-pNA), and Spectrozyme-TH (H-D-HHT-Ala-Arg-pNA) in 0.15 M NaCl buffered with 10 mM HEPES ancTlO mM Tris-HCT at pH 7.4, 37°C. Spontaneous hydrolysis was insignificant for these substrates. Both S-2238 and Spectrozyme-TH exhibited limiting solubilities at ∼35 μM, while Chromozym-TH did not do so up to 50 μM. From initial estimates, Km's and k t's were refined by computer Gause-Newton iterations or nonlinearneast-square fits for the concentration of p-nitroanalide formed per second versus the initial substrate concentration. No major differences were found between a-thrombin (99% α, 91% esterolytically active enzyme, and > 3,500 kilo-U.S. clotting units/g with fibrinogen) and Yγ-thrombin (98% γ. 89% active enzyme, and < 10 kilo-units/g). For α- versus γ-thrombin and the three substrates, respectively, the Km 's were 6.75 ± 0.13 vs 7.62 ± 0.30, 18.4 ± 0.4 vs 23.0 ± 0.3, and 2.53 ± 0.02 vs 3.85 ± 0.51 μM; the kcat> s were 125 ± 1 vs 134 ± 2, 181 ± 2 vs 130 ± 1, and 35.5 ± 0.2 vs 52.4 ± 2.0 s−1 ; and the kcat/Km 's were 18.5 vs 17.6, 9.84 vs 5.65, and 10.1 vs 13.6 s−1 μm . These values closely approximate those of 7.2 ± 0.9 μM, 84 ± 4 s−1 , and 11.7 s−1 μM−1 determined by Higgins, Lewis, and Shafer (J. Biol. Chem. 258:9276-9282, 1983) for the Aα cleavage of human fibrinogen by human α-thrombin under physiologically relevant conditions. Thus, these chromogenic substrates have thrombin specificities similar to that of fibrinogen, although their amidolytic activities are independent of additional active-site regions required for fibrinogen clotting activity (α vs γ-thrombin). Fibrinogen interactions with such active-site regions might account for why the fibrinogen Aa site is an atypical thrombin susceptible bond and the high species variability of fibrinopeptides. (Supported in part by NIH grant HL-13160).
FACTORS XI ANDXII - MICROTITRE AMIDOLYTIC ASSAYS VERSUS CLOTTING ASSAYS.
Authors: Walshe K , Mackie I J , Gallimore M , Machin S J
Published: August 2018

Coagulation assays for FXI and FXII are technically difficult and require expensive human substrate plasmas with a risk of viral contamination: these factors have hampered the study of the contact system in thrombosis and shock. Amidolytic microtitre plate assays have been developed and compared with 1-stage clotting assays. The FXII assay utilises acetone removal of FXIIa inhibitors, blockade of kallikrein and α-2-macroglobulin and S-2222. The FXI assay involves, inactivation of FXI inhibitors, blockade of kallikrein and FXIIa, and measurement of FXI with S-2366. The FXII amidolytic assay was linear up to 200%, and sensitive to < 1% FXII, with a cv of 5%. It was not affected by decreased levels of FXI, PKK, or FVII. Normal and deficient patients gave an excellent correlation with clotting assays (r=0.96). The normal range (mean +/- 2SD) for FXII was 0.73-1.45 and 0.56-1.48 in amidolytic and clotting assays respectively. Females gave higher values than men in both assay types. The FXI amidolytic assay showed similar linearity, sensitivity and reproducibility and was unaffected by decreased levels of FXII and PKK. 2 0 normals showed a good correlation with the clotting assay (r=0.75), although the latter gave consistently, and significantly higher values (p< 0.05). The normal range was 0.61-1.41 and 0.60-1.42 in the amidolytic and clotting assays respectively, females again giving higher values than males. Both amidolytic assays are more sensitive, reproducible and economical, using 25% of the reagents for ’tube-type’ amidolytic assay, and cheaper reagents than clotting assays. A human substrate plasma is not required, making them safer; large numbers of samples may be rapidly tested. The higher levels of FXI and FXII seen in females may be related to the oral contraceptive pill.
AN INTERLOCKING SINGLE-STRAND MODEL FOR FIBRIN POLYMERIZATION.
Authors: Hunziker E B , Straub P W , Haeberli A
Published: August 2018

The early stages of fibrin polymerization were investigated by rotatory shadowing and electron microscopy. Individual molecules within initial oligomers were found to be unaligned and contacted neighbouring molecules by single E + D and D + E contacts, suggesting an intermediate phase of activation (des A-fibrin). The interacting molecular domains were separated by a distance of 2 to 3 nm, indicating that (both or at least one) binding sites are located on protruding segments of the polypeptide chains. Upon completion of fibrin activation (des AA-fibrin), molecules within the early oligomers aligned to form single-stranded polymers,o these being characterized by repeating trinodular units of 230 A in length. Based upon these findings, a new interlocking single-stranded model for fibrin polymerization was designed and tested. The model is consistent with previous experimental data on fibrin polymerization such as that obtained by X-ray diffraction and negative staining. Moreover, early branching and lateral association phenomena are easily explained.
THE PEPTIDE β43-47WHICH INCREASES MICROVASCULAR PERMEABILITY IS RELEASED BY PLASMIN DURIIG CLEAVAGE OF FRAGMENT Y OF FIBRINOGEN
Authors: Cierniewski C S , Poniatowski J , Urbanczyk J
Published: August 2018

Pentapeptide Ala-Arg-Prv-Ala-Lys corresponding to β43-47 is known to increase microvascular permeability and is supposed to be involved in the establishment of pulmonary edema in conditions with fibrin accumulation in the lungs (Belew, et al., Thromb. Res. 13, 983-994, 1978). This peptide also induced dilation of bovine mesenteric arteries and caused a release of prostacyclin as well as an increase in cyclic AMP in blood vessels (Anderson, et al., Thromb. Res. 30, 213-218, 1983). The aim of this report was to demonstrate at which step of plasm in degradation this physiologically active peptide is released from fibrin(ogen) to the environment. For this purpose the pentapeptide and its analog Tyr-Ala-Arg-Pro-Ala-Lys were synthesized and used to produce specific antibodies in three rabbits. Antibody titers were determined by radioimmunoassay using radioiodinated Tyr-Ala-Arg-Prtr-Ala-Lys and fibrinogsn and the highest titer antisera from each rabbit were selected. Antibodies raised against β43-47 recognized during immunoblotting intact fibrinogen, fragments X and Y as well as the Bp chain. Since fragment Y is the last product which reacts with anti-β43-47 antibodies, splitting of fragment Y into fragment D and fragment E must be accompanied by plasmin cleavage of the peptide bond βLys47-Ala48. Anti-β43-47 antibodies precipitated also radioiodinated fibrinogen which was not exposed to sodium dodecyl sulfate confirming our previous observations (Cierniewski and Edgington, Thromb. Res. 14, 747-764, 1979) that sequence 43-53 of the β chain is exposed on the hydrated surface of intact fibrinogen molecule.
A NEW CASE OF A CONGENITAL COMBINED HYPO-DYSFIBRINOGENEMIA?
Authors: Hamulyák K , Muller A D , Hemker H C
Published: August 2018

A laboratory screening was performed in the case of a one month old female child because of a mild umbilical stump haemorrhage. This screening showed a low amount of thrombin clottable fibrinogen (method according to Clauss: 0.2 gr/l) but no other abnormalities. Fibrin(ogen) degradation products could not be detected; factor XIII: 30% ; the amount of fibrinogen related antigen (rocketimmunoelectrophoresis) was at least 3 fold higher than the amount of thrombin clottable fibrinogen. In "heat defibrinated" samples this discrepancy was much more pronounced. Crossed immunoelectrophoresis in the absence and presence of calcium showed a slightly abnormal form and position of the precipitation arc. Reptilase induced fibrin clot formation appeared to be less abnormal than thrombin induced fibrin clot formation. These preliminary results provide evidence for the presence of a combined hypo-dysfibrinogenemia. These results do not allow us to conclude that our case can be distinghuished from other abnormal fibrinogens. We have planned In the near future the definitive purification and characterization. A remarkable point in our case is, that the family history appeared to be negative, as well as the preliminary results of the laboratory investigations of the father, mother and sister. Consanguinity between the parents was absent. We assume that our case is an example of a spontaneous mutation, in contrast to most of the described hypo- and dysfibrinogenemias, which follow a dominant inheritance pattern.
FIBRINOGENS NEW YORK II, III, AND IV : RELATIONSHIP BETWEEN ABNORMAL FIBRIN MONOMER POLYMERIZATION, BLEEDING TENDENCY, AND THROMBOTIC TENDENCY
Authors: Karp George , Liu Chung Y
Published: August 2018

It has been proposed that a normal fibrin monomer polymerization is required for a normal fibrin potentiation of tPA-induced plasminogen activation. Accordingly, an abnormal fibrin moncmer polymerization may contribute to a thrombotic tendency. To investigate this possibility, three abnormal plasma fibrinogens (dysfibrinogenemia) have been studied and tentatively designated New York II (NY-II), New York III (NY-III), and New York IV (NY-IV) after the city where they were described.

Each of these patients has a long thrombin clotting time (TCT), a long Reptilase clotxing time, and an abnormal fibrin monomer polymerization. The abnormal fibrin moncmer polymerization can be corrected by addition of Ca2+. NY-II is characterized by a normal level of plasma fibrinogen, but delayed fibrino-peptide release and marked prolongation of the TCI; NY-III, by a normal level of plasma fibrinogen and a normal fibririopeptide release; and NY-IV, by a low level of plasma fibrinogen, a normal fibrinopeptide release, and marked prolongation of the TCT. Each of these cases appears to be congenital, not acquired although the absence of data on relatives prevents definite conclusions. SDS-PAGE analysis of the mobility of the reduced abnormal fibrinogens shows no detectable abnormality, indicating no significantly altered molecular size of Aα, Bβ, and γ chains.

No patient appears to be associated with either a bleeding or a thrombotic tendency. Thus, no direct association is observed between an abnormal fibrin monomer polymerization and a thrombotic tendency. Accordingly, fibrin monomer polymerization is not a function absolutly required for potentiation of plasminogen activation. However, based on the present observations and the observations previously described of abnormal fibrinogens with abnormal fibrin monomer polymerization from patients associated with bleeding and/or thrombotic tendency, a bleeding tendency seems to be correlated with a condition in which both a long TCT and an abnormal fibrin moncmer polymerization can not be normalized by addition of Ca2+.
FIBRINOGEN LONG BEACH
Authors: Pirkle H , Theodor I , Vukasin P , Nandi B , Miyada D , Baker J , Dukes M
Published: August 2018

We find that, in addition to a normal fibrinopeptide A (FPA), fibrinogen Long Beach releases an abnormal FPA whose HPLC behavior suggests and whose amino acid analysis confirms the substitution of His for Arg at position 16, the thrombin cleavage site. However, this dysfibrinogen displays properties strikingly different from those previously reported to be associated with Aa 16 Arg → His substitutions. These properties include a limited release of normal FPA (18-20%) by both thrombin and batroxobin, an essentially full release of abnormal FPA (40-50%) by thrombin (1.5 NIH u/ml) in contrast to a 7-8% release of abnormal FPA by batroxobin, and a complete failure of highly purified batroxobin to clot the dysfibrinogen while generating a fragment which migrates between the B8 and y chains on SDS gel electrophoresis. The basis for these differing properties is not yet clear.
MICROCARRIER CULTURE OF HUMAN ENDOTHELIAL CELL TYPES - A SOURCE OF METABOLITES
Authors: Hunter N R , MacGregor I R , Dawes J , Pepper D S
Published: August 2018

The production of human endothelial cell secretory products in amounts sufficient for biochemical studies is largely restricted by the culture growth area. Conventional flat bed systems yield at best 20-30 x 106 cells per 180cm2 culture flask. To overcome this problem, cells may be grown on Cytodex 3 microcarriers allowing large numbers of cells to be grown and conditioned in small culture volumes. A typical microcarrier unit will contain 200-300 x 106 cells and may be expanded in excess of 1000 x 106 cells at confluence. High viability (95%) and recovery (70-80%) in sub-culturing of microcarrier to microcarrier culture can be achieved with careful management of culture conditions and brief exposure to enzymes.

Human umbilical artery and vein, and saphenous vein endothelial cells were prepared and grogn on microcarrier cultures to cell populations of 200-450 x 106 cells and conditioned for 14 day periods in serum-free media.

The production profiles of several endothelial cell proteins including thrombospondin (TSP), von Willebrand Factor (vWF) and issue plasminogen activator (t-PA) were measured by radioimmunoassay under these conditions, and demonstrate the use of microcarrier cultures in producing milligram quantities of engothelial cell protein. For example, a HUVEC culture of 200 x 106 cells conditioned with serum-free media for 14 days yielded a total of 6.9mg TSP, 0.7mg vWF and 48.9ug t-PA. In this laboratory one such application of the system was the purification of endothelial proteins in amounts sufficient for immunisation of mice prior to the production of monoclonal antibodies and for subsequent characterisation.
PEROXIDE STIMULATION OF PGI3 AND DIHOMO-PGI2 IN ENDOTHELIUM
Authors: Bordet J C , Guichardant M , Lagarde M
Published: August 2018

Human umbilical endothelial cell (EC) monolayers incubated with eicosapentaenoic acid (EPA) produce small amounts of prostaglandin E3 (PGI3). We have previously shown that this metabolite is markedly enhanced in EC supernatant by co-incubating EPA with arachidonic acid (AA) (BBRC 135, 403, 1986). Moreover we found that PGF3a and PGE3 were similarly enhanced, and we concluded that such a stimulation occured at the cyclooxygenase rather than at the prostacyclin synthase level. It is generally assumed that cyclooxygenase is a peroxide-dependent enzyme and the present study shows that the potentiating effect of AA on EPA cyclooxygenation may be due to its hydroperoxy derivative, 15-HPETE. This has been established by measuring prostanoids of the trienoic series from (14-C)EPA and by detection of their metoxy-pentafluorobenzyl-trimethyl silyl derivatives from unlabelled EPA by gas chromatography-mass spectrometry. The potentiating effect of n-6 hydroperoxy derivative of linoleic acid (13-HPODE) was even higher than that of 15-HPETE. In addition, the cyclooxygenation of docosatetraenoic acid (DTA) or adrenic acid, was found to be also potentiated by 15-HPETE and 13-HPODE, but higher concentrations were required for the efficient synthesis of dihomo-PGI2. Concentrations of peroxides required for such potentiations were however far lower (−2μM) than those inhibiting prostacyclin synthase (≥100μM under our conditions). EPA and DTA, as competitive inhibitors of AA cyclooxygenation, appeared to need a higher peroxide tone than AA for their own metabolism. The biological relevance of DTA is not proved at this day, and dihomo-PGI2 has been found less active than PGI2. In contrast, PGI3 has been assumed to exhibit similar antiaggregatory effect than PGI2. EPA may then beneficially enhance the prostacyclin potential of vascular endothelium especially in conditions where a high peroxide tone is suspected like ageing or diabetes